Inherent dynamics within the Crimean-Congo Hemorrhagic fever virus protease are localized to the same region as substrate interactions
Elan Z. Eisenmesser,1* Glenn C. Capodagli,2 Geoffrey S. Armstrong,3 Michael J. Holliday,1 Nancy G. Isern,4 Fengli Zhang,5 and Scott D. Pegan2* 1
Department of Biochemistry and Molecular Genetics, School of Medicine, University of Colorado Denver, Aurora, Colorado 80224
2
Department of Pharmaceutical and Biomedical Sciences, University of Georgia, Athens, Georgia 30602 Department of Chemistry and Biochemistry, University of Colorado at Boulder, Boulder, Colorado 803093
3 4
WR Wiley Environmental Molecular Sciences Laboratory, High Field NMR Facility, Washington 99532
5
National High Magnetics Field Laboratory, Tallahassee, Florida 32310
Received 26 September 2013; Accepted 30 December 2014 DOI: 10.1002/pro.2637 Published online 6 January 2015 proteinscience.org
Abstract: Crimean-Congo Hemorrhagic fever virus (CCHFV) is one of several lethal viruses that encodes for a viral ovarian tumor domain (vOTU), which serves to cleave and remove ubiquitin (Ub) and interferon stimulated gene product 15 (ISG15) from numerous proteins involved in cellular signaling. Such manipulation of the host cell machinery serves to downregulate the host response and, therefore, complete characterization of these proteases is important. While several structures of the CCHFV vOTU protease have been solved, both free and bound to Ub and ISG15, few structural differences have been found and little insight has been gained as to the structural plasticity of this protease. Therefore, we have used NMR relaxation experiments to probe the dynamics of CCHFV vOTU, both alone and in complex with Ub, discovering a highly dynamic protease that exhibits conformational exchange within the same regions found to engage its Ub substrate. These experiments reveal a structural plasticity around the N-terminal regions of CCHFV vOTU, which are unique to vOTUs, and provide a rationale for engaging multiple substrates with the same binding site. Keywords: CCHFV; vOTU; protein dynamics; protein–protein interactions
Additional Supporting Information may be found in the online version of this article. NMR experiments were collected at multiple facilities. Part of the data was acquired at the High Magnetic Field Laboratory (NHMFL), which is supported by cooperative agreement DMR 0654118 between the National Science Foundation and the State of Florida. A portion of NMR data collection was performed at the Environmental Molecular Sciences Laboratory, a national scientific user facility sponsored by the Department of Energy’s Office of Biological and Environmental Research and located at Pacific Northwest National Laboratory. The content of this report is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Grant sponsor: The Rocky Mountain 900 Facility (used for NMR data collection); Grant number: NIHGM68928; Grant sponsor: NIH; Grant numbers: 1F31CA183206-01A1 (to MJH), GM107262-01A1 (to EZE), and Grant numbers: 1R03MH097507-01A1 and 1R01Al109008-01 (to SDP). *Correspondence to: Elan Zohar Eisenmesser, Department of Biochemistry and Molecular Genetics, School of Medicine, University of Colorado Denver, Aurora, CO 80224. E-mail: [email protected] or Scott D. Pegan Department of Pharmaceutical and Biomedical Sciences, University of Georgia, Athens, GA 30602. E-mail: [email protected]
C 2015 The Protein Society Published by Wiley-Blackwell. V
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Introduction Crimean-Congo Hemorrhagic fever virus (CCHFV) is a negative-sense, single-stranded RNA virus that belongs to the Nairovirus genus within the Bunyaviridae family. CCHFV is among several ticktransmitted viruses that have garnered increasing interest due to their emergence in Europe, which is at least partially blamed on global climate change.1 Like many viruses, CCHFV has adapted mechanisms to manipulate the host cell machinery for its own pathogenesis. For example, several RNA viruses, which include nairoviruses such as CCHFV, encode for a viral ovarian tumor domain (vOTU) that removes ubiquitin (Ub) and Ub-like interferonstimulated gene product 15 (ISG15) from proteins and thereby, downregulates the innate immune response.2 Interestingly, CCHFV vOTU protease is relatively unique in this dual substrate specificity. Specifically, while most known deubiquitinating enzymes including vOTUs target either Ub or ISG15, CCHFV vOTU appears to target both.3,4 Conjugation of Ub and ISG15 to host proteins plays a critical role in regulating antiviral proteins of the interferon (IFN) type 1 response.5,6 As a result, vOTUs, particularly CCHFV vOTU from the most lethal human nairovirus CCHFV, are currently considered potent nairovirus virulence factors because of their perceived role in subverting the host antiviral response.2,4,7,8 X-ray crystal structures have been solved of CCHFV vOTU alone,7 in complex with Ub,7–9 and in complex with ISG15,7,8 which revealed a conserved OTU-like fold of its eukaryotic counterparts. Complexes were formed utilizing recombinant Ub and ISG15 proteins derivatized with 3-bromopropylamine hydrobromide, which are suicide substrates that result in covalent bonds to the CCHFV vOTU catalytic Cys residue, which is C40. The primary difference between CCHFV vOTU and its eukaryotic homologues is the inclusion of two extra N-terminal b-strands that form an extended b-sheet, which is also present within the Dugbe virus protease (DUGV vOTU).3,4 This allows a 30 twist of the bound Ub or Ub-like domain within ISG15. However, CCHFV vOTU is nearly identical within all of the structures determined to date, whether alone or in complex with either Ub or ISG15, and there is no information regarding the potential structural plasticity of CCHFV vOTU. Additionally, the sole substrate-free structure of CCHFV vOTU has its active site obscured by an a-helix originating from a crystal contact.7 This further suggests that the direct utilization of X-ray crystallographic techniques for structural information related to CCHFV vOTU can benefit from an alternative NMR based approach. Atomic resolution investigations regarding dynamics have been conducted for other viral proteases, which include the Norwalk virus 3CLpro,10
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which is a cysteine protease, and many pivotal studies of the human immunodeficiency virus type I protease,11,12 which is an aspartyl protease. While these highly divergent proteases have been shown to exhibit dynamics on multiple timescales, a defining feature of both of these enzymes is the micro–millisecond motions observed within their active sites. Such inherent motions within macromolecules, and particularly enzymes, support the theory of conformational selection whereby molecules sample important conformations that allow for substrate binding and potentially catalysis.13 However, such conformational sampling has recently been shown to be much more complex than previously thought with highly localized “dynamic segments” nonetheless communicating with each other over large distances.14 For example, allosteric pathways in enzymes such as CheY15 and cyclophilin-A16 have been identified. Thus, our aim in this study was to first discern if the solution structure of CCHFV vOTU was similar to that in the X-ray crystal structures and then to characterize the dynamics of CCHFV vOTU in solution, in order to determine whether regions involved in substrate recognition were inherently dynamic. By utilizing NMR, the backbone assignments of substrate-free CCHFV vOTU and both the enzyme and Ub within the CCHFV vOTU/Ub complex were determined. While we were able to confirm that the average structure of CCHFV vOTU was indeed similar to that in the crystals and the average structure was unperturbed upon binding Ub, the dynamics were significantly different on the ms timescale but highly similar on the ms timescale. Of particular importance, there was a remarkable correlation between the inherent dynamics of CCHFV vOTU (i.e., motions prior to substrate engagement) and the site of engaging Ub. Thus, this study has revealed the structural plasticity of CCHFV vOTU and indicates that dynamics within the protease are largely relegated to the catalytic region.
Results The average CCHFV vOTU structure is unperturbed upon complex formation with Ub We sought to determine whether a lack of structural changes between free and bound CCHFV vOTU in the X-ray crystal structures was reflective of the solution behavior of CCHFV vOTU upon binding Ub as well. Specifically, the structures of CCHFV vOTU within the X-ray crystal structures of free CCHFV vOTU, CCHFV vOTU/Ub, and CCHFV vOTU/ISG15 exhibit an RMSD of backbone atoms of less than 0.7 ˚ and are nearly indistinguishable [Fig. 1(A)]. IsoA thermal titration calorimetry (ITC) confirmed that the affinity of CCHVF vOTU Ub for was within a range that could be used to interrogate the complex, which was 11.4 6 0.7 lM [Fig. 1(B)]. Thus, we
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vOTU is not significantly perturbed upon binding Ub [Fig. 1(C)]. We also addressed any potential structural changes between both free and bound Ub by reversing the labeling scheme and monitoring the CA chemical shift propensities for Ub in both states (Supporting Information Fig. S1). Analogous to CCHFV vOTU, Ub also showed few changes to its chemical shift propensities apart from the C-terminal region that exhibits slightly more negative values that are consistent with a more extended, b-strand like structure upon insertion into CCHFV vOTU. Thus, these NMR data are consistent with the X-ray crystal structure data and indicate little is changed for both CCHFV vOTU and Ub upon binding.
CCHFV vOTU exhibits dynamics on multiple timescales
Figure 1. The average structure of CCHFV vOTU is not significantly perturbed upon binding Ub in solution. (A) The Xray crystal structure of CCHFV vOTU alone (white, PDB accession 3PHU) and in complex with Ub (blue with Ub in green, PDB accession 3PRP). The backbone atoms and all heavy atoms of CCHFV vOTU exhibit an RMSD of less than 0.7 A˚ and 1.8 A˚, respectively. (B) ITC binding of CCHF vOTU to Ub. The top panel represents the raw heat data from 25 injections at 25 C, and the bottom panel shows the integrated heat peak areas plotted versus the molar ratio of Ub to CCHF vOTU. The line represents the best fit to an independent model after subtracting the heat generated by titrating Ub into buffer alone to account for the heat of dilution. This figure was generated using the NanoAnalyze software provided by TA Instruments. Kd 5 11.4 6 0.7 lM, DH 5 28.6 6 0.7 kJ mol21, TDS 5 57.3 6 0.6 kJ mol21, DG 5 227.7 6 0.1 kJ mol21, and n 5 1.2 1 0.04. (C) CA chemical shift propensities relative to their respective unfolded resonances are shown for both free CCHFV vOTU (white) and bound CCHFV vOTU (blue).
assigned the backbone resonances of CCHFV vOTU in both its free state as well as in the presence of Ub in order to compare the chemical shift propensities in both states. Chemical shift propensities are highly sensitive measures of secondary structure, where CA has been shown to be the most sensitive of these.17 CA chemical shift propensities were calculated by subtracting the random coil chemical shifts for each residue type from the chemical shifts measured here. Indeed, the chemical shift propensities for CA resonances were nearly identical between free CCHFV vOTU and CCHFV vOTU bound to Ub indicating that the average structure of CCHFV
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Initial analysis of R2 relaxation rates for free CCHFV vOTU at 20 C revealed that most of the elevated rates are localized to either the active site or are adjacent to the active site where the C-terminal region of Ub is known to bind [Fig. 2(A) black with those atoms exhibiting elevated R2 relaxation rates shown in red]. Since elevated R2 relaxation rates are associated with relatively slow motions on the ms-ms timescales, this is the first experimental proof that CCHFV vOTU undergoes conformational exchange prior to substrate engagement. For example, Residues 36–41 of CCHFV vOTU that includes the catalytic C40 all exhibited elevated R2 relaxation rates [Fig. 2(A), inset]. Interestingly, the amide of G38 exhibits the highest R2 relaxation rate of all amides, which may be indicative of elevated chemical exchange due to the catalytic C40 side chain that points directly toward this residue within the loop that forms the oxyanion hole. Additional residues that exhibited elevated R2 relaxation rates include T10, N11, I13, and A14, all of which reside on the Nterminal b-strand that is involved in binding both Ub and ISG15.8 While elevated R2 relaxation rates indicate that conformational exchange occurs within the ms–ms timescale (due to chemical exchange), comparisons to an R1rho experiment that employs a highpowered refocusing field can help narrow down this timescale. For example, many residues that exhibit elevated R2 relaxation rates exhibit reduced R1rho relaxation rates, which include the entire stretch of Residues 31–41 (Fig. 2, inset). This is consistent with chemical exchange phenomena that occur lower than approximately 104/s, since they are quenched upon employing a high-powered refocusing pulse within the R1rho experiment. Conversely, Q149 exhibits an elevated R2 and R1rho relaxation rate that are identical. In other words, the chemical exchange phenomena for this residue occurs on a much faster timescale than the other residues (i.e.,
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Figure 2. Relaxation rates of free CCHFV vOTU indicate that dynamics occur on multiple timescales. (A) Left, both R2 (black) and R1rho (red) relaxation rates for free CCHFV vOTU. The average R2 relaxation rate was 23.4 Hz with 0.5 SD of 2.1 Hz (red line indicates the sum of these). Right, residues that exhibit R2 relaxation rates higher than 0.5 SD from the average are shown in red. CCHFV vOTU is shown as a cartoon (white) with Ub shown as a transparent cartoon (green) to orient the reader. The catalytic Cys, C40, is shown as yellow and several residues that include G38 and Q149 are highlighted and described in more detail within the main text. Inset, blow-up of residues around C40 that exhibit suppressed relaxation rates with the higher field imparted in the R1rho experiment, indicating exchange occurs on a timescale of ms. (B) Left, R1 relaxation rates for free CCHFV vOTU. The average R1 relaxation rate was 0.77 Hz with 0.5 SD of 0.08 Hz (red line indicates the sum of these). Right, residues that exhibit R1 relaxation rates higher than 0.5 SD from the average are shown in red. These elevated R1 relaxation rates are primarily confined to loops, except for N39, Y58, and H59 that are within structural proximity and all have their side chains pointed within the same direction. All relaxation data were collected at 20 C at 900 MHz on 15N-labeled CCHFV vOTU using standard BioBack sequences.
submicrosecond), since it is unperturbed. Such experiments already highlight an important finding within recent years that ms-ms dynamics are often present within enzyme active sites, suggesting that such macromolecules are poised for function (i.e., “flexible for function”). However, it is also important to note these R2 and R1rho experiments also reveal differential dynamics within the CCHFV vOTU active site on the ms-ms timescales within CCHFV vOTU. R1 relaxation rates were also measured for CCHFV vOTU and offer insight into the dynamics on the much faster timescales of ps–ns [Fig. 2(B)]. Specifically, most residues exhibiting elevated R1 relaxation rates are confined to loop regions within CCHFV vOTU, which simply indicates that these loops are relatively flexible. Moreover, much of these residues may simply be locally disordered, as they
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are distant from one another. However, two regions structurally juxtaposed to the catalytic C40 have multiple residues with elevated R1 relaxation rates, which include N39, Y58, and H59 (Fig. 2, right). Even adjacent residues to these exhibit higher than average R1 relaxation rates, such as Residues 36–38 and 54–60. Thus, extended flexible regions even within the faster timescales are relegated to close proximity of the CCHFV vOTU active site.
Chemical exchange within free CCHFV vOTU is localized to its site of substrate engagement Considering that the dynamics monitored by R2 and R1rho relaxation rates above indicate that conformational exchange occurs within the active site of CCHFV vOTU, we sought to further characterize these dynamics through R2-CPMG (Carr-PurcellMeiboom-Gill) dispersion experiments. R2-CPMG
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Figure 3. R2-CPMG dispersion measurements reveal that CCHFV vOTU exhibits several conformational exchange phenomena. (A) Representative R2-CPMG dispersion curves collected on free CCHFV vOTU at 20 C at both 900 MHz (red) and 600 MHz (orange) were simultaneously fit as described in Material and Methods with the resulting kex given above. (B) R2-CPMG dispersion curves are shown for the same residues, but collected at 10 C at 900 MHz (blue) and 600 MHz (cyan). (C) The cocrystal complex of CCHFV vOTU (white) is shown with transparent Ub (green) to orient the reader. Residues exhibiting measurable exchange within R2-CPMG dispersions at both 20 C and 10 C are shown (red) along with residues that only exhibit measurable exchange at 10 C (blue). All data here were collected at 900 MHz at the indicated temperatures using 2H15N-labeled CCHFV vOTU measured using a TROSY-CPMG sequence.30
dispersion experiments are particularly useful in quantifying ms-ms dynamics, which is the same timescales critical for turnover of multiple enzymes as others and we have described.16,18,19 Specifically, R2-CPMG dispersion quantifies chemical exchange (i.e., Rex) as a function of an applied refocusing field (i.e., mcpmg) and can be used to obtain multiple biophysical parameters associated with conformational exchange that includes the kinetic rates of exchange (i.e., kex). Two static fields are both necessary and sufficient for accurate extraction of the biophysical parameters as previously shown20 and thus we collected R2-CPMG dispersions at both 900 MHz and 600 MHz. Moreover, since different
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dynamic processes may exhibit different temperature dependencies, we have previously employed R2-CPMG dispersion experiments at multiple temperatures16 and do so here at both 20 C and 10 C (Fig. 3). To ensure the dynamics monitored here were reflective of internal motions, and not induced by oligomeric interactions, we also performed R2CPMG dispersion experiments at multiple concentrations of CCHFV vOTU (Supporting Information Fig. S2). There were no concentration-dependent changes in the chemical exchange contributions (i.e., Rex), thereby indicating that the monitored chemical exchange was indeed a measure of internal dynamics.
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Simultaneously fitting the two R2-CPMG dispersions collected at 900 MHz and 600 MHz reveal multiple conformational exchange processes at 20 C [Fig. 3(A), top]. Specifically, several residues within the first two b-strands of CCHFV vOTU exhibit exchange such as T10, A14, and S19. Of interest, these two b-strands are the distinguishing structural features of vOTUs from their mammalian counterparts and are found in both CCHFV vOTU and DUGV vOTU.3 Thus, our finding that identify this region as being conformationally flexible is consistent with the fact that this structural feature has been implicated in the diversity of binding both Ub and ISG15. The flexibility of these two b-strands may allow CCHFV vOTU to engage both substrates. In contrast to this N-terminal region, the calculated rates of exchange processes for residues within the active site are much higher, which included Residues 36–38 [Fig. 3(A), bottom]. This finding implies that the associated dynamic exchange process of CCHFV vOTU is not uniform, but are instead segmental in nature. Such segmental dynamics have recently been shown for many proteins, which include the HIV-1 protease,12 CheY,15 as well as cyclophilin-A.16 Individual R2 relaxation rates in CCHFV vOTU were much higher for Residues 38– 42, which includes the catalytic Cys, C40. Linebroadening for these residues was extensive and only G38 could be reliably fit (Fig. 3). However, even with the highest refocusing frequency imparted, (i.e., mcpmg 5 1000 Hz), these R2 relaxation rates remained relatively high, suggesting these residues also exhibited fast rates of exchange similar to Residues 36–38 or possibly faster rates of exchange. The comparison between R2-CPMG dispersions collected at 20 C and 10 C further illustrate the differential response to temperature between these distal regions of CCHFV vOTU [Fig. 3(B)]. Specifically, residues within the N-terminal region, T10, A14, and S19, exhibit very similar exchange between the two temperatures while residues within the active site, Residues 36–38, exhibit higher exchange at 10 C than 20 C. Such a differential response to the exchange contributions (i.e., the amplitudes in R2CPMG dispersion) is characteristic of the different exchange regimes.21 Namely, residues undergoing fast exchange, such as the active site residues, exhibit an increase in exchange contributions with lower temperatures that produce lower exchange rates (i.e., Rex is inversely proportional to temperature). Conversely, residues undergoing slow exchange exhibit lower exchange contributions at lower temperatures while residues at intermediate exchange, such as the N-terminal residues of CCHFV vOTU, are not drastically affected. Finally, and likely the most important outcome of these studies here, is that the inherent dynamics within the ms-ms timescale is largely relegated to
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the interaction surface of CCHFV vOTU [Fig. 3(C)]. Although R2-CPMG dispersions reveal a complicated collection of conformational exchange processes, an overly simplistic view of a rigid CCHFV vOTU engaging its substrate based on structural studies alone is incorrect as evidenced here by multiple relaxation experiments.
The dynamics of CCHFV vOTU is altered upon binding with Ub Probing dynamics within the CCHFV vOTU/Ub complex proved to be somewhat problematic, which was largely due to the relatively low binding affinity of 11.4 mM [Fig. 1(B)] and further described here (Fig. 4). Such a low affinity complex is not necessarily surprising, since this CCHFV vOTU bound to monomeric Ub represents the product complex after cleavage of conjugated Ub.4 Nonetheless, large chemical shift differences between free CCHFV vOTU and stoichiometric amounts of Ub are consistent with slow exchange [Fig. 4(A,B) left]. In contrast, above stoichiometric concentrations of the complex, the CCHFV vOTU/Ub complex is in fast exchange with the respective free forms. This exchange to the free form from the bound complex is shown by a comparison of both chemical shifts with R2-CPMG dispersion data with increasing amounts of Ub. Specifically, we used three concentrations of added Ub where the binding constant of 11.4 mM determined above [see Fig. 1(B)] can be used to calculate the percent of bound enzyme at 0.8 mM (92.3% bound), 1.5 mM (98.6% bound), and 4.3 mM (99.7% bound). While there is a lack of observable chemical shift changes under these bound conditions [Fig. 4(A,B) middle], the exchange contributions to R2-CPMG dispersion profiles diminished with increasing Ub concentrations [Fig. 4(A,B) right]. Such a concentration-dependence suggests that the monitored exchange contribution to the R2-CPMG dispersions are due to the changing environment between bound CCHFV vOTU to free CCHFV vOTU, rather than actual internal dynamics of the enzyme. The underlying reason is that chemical shifts are dominated by the bound complex while R2-CPMG are highly sensitive to monitoring exchange to minor populations as low as 0.5%, which in this case is the free enzyme (i.e, chemical exchange between bound and free enzyme). R2CPMG dispersion profiles are essentially flat with the highest concentration of Ub that we were able to monitor [Fig. 4(A,B)], that is, 4.3 mM Ub (99.7% bound), which suggests that there is a relative quenching of internal ms dynamics for CCHFV vOTU within the complex. Although flat R2-CPMG dispersion profiles are suggestive of a loss of ms dynamics within the complex, overall elevated R2 rates that are insensitive to CPMG refocusing are indicative of faster
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exchange phenomena that persist. In other words, despite the lack of R2-CPMG dispersions for the enzyme within the CCHFV vOTU/Ub complex, which are primarily a measure of ms dynamics, the absolute R2 relaxation rates remain elevated for specific regions, which indicate ms timescale motions are still present. Thus, dynamic regions can still be identified here on the faster ms timescale by evaluating the R2 relaxation rates at the highest refocusing field imparted during R2-CPMG dispersion experiments, that is, mcpmg of 1000 Hz [Fig 4(C), left]. It should be noted that these R2 relaxation rates in Figure 4(C), extracted from R2-CPMG dispersions, were monitored on the TROSY component of 2H,15Nlabeled CCHFV vOTU, which is in contrast to standard R2 relaxation rates shown in Figure 2 monitored using 15N-labeled CCHFV vOTU. Chemical exchange on this faster timescale of ms is preferentially consolidated within the active site of CCHFV vOTU both in the free state and upon Ub binding [Fig. 4(C), right]. Interestingly, there is a slight shift of a dynamic region from free to bound CCHFV vOTU that comprises Residues 95–101 [Fig. 4(C), red] to Residues 99–106 [Fig. 4(C), black], respectively, which appears to consolidate around the Cterminus of Ub. Thus, while dynamics on the ms timescale are predominantly quenched within the CCHFV vOTU/Ub complex, faster ms timescale motions persist near or adjacent to the active site. It should be noted that while R2-CPMG dispersions in the bound CCHFV vOTU/Ub complex failed to show measurable exchange near the active site residue C40, there are indications that chemical exchange here also persists on the ms timescale. For example, Residues 36–42 exhibit extreme line broadening within the complex and the resonance for C40 itself is not even observed in the complex, indicating that these residues do indeed exhibit exchange. Moreover, the R2 values at the mcpmg of 1000 Hz for this region surrounding the catalytic C40 remains high in both the free and bound forms [Fig. 4(C)].
Discussion and Conclusions NMR relaxation studies employed here indicate that despite the similarities in the static structural descriptions of CCHFV vOTU, both in the X-ray crystal and in solution (Fig. 1), CCHFV vOTU is a highly dynamic molecule on multiple timescales (Fig. 2). R2-CPMG dispersion experiments revealed multiple dynamic processes, which included slower processes likely involved in CCHFV vOTU substrate recognition and faster processes within the active site (Fig. 3). Despite the high noise of the relaxation data collected on the CCHFV vOTU/Ub complex, the R2-CPMG dispersion data collected using multiple concentrations of added Ub indicate that most of the exchange contributions on the slower timescales of milliseconds are diminished with increasing Ub,
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Figure 4. Probing dynamics within the CCHFV vOTU/Ub complex. (A) Left, overlay of 15N-HSQC spectra showing Q11 amide resonance of 0.7 mM CCHFV vOTU alone (red), with 0.8 mM Ub (blue), with 1.5 mM Ub (green), and with 4.3 mM Ub (black). Middle, blow up of the bound Q11 resonances. Right, R2-CPMG dispersions of the same bound concentrations of CCHFV vOTU and color-coded the same. (B) The same as (A) with A14. (C) R2 relaxation rates at the maximum applied field for R2-CPMG dispersion experiments are shown (mcpmg 5 1000 Hz) for both CCHFV vOTU alone (red) and CCHFV vOTU in complex with 4.3mM Ub (black). For CCHFV vOTU alone, the average R2 relaxation rate was 8.8 Hz with 0.5 SD of 1.5 Hz (red line indicates the sum of these) and for CCHFV vOTU in complex with Ub, the average R2 relaxation rate was 14.8 Hz with 0.5 SD of 2.4 Hz (black line indicates the sum of these). Elevated R2 values are mapped onto the complex for each, which are Residues 35–42, 54–58, 71–74, 95–101, and 148–151 for CCHFV vOTU alone (red balls), and Residues 34–44, 54–58, 78–79, 99–106, and 149–152 for CCHFV vOTU in complex (black balls). All data here were collected at 900 MHz at 20 C on 2H15N-labeled CCHFV vOTU measured using a TROSY-based and TROSY-CPMG sequence,30 which is in contrast to those data collected in Figure 2 on 15N-labeled CCHFV vOTU using standard BioPack non-TROSY sequences. CCHFV vOTU free spectra shown here were collected with 128 indirect points in 15N while bound spectra were collected with 420 indirect points in 15N for added resolution.
especially for residues implicated in binding Ub and ISG15 [Fig. 4(A)]. This is not surprising as the bound Ub likely represents the product complex after CCHFV vOTU cleavage of Ub from the respective Ub substrate or from another Ub linkage. In other words, if Ub is simply acting as an inhibitor or ligand then the sampled conformations may be significantly shifted to a more confined (i.e., similar)
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Figure 5. The inherent chemical exchange of CCHFV vOTU maps to the same region as Ub binding. (A) Chemical exchange for substrate-free CCHFV vOTU monitored through R2-CPMG dispersions at 900 MHz at 20 C (Rex, red) and the absolute change in amide chemical shifts upon CCHFV vOTU binding to Ub (|Ddfree-bound|, purple) are shown. The average |Ddfree-bound| was 0.82 ppm and 0.5 SD was 0.51 ppm (the purple line indicates the sum of these). (B) Residues exhibiting Rex values greater than 0.5 Hz (left, red) and |Ddfree-bound| greater than 0.5 SD of above the mean (right, purple) are shown as a surface representations. All data shown here were determined from R2-CPMG dispersions at both 600 MHz and 900 MHz collected at 20 C and simultaneously fit with the associated 900 MHz Rex values calculated from these fits shown in (A).
set of major conformers that would result in a quenching of the measured R2-CPMG dispersions. Similar quenching of dynamic exchange has been observed for other enzymes such as cyclophilin-A in complex with its inhibitor cyclosporine18 as well as proteins such as the Major Urinary Protein binding to a pheromone.22 The existence of inherent dynamics within the binding site of CCHFV vOTU as well as the subsequent shift in dynamic equilibria upon Ub binding that culminate in substantial quenching of chemical exchange are consistent with the theory of conformational selection and the Monod-Wyman-Changeux model of allostery.13 In fact, the remarkable correlation between inherent flexibility of CCHFV vOTU and substrate engagement is shown here by plotting the chemical exchange contributions extracted from R2-CPMG dispersion fits along with those regions that exhibit chemical shift differences upon binding Ub [Fig. 5(A)]. In other words, conformational selection is likely to play a significant role in substrate binding as inherent exchange maps very well to those regions exhibiting changes to their chemical environment [Fig. 5(B)]. In contrast, there are several regions that appear to exhibit an increase in dynamics within the CCHFV vOTU/Ub complex
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relative to CCHFV vOTU alone, at least within the faster regime of chemical exchange [Fig. 4(C)]. Substrate binding has been shown to increase measured ms-ms timescale dynamics within a subset of systems, such as that of glutaredoxin, which is consistent with an “induced fit” or Koshland-MemethyFilmer model.23 Thus, CCHFV vOTU exhibits a complicated set of dynamics as well as dynamic changes upon binding Ub. However, it is clear from these studies that dynamic excursions from the average structure do occur and CCHFV vOTU is flexible for function. Several recent findings have shown that protein dynamics are not necessarily a single cooperative process as previously reported.18,24 Instead, protein motions appear to be a collection of “dynamic segments” that are partially correlated to form dynamic networks as more recently proposed.14 For example, our group identified several conformational exchange processes around the active site of substrate-free cyclophilin-A that exhibit a 10-fold difference in their rates of movements within the ms–ms timescale.16 These findings of segmental dynamics are in contrast to previous studies that employed a global fit of exchange measured at only one static field,18,24 which was shown to be inaccurate by Loria and coworkers.20 In fact, the widespread application of global fitting to dynamics data forfeits the inherent atomic resolution provided by NMR experiments and, in many cases such as with cyclophilin-A, may be obscuring the true motions of the system. The presence of localized dynamics are further illustrated by recent findings from several groups that have led to the identification of segmental motions in the allosteric protein CheY that differ by approximately 10-fold15 and the HIV-1 protease that differ by approximately fourfold.12 For CCHFV vOTU probed here, there is also approximately an order of magnitude difference between the active site dynamics and dynamics within the N-terminal region (Fig. 3). In fact, the dynamic rates of exchange extracted from R2-CPMG dispersions of residues within the CCHFV vOTU active site are all orders of magnitude faster than the rate of turnover.4 Therefore, the notion that proteins, and in particular enzymes, may be dynamically fine-tuned machines intricately designed for function may be true, but this does not necessarily imply that enzymes undergo a single cooperative process that represents catalysis. Instead, enzyme function and allostery has been shown to comprise a plethora of underlying dynamic phenomena that include thermodynamic coupling.25 As new technologies emerge, it will likely become possible to utilize NMR to probe active enzyme systems that catalyze irreversible reactions such as those with CCHFV vOTU. Thus, applying NMR relaxation experiments during turnover would provide invaluable information to help
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further bridge our understanding between dynamics and function.
CCHF vOTU present. Data sets were analyzed with the NanoAnalyze software and fit to an independent model.
Material and Methods Protein expression and purification
NMR spectroscopy and data analysis
For CCHFV vOTU, the sequence encoding the protease Residues 1–169 was cloned into a pET15b vector (Novagen, Madison, WI) encoding an N-terminal 6xHis tag for subsequent expression in BL21/DE3 cells. These residues have been shown to be both necessary and sufficient for CCHFV vOTU structural integrity and activity.9 CCHFV vOTU was grown in LB or M9 minimal media supplemented with 15N-ammonium chloride for 15N-labeled samples. For 13C,15N,2H-labeled CCHFV vOTU in M9 minimal media, 13C-glucose with D2O was used instead of unlabeled glucose in H2O and spun cell pellets were refolded in their entirety as previously described for several proteins in order to exchange amide deuterons for protons.16,26–28 Cells were lysed via sonication directly in Ni Buffer (50 mM Na3PO4, 500 mM NaCl pH 7.5, and 10 mM imidazole) or refolded protein was dialyzed into Ni buffer and applied to a Ni-affinity column (GE Healthcare). Elutions were concentrated and cleaved with thrombin to remove the 6xHis tag. Lastly, size exclusion chromatography using a Superose 75 column (GE Healthcare) was used to further purify CCHFVp using NMR Buffer (50 mM Na3PO4, 150 mM NaCl pH 6.5, 1 mM DTT). All purifications were conducted on an AKTA FPLC system (GE Healthcare). For Ub, the sequence encoding the full-length protein of Residues 1–76 was cloned into a pET28b vector (Novagen), yet the 6xHis tag was removed as it was found unnecessary using the following purification that we developed based on the relative high stability of Ub. Specifically labeled Ub proteins were prepared as described above. Cells were lysed in Ub SP Buffer (50 mM NaOAc, pH 5.0) or refolded protein was dialyzed into Ub SP Buffer. Protein was applied to an SP-Sepharose column (GE Healthcare) and eluted with Ub SP Buffer plus 1 M NaCl. Eluted fractions containing Ub were concentrated and further purified over a Superose-75 in NMR Buffer.
For assignments, a 1-mM 13C,15N,2H-labeled CCHFV vOTU sample and 3 mM 13C,15N,2H-labeled Ub sample were used to assign each protein alone in the same NMR Buffer (50 mM Na3PO4, 150 mM NaCl pH 6.5, 1 mM DTT). For the complexes, samples of the bound conformation of each comprised 0.7 mM 13C,15N,2H-labeled protein with 0.8 mM unlabeled of the other. All assignment spectra were collected on either a Varian 600 MHz or Varian 800 MHz spectrometer equipped with a Cold Probe, except for 13C,15N,2H-labeled Ub that was collected on a Bruker 700 MHz equipped with a Cold Probe. Standard 3D HNCACB and HNcoCACB pulse sequences were used to assign the free proteins and a 15N-edited NOESY-HSQC was also used to help assign sequential amides for CCHFV vOTU. For the complexes, TROSY versions of HNCA, HNcoCA, HNCACB, and HNcoCACB were used with the latter two optimized to transfer magnitization to the CB using a tauCC of 6 ms. For relaxation experiments, standard R2, R1rho, and R1 relaxation experiments that comprise the BioPack software were used on a Varian 900 MHz spectrometer for a 1 mM 15N-labeled CCHFV vOTU. Relaxation times for R2 and R1rho were 0.01, 0.03, 0.05, 0.07, and 0.09 s while relaxation times for R1 were 0.01, 0.1, 0.3, 0.5, 0.7, 0.9, and 1.1 s. Relaxation rates were obtained for each data set using nmrPipe software fitting to a single exponential. For dispersion experiments, Transverse Relaxation Optimized SpectroscopY (TROSY)-based 15N-R2-CPMG pulse sequences were to either 1 mM 2H,15N-labeled proteins alone or complexes with concentrations described in the main text at both 600 MHz and 900 MHz29 and analyzed using the least squares fitting program CPMG_FIT, kindly provided by Dr. Dmitry Korzhnev (Departments of Molecular & Medical Genetics & Biochemistry, University of Toronto, Ontario, Canada). This program employs an analytical solution of the data to the full Carver-Richards equations as we have previously described.16,28
Isothermal titration calorimetry ITC experiments were performed in duplicate using a NanoITC system (TA Instruments, Lindon, UT). To assess the interaction between CCHF vOTU and Ub, a solution of 3.5 mM Ub was titrated into 0.65 mM CCHF vOTU. ITC runs were performed at 25 C and comprised of one injection of 1 lL followed by 24 injections of 2 lL for a total of 25 injections. Each injection was spaced 200 s apart. To account for the heat of dilution, 3.5 mM Ub was titrated in identical manner into the dialysis buffer with no
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References 1. Estrada-Pena A, de la Fuente J (2014) The ecology of ticks and epidemiology of tick-borne viral diseases. Antiviral Res 108C:104–128. 2. Frias-Staheli N, Giannakopoulos NV, Kikkert M, Taylor SL, Bridgen A, Paragas J, Richt JA, Rowland RR, Schmaljohn CS, Lenschow DJ, Snijder EJ, GarciaSastre A, Virgin HW (2007) Ovarian tumor domaincontaining viral proteases evade ubiquitin- and ISG15dependent innate immune responses. Cell Host Microbe 2:404–416.
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3. Bailey-Elkin BA, van Kasteren PB, Snijder EJ, Kikkert M, Mark BL (2014) Viral OTU deubiquitinases: a structural and functional comparison. PLoS Pathog 10, doi: 10.1371/journal.ppat.1003894. 4. Capodagli GC, Deaton MK, Baker EA, Lumpkin RJ, Pegan SD (2013) Diversity of ubiquitin and ISG15 specificity among nairoviruses’ viral ovarian tumor domain proteases. J Virol 87:3815–3827. 5. Weber F, Mirazimi A (2008) Interferon and cytokine responses to Crimean Congo hemorrhagic fever virus; an emerging and neglected viral zonoosis. Cytokine Growth Factor Rev 19:395–404. 6. Zhao C, Denison C, Huibregtse JM, Gygi S, Krug RM (2005) Human ISG15 conjugation targets both IFNinduced and constitutively expressed proteins functioning in diverse cellular pathways. Proc Natl Acad Sci USA 102:10200–10205. 7. Akutsu M, Ye Y, Virdee S, Chin JW, Komander D (2011) Molecular basis for ubiquitin and ISG15 crossreactivity in viral ovarian tumor domains. Proc Natl Acad Sci USA 108:2228–2233. 8. James TW, Frias-Staheli N, Bacik JP, Macleod JML, Khajehpour M, Garcia-Sastre A, Mark BL (2011) Structural basis for the removal of ubiquitin and interferon-stimulated gene 15 by a viral ovarian tumor domain-containing protease. Proc Natl Acad Sci USA 108:2222–2227. 9. Capodagli GC, McKercher MA, Baker EA, Masters EM, Brunzelle JS, Pegan SD (2011) Structural analysis of a viral ovarian tumor domain protease from the CrimeanCongo hemorrhagic fever virus in complex with covalently bonded ubiquitin. J Virol 85:3621–3630. 10. Takahashi D, Hiromasa Y, Kim Y, Anbanandam A, Yao XL, Chang KO, Prakash O (2013) Structural and dynamics characterization of norovirus protease. Protein Sci 22:347–357. 11. Ishima R, Wingfield PT, Stahl SJ, Kaufman JD, Torchia DA (1998) Using amide H-1 and N-15 transverse relaxation to detect millisecond time-scale motions in perdeuterated proteins: application to HIV-1 protease. J Am Chem Soc 120:10534–10542. 12. Torbeev VY, Raghuraman H, Hamelberg D, Tonelli M, Westler WM, Perozo E, Kent SBH (2011) Protein conformational dynamics in the mechanism of HIV-1 protease catalysis. Proc Natl Acad Sci USA 108:20982–20987. 13. Changeux JP (2012) Allostery and the Monod-WymanChangeux model after 50 years. Ann Rev Biophys 41: 103–133. 14. Csermely P, Palotai R, Nussinov R (2010) Induced fit, conformational selection and independent dynamic segments: an extended view of binding events. TIBS 35: 539–546. 15. McDonald LR, Boyer JA, Lee AL (2012) Segmental motions, not a two-state concerted switch, underlie allostery in CheY. Structure 20:1363–1373. 16. Schlegel J, Armstrong GS, Redzic JS, Zhang FL, Eisenmesser EZ (2009) Characterizing and controlling
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the inherent dynamics of cyclophilin-A. Protein Sci 18: 811–824. Berjanskii MV, Wishart DS (2005) A simple method to predict protein flexibility using secondary chemical shifts. J Am Chem Soc 127:14970–14971. Eisenmesser EZ, Millet O, Labeikovsky W, Korzhnev DM, Wolf-Watz M, Bosco DA, Skalicky JJ, Kay LE, Kern D (2005) Intrinsic dynamics of an enzyme underlies catalysis. Nature 438:117–121. Kovrigin EL, Loria JP (2006) Enzyme dynamics along the reaction coordinate: critical role of a conserved residue. Biochemistry 45:2636–2647. Kovrigin EL, Kempf JG, Grey MJ, Loria JP (2006) Faithful estimation of dynamics parameters from CPMG relaxation dispersion measurements. J Magn Reson 180:93–104. Millet O, Loria JP, Kroenke CD, Pons M, Palmer AG (2000) The static magnetic field dependence of chemical exchange linebroadening defines the NMR chemical shift time scale. J Am Chem Soc 122:2867–2877. Perazzolo C, Verde M, Homans SW, Bodenhausen G (2007) Evidence of chemical exchange in recombinant Major Urinary Protein and quenching thereof upon pheromone binding. J Biomol NMR 38:3–9. Jensen KS, Winther JR, Teilum K (2011) Millisecond dynamics in glutaredoxin during catalytic turnover is dependent on substrate binding and absent in the resting states. J Am Chem Soc 133:3034–3042. Fraser JS, Clarkson MW, Degnan SC, Erion R, Kern D, Alber T (2009) Hidden alternative structures of proline isomerase essential for catalysis. Nature 462:669– U149. Motlagh HN, Wrabl JO, Li J, Hilser VJ (2014) The ensemble nature of allostery. Nature 508:331–339. Schlegel J, Redzic JS, Porter C, Yurchenko V, Bukrinsky M, Armstrong GS, Zhang FL, Isern NG, DeGregori J, Hodges R, Eisenmesser EZ (2009) Solution characterization of the extracellular region of CD147 and its interaction with its enzyme ligand cyclophilin-A. J Mol Biol 391:518–535. Redzic JS, Armstrong GS, Isern NG, Jones DNM, Kieft SK, Eisenmesser E (2011) The retinal specific EMMPRIN/CD147 domain: from molecular structure to biological activity. J Mol Biol 411:68–82. Kendrick AA, Holliday M, Isern NG, Zhang F, Camilloni C, Huynh C, Vendruscolo M, Armstrong GS, Eisenmesser EZ (2014) The dynamics of interleukin-8 and its interaction with human CXC receptor I peptide. Protein Sci 23:464–480. Loria JP, Rance M, Palmer AG (1999) A TROSY CPMG sequence for characterizing chemical exchange in large proteins. J Biomol NMR 15:151–155. Loria JP, Rance M, Palmer AG (1999) A relaxationcompensated Carr-Purcell-Meiboom-Gill sequence for characterizing chemical exchange by NMR spectroscopy. J Am Chem Soc 121:2331–2332.
Inherent Dynamics Within CCHFV vOTO
Elan Z. Eisenmesser,1* Glenn C. Capodagli,2 Geoffrey S. Armstrong,3 Michael J. Holliday,1 Nancy G. Isern,4 Fengli Zhang,5 and Scott D. Pegan2* 1
Department of Biochemistry and Molecular Genetics, School of Medicine, University of Colorado Denver, Aurora, Colorado 80224
2
Department of Pharmaceutical and Biomedical Sciences, University of Georgia, Athens, Georgia 30602 Department of Chemistry and Biochemistry, University of Colorado at Boulder, Boulder, Colorado 803093
3 4
WR Wiley Environmental Molecular Sciences Laboratory, High Field NMR Facility, Washington 99532
5
National High Magnetics Field Laboratory, Tallahassee, Florida 32310
Received 26 September 2013; Accepted 30 December 2014 DOI: 10.1002/pro.2637 Published online 6 January 2015 proteinscience.org
Abstract: Crimean-Congo Hemorrhagic fever virus (CCHFV) is one of several lethal viruses that encodes for a viral ovarian tumor domain (vOTU), which serves to cleave and remove ubiquitin (Ub) and interferon stimulated gene product 15 (ISG15) from numerous proteins involved in cellular signaling. Such manipulation of the host cell machinery serves to downregulate the host response and, therefore, complete characterization of these proteases is important. While several structures of the CCHFV vOTU protease have been solved, both free and bound to Ub and ISG15, few structural differences have been found and little insight has been gained as to the structural plasticity of this protease. Therefore, we have used NMR relaxation experiments to probe the dynamics of CCHFV vOTU, both alone and in complex with Ub, discovering a highly dynamic protease that exhibits conformational exchange within the same regions found to engage its Ub substrate. These experiments reveal a structural plasticity around the N-terminal regions of CCHFV vOTU, which are unique to vOTUs, and provide a rationale for engaging multiple substrates with the same binding site. Keywords: CCHFV; vOTU; protein dynamics; protein–protein interactions
Additional Supporting Information may be found in the online version of this article. NMR experiments were collected at multiple facilities. Part of the data was acquired at the High Magnetic Field Laboratory (NHMFL), which is supported by cooperative agreement DMR 0654118 between the National Science Foundation and the State of Florida. A portion of NMR data collection was performed at the Environmental Molecular Sciences Laboratory, a national scientific user facility sponsored by the Department of Energy’s Office of Biological and Environmental Research and located at Pacific Northwest National Laboratory. The content of this report is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Grant sponsor: The Rocky Mountain 900 Facility (used for NMR data collection); Grant number: NIHGM68928; Grant sponsor: NIH; Grant numbers: 1F31CA183206-01A1 (to MJH), GM107262-01A1 (to EZE), and Grant numbers: 1R03MH097507-01A1 and 1R01Al109008-01 (to SDP). *Correspondence to: Elan Zohar Eisenmesser, Department of Biochemistry and Molecular Genetics, School of Medicine, University of Colorado Denver, Aurora, CO 80224. E-mail: [email protected] or Scott D. Pegan Department of Pharmaceutical and Biomedical Sciences, University of Georgia, Athens, GA 30602. E-mail: [email protected]
C 2015 The Protein Society Published by Wiley-Blackwell. V
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Introduction Crimean-Congo Hemorrhagic fever virus (CCHFV) is a negative-sense, single-stranded RNA virus that belongs to the Nairovirus genus within the Bunyaviridae family. CCHFV is among several ticktransmitted viruses that have garnered increasing interest due to their emergence in Europe, which is at least partially blamed on global climate change.1 Like many viruses, CCHFV has adapted mechanisms to manipulate the host cell machinery for its own pathogenesis. For example, several RNA viruses, which include nairoviruses such as CCHFV, encode for a viral ovarian tumor domain (vOTU) that removes ubiquitin (Ub) and Ub-like interferonstimulated gene product 15 (ISG15) from proteins and thereby, downregulates the innate immune response.2 Interestingly, CCHFV vOTU protease is relatively unique in this dual substrate specificity. Specifically, while most known deubiquitinating enzymes including vOTUs target either Ub or ISG15, CCHFV vOTU appears to target both.3,4 Conjugation of Ub and ISG15 to host proteins plays a critical role in regulating antiviral proteins of the interferon (IFN) type 1 response.5,6 As a result, vOTUs, particularly CCHFV vOTU from the most lethal human nairovirus CCHFV, are currently considered potent nairovirus virulence factors because of their perceived role in subverting the host antiviral response.2,4,7,8 X-ray crystal structures have been solved of CCHFV vOTU alone,7 in complex with Ub,7–9 and in complex with ISG15,7,8 which revealed a conserved OTU-like fold of its eukaryotic counterparts. Complexes were formed utilizing recombinant Ub and ISG15 proteins derivatized with 3-bromopropylamine hydrobromide, which are suicide substrates that result in covalent bonds to the CCHFV vOTU catalytic Cys residue, which is C40. The primary difference between CCHFV vOTU and its eukaryotic homologues is the inclusion of two extra N-terminal b-strands that form an extended b-sheet, which is also present within the Dugbe virus protease (DUGV vOTU).3,4 This allows a 30 twist of the bound Ub or Ub-like domain within ISG15. However, CCHFV vOTU is nearly identical within all of the structures determined to date, whether alone or in complex with either Ub or ISG15, and there is no information regarding the potential structural plasticity of CCHFV vOTU. Additionally, the sole substrate-free structure of CCHFV vOTU has its active site obscured by an a-helix originating from a crystal contact.7 This further suggests that the direct utilization of X-ray crystallographic techniques for structural information related to CCHFV vOTU can benefit from an alternative NMR based approach. Atomic resolution investigations regarding dynamics have been conducted for other viral proteases, which include the Norwalk virus 3CLpro,10
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which is a cysteine protease, and many pivotal studies of the human immunodeficiency virus type I protease,11,12 which is an aspartyl protease. While these highly divergent proteases have been shown to exhibit dynamics on multiple timescales, a defining feature of both of these enzymes is the micro–millisecond motions observed within their active sites. Such inherent motions within macromolecules, and particularly enzymes, support the theory of conformational selection whereby molecules sample important conformations that allow for substrate binding and potentially catalysis.13 However, such conformational sampling has recently been shown to be much more complex than previously thought with highly localized “dynamic segments” nonetheless communicating with each other over large distances.14 For example, allosteric pathways in enzymes such as CheY15 and cyclophilin-A16 have been identified. Thus, our aim in this study was to first discern if the solution structure of CCHFV vOTU was similar to that in the X-ray crystal structures and then to characterize the dynamics of CCHFV vOTU in solution, in order to determine whether regions involved in substrate recognition were inherently dynamic. By utilizing NMR, the backbone assignments of substrate-free CCHFV vOTU and both the enzyme and Ub within the CCHFV vOTU/Ub complex were determined. While we were able to confirm that the average structure of CCHFV vOTU was indeed similar to that in the crystals and the average structure was unperturbed upon binding Ub, the dynamics were significantly different on the ms timescale but highly similar on the ms timescale. Of particular importance, there was a remarkable correlation between the inherent dynamics of CCHFV vOTU (i.e., motions prior to substrate engagement) and the site of engaging Ub. Thus, this study has revealed the structural plasticity of CCHFV vOTU and indicates that dynamics within the protease are largely relegated to the catalytic region.
Results The average CCHFV vOTU structure is unperturbed upon complex formation with Ub We sought to determine whether a lack of structural changes between free and bound CCHFV vOTU in the X-ray crystal structures was reflective of the solution behavior of CCHFV vOTU upon binding Ub as well. Specifically, the structures of CCHFV vOTU within the X-ray crystal structures of free CCHFV vOTU, CCHFV vOTU/Ub, and CCHFV vOTU/ISG15 exhibit an RMSD of backbone atoms of less than 0.7 ˚ and are nearly indistinguishable [Fig. 1(A)]. IsoA thermal titration calorimetry (ITC) confirmed that the affinity of CCHVF vOTU Ub for was within a range that could be used to interrogate the complex, which was 11.4 6 0.7 lM [Fig. 1(B)]. Thus, we
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vOTU is not significantly perturbed upon binding Ub [Fig. 1(C)]. We also addressed any potential structural changes between both free and bound Ub by reversing the labeling scheme and monitoring the CA chemical shift propensities for Ub in both states (Supporting Information Fig. S1). Analogous to CCHFV vOTU, Ub also showed few changes to its chemical shift propensities apart from the C-terminal region that exhibits slightly more negative values that are consistent with a more extended, b-strand like structure upon insertion into CCHFV vOTU. Thus, these NMR data are consistent with the X-ray crystal structure data and indicate little is changed for both CCHFV vOTU and Ub upon binding.
CCHFV vOTU exhibits dynamics on multiple timescales
Figure 1. The average structure of CCHFV vOTU is not significantly perturbed upon binding Ub in solution. (A) The Xray crystal structure of CCHFV vOTU alone (white, PDB accession 3PHU) and in complex with Ub (blue with Ub in green, PDB accession 3PRP). The backbone atoms and all heavy atoms of CCHFV vOTU exhibit an RMSD of less than 0.7 A˚ and 1.8 A˚, respectively. (B) ITC binding of CCHF vOTU to Ub. The top panel represents the raw heat data from 25 injections at 25 C, and the bottom panel shows the integrated heat peak areas plotted versus the molar ratio of Ub to CCHF vOTU. The line represents the best fit to an independent model after subtracting the heat generated by titrating Ub into buffer alone to account for the heat of dilution. This figure was generated using the NanoAnalyze software provided by TA Instruments. Kd 5 11.4 6 0.7 lM, DH 5 28.6 6 0.7 kJ mol21, TDS 5 57.3 6 0.6 kJ mol21, DG 5 227.7 6 0.1 kJ mol21, and n 5 1.2 1 0.04. (C) CA chemical shift propensities relative to their respective unfolded resonances are shown for both free CCHFV vOTU (white) and bound CCHFV vOTU (blue).
assigned the backbone resonances of CCHFV vOTU in both its free state as well as in the presence of Ub in order to compare the chemical shift propensities in both states. Chemical shift propensities are highly sensitive measures of secondary structure, where CA has been shown to be the most sensitive of these.17 CA chemical shift propensities were calculated by subtracting the random coil chemical shifts for each residue type from the chemical shifts measured here. Indeed, the chemical shift propensities for CA resonances were nearly identical between free CCHFV vOTU and CCHFV vOTU bound to Ub indicating that the average structure of CCHFV
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Initial analysis of R2 relaxation rates for free CCHFV vOTU at 20 C revealed that most of the elevated rates are localized to either the active site or are adjacent to the active site where the C-terminal region of Ub is known to bind [Fig. 2(A) black with those atoms exhibiting elevated R2 relaxation rates shown in red]. Since elevated R2 relaxation rates are associated with relatively slow motions on the ms-ms timescales, this is the first experimental proof that CCHFV vOTU undergoes conformational exchange prior to substrate engagement. For example, Residues 36–41 of CCHFV vOTU that includes the catalytic C40 all exhibited elevated R2 relaxation rates [Fig. 2(A), inset]. Interestingly, the amide of G38 exhibits the highest R2 relaxation rate of all amides, which may be indicative of elevated chemical exchange due to the catalytic C40 side chain that points directly toward this residue within the loop that forms the oxyanion hole. Additional residues that exhibited elevated R2 relaxation rates include T10, N11, I13, and A14, all of which reside on the Nterminal b-strand that is involved in binding both Ub and ISG15.8 While elevated R2 relaxation rates indicate that conformational exchange occurs within the ms–ms timescale (due to chemical exchange), comparisons to an R1rho experiment that employs a highpowered refocusing field can help narrow down this timescale. For example, many residues that exhibit elevated R2 relaxation rates exhibit reduced R1rho relaxation rates, which include the entire stretch of Residues 31–41 (Fig. 2, inset). This is consistent with chemical exchange phenomena that occur lower than approximately 104/s, since they are quenched upon employing a high-powered refocusing pulse within the R1rho experiment. Conversely, Q149 exhibits an elevated R2 and R1rho relaxation rate that are identical. In other words, the chemical exchange phenomena for this residue occurs on a much faster timescale than the other residues (i.e.,
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Figure 2. Relaxation rates of free CCHFV vOTU indicate that dynamics occur on multiple timescales. (A) Left, both R2 (black) and R1rho (red) relaxation rates for free CCHFV vOTU. The average R2 relaxation rate was 23.4 Hz with 0.5 SD of 2.1 Hz (red line indicates the sum of these). Right, residues that exhibit R2 relaxation rates higher than 0.5 SD from the average are shown in red. CCHFV vOTU is shown as a cartoon (white) with Ub shown as a transparent cartoon (green) to orient the reader. The catalytic Cys, C40, is shown as yellow and several residues that include G38 and Q149 are highlighted and described in more detail within the main text. Inset, blow-up of residues around C40 that exhibit suppressed relaxation rates with the higher field imparted in the R1rho experiment, indicating exchange occurs on a timescale of ms. (B) Left, R1 relaxation rates for free CCHFV vOTU. The average R1 relaxation rate was 0.77 Hz with 0.5 SD of 0.08 Hz (red line indicates the sum of these). Right, residues that exhibit R1 relaxation rates higher than 0.5 SD from the average are shown in red. These elevated R1 relaxation rates are primarily confined to loops, except for N39, Y58, and H59 that are within structural proximity and all have their side chains pointed within the same direction. All relaxation data were collected at 20 C at 900 MHz on 15N-labeled CCHFV vOTU using standard BioBack sequences.
submicrosecond), since it is unperturbed. Such experiments already highlight an important finding within recent years that ms-ms dynamics are often present within enzyme active sites, suggesting that such macromolecules are poised for function (i.e., “flexible for function”). However, it is also important to note these R2 and R1rho experiments also reveal differential dynamics within the CCHFV vOTU active site on the ms-ms timescales within CCHFV vOTU. R1 relaxation rates were also measured for CCHFV vOTU and offer insight into the dynamics on the much faster timescales of ps–ns [Fig. 2(B)]. Specifically, most residues exhibiting elevated R1 relaxation rates are confined to loop regions within CCHFV vOTU, which simply indicates that these loops are relatively flexible. Moreover, much of these residues may simply be locally disordered, as they
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are distant from one another. However, two regions structurally juxtaposed to the catalytic C40 have multiple residues with elevated R1 relaxation rates, which include N39, Y58, and H59 (Fig. 2, right). Even adjacent residues to these exhibit higher than average R1 relaxation rates, such as Residues 36–38 and 54–60. Thus, extended flexible regions even within the faster timescales are relegated to close proximity of the CCHFV vOTU active site.
Chemical exchange within free CCHFV vOTU is localized to its site of substrate engagement Considering that the dynamics monitored by R2 and R1rho relaxation rates above indicate that conformational exchange occurs within the active site of CCHFV vOTU, we sought to further characterize these dynamics through R2-CPMG (Carr-PurcellMeiboom-Gill) dispersion experiments. R2-CPMG
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Figure 3. R2-CPMG dispersion measurements reveal that CCHFV vOTU exhibits several conformational exchange phenomena. (A) Representative R2-CPMG dispersion curves collected on free CCHFV vOTU at 20 C at both 900 MHz (red) and 600 MHz (orange) were simultaneously fit as described in Material and Methods with the resulting kex given above. (B) R2-CPMG dispersion curves are shown for the same residues, but collected at 10 C at 900 MHz (blue) and 600 MHz (cyan). (C) The cocrystal complex of CCHFV vOTU (white) is shown with transparent Ub (green) to orient the reader. Residues exhibiting measurable exchange within R2-CPMG dispersions at both 20 C and 10 C are shown (red) along with residues that only exhibit measurable exchange at 10 C (blue). All data here were collected at 900 MHz at the indicated temperatures using 2H15N-labeled CCHFV vOTU measured using a TROSY-CPMG sequence.30
dispersion experiments are particularly useful in quantifying ms-ms dynamics, which is the same timescales critical for turnover of multiple enzymes as others and we have described.16,18,19 Specifically, R2-CPMG dispersion quantifies chemical exchange (i.e., Rex) as a function of an applied refocusing field (i.e., mcpmg) and can be used to obtain multiple biophysical parameters associated with conformational exchange that includes the kinetic rates of exchange (i.e., kex). Two static fields are both necessary and sufficient for accurate extraction of the biophysical parameters as previously shown20 and thus we collected R2-CPMG dispersions at both 900 MHz and 600 MHz. Moreover, since different
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dynamic processes may exhibit different temperature dependencies, we have previously employed R2-CPMG dispersion experiments at multiple temperatures16 and do so here at both 20 C and 10 C (Fig. 3). To ensure the dynamics monitored here were reflective of internal motions, and not induced by oligomeric interactions, we also performed R2CPMG dispersion experiments at multiple concentrations of CCHFV vOTU (Supporting Information Fig. S2). There were no concentration-dependent changes in the chemical exchange contributions (i.e., Rex), thereby indicating that the monitored chemical exchange was indeed a measure of internal dynamics.
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Simultaneously fitting the two R2-CPMG dispersions collected at 900 MHz and 600 MHz reveal multiple conformational exchange processes at 20 C [Fig. 3(A), top]. Specifically, several residues within the first two b-strands of CCHFV vOTU exhibit exchange such as T10, A14, and S19. Of interest, these two b-strands are the distinguishing structural features of vOTUs from their mammalian counterparts and are found in both CCHFV vOTU and DUGV vOTU.3 Thus, our finding that identify this region as being conformationally flexible is consistent with the fact that this structural feature has been implicated in the diversity of binding both Ub and ISG15. The flexibility of these two b-strands may allow CCHFV vOTU to engage both substrates. In contrast to this N-terminal region, the calculated rates of exchange processes for residues within the active site are much higher, which included Residues 36–38 [Fig. 3(A), bottom]. This finding implies that the associated dynamic exchange process of CCHFV vOTU is not uniform, but are instead segmental in nature. Such segmental dynamics have recently been shown for many proteins, which include the HIV-1 protease,12 CheY,15 as well as cyclophilin-A.16 Individual R2 relaxation rates in CCHFV vOTU were much higher for Residues 38– 42, which includes the catalytic Cys, C40. Linebroadening for these residues was extensive and only G38 could be reliably fit (Fig. 3). However, even with the highest refocusing frequency imparted, (i.e., mcpmg 5 1000 Hz), these R2 relaxation rates remained relatively high, suggesting these residues also exhibited fast rates of exchange similar to Residues 36–38 or possibly faster rates of exchange. The comparison between R2-CPMG dispersions collected at 20 C and 10 C further illustrate the differential response to temperature between these distal regions of CCHFV vOTU [Fig. 3(B)]. Specifically, residues within the N-terminal region, T10, A14, and S19, exhibit very similar exchange between the two temperatures while residues within the active site, Residues 36–38, exhibit higher exchange at 10 C than 20 C. Such a differential response to the exchange contributions (i.e., the amplitudes in R2CPMG dispersion) is characteristic of the different exchange regimes.21 Namely, residues undergoing fast exchange, such as the active site residues, exhibit an increase in exchange contributions with lower temperatures that produce lower exchange rates (i.e., Rex is inversely proportional to temperature). Conversely, residues undergoing slow exchange exhibit lower exchange contributions at lower temperatures while residues at intermediate exchange, such as the N-terminal residues of CCHFV vOTU, are not drastically affected. Finally, and likely the most important outcome of these studies here, is that the inherent dynamics within the ms-ms timescale is largely relegated to
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the interaction surface of CCHFV vOTU [Fig. 3(C)]. Although R2-CPMG dispersions reveal a complicated collection of conformational exchange processes, an overly simplistic view of a rigid CCHFV vOTU engaging its substrate based on structural studies alone is incorrect as evidenced here by multiple relaxation experiments.
The dynamics of CCHFV vOTU is altered upon binding with Ub Probing dynamics within the CCHFV vOTU/Ub complex proved to be somewhat problematic, which was largely due to the relatively low binding affinity of 11.4 mM [Fig. 1(B)] and further described here (Fig. 4). Such a low affinity complex is not necessarily surprising, since this CCHFV vOTU bound to monomeric Ub represents the product complex after cleavage of conjugated Ub.4 Nonetheless, large chemical shift differences between free CCHFV vOTU and stoichiometric amounts of Ub are consistent with slow exchange [Fig. 4(A,B) left]. In contrast, above stoichiometric concentrations of the complex, the CCHFV vOTU/Ub complex is in fast exchange with the respective free forms. This exchange to the free form from the bound complex is shown by a comparison of both chemical shifts with R2-CPMG dispersion data with increasing amounts of Ub. Specifically, we used three concentrations of added Ub where the binding constant of 11.4 mM determined above [see Fig. 1(B)] can be used to calculate the percent of bound enzyme at 0.8 mM (92.3% bound), 1.5 mM (98.6% bound), and 4.3 mM (99.7% bound). While there is a lack of observable chemical shift changes under these bound conditions [Fig. 4(A,B) middle], the exchange contributions to R2-CPMG dispersion profiles diminished with increasing Ub concentrations [Fig. 4(A,B) right]. Such a concentration-dependence suggests that the monitored exchange contribution to the R2-CPMG dispersions are due to the changing environment between bound CCHFV vOTU to free CCHFV vOTU, rather than actual internal dynamics of the enzyme. The underlying reason is that chemical shifts are dominated by the bound complex while R2-CPMG are highly sensitive to monitoring exchange to minor populations as low as 0.5%, which in this case is the free enzyme (i.e, chemical exchange between bound and free enzyme). R2CPMG dispersion profiles are essentially flat with the highest concentration of Ub that we were able to monitor [Fig. 4(A,B)], that is, 4.3 mM Ub (99.7% bound), which suggests that there is a relative quenching of internal ms dynamics for CCHFV vOTU within the complex. Although flat R2-CPMG dispersion profiles are suggestive of a loss of ms dynamics within the complex, overall elevated R2 rates that are insensitive to CPMG refocusing are indicative of faster
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exchange phenomena that persist. In other words, despite the lack of R2-CPMG dispersions for the enzyme within the CCHFV vOTU/Ub complex, which are primarily a measure of ms dynamics, the absolute R2 relaxation rates remain elevated for specific regions, which indicate ms timescale motions are still present. Thus, dynamic regions can still be identified here on the faster ms timescale by evaluating the R2 relaxation rates at the highest refocusing field imparted during R2-CPMG dispersion experiments, that is, mcpmg of 1000 Hz [Fig 4(C), left]. It should be noted that these R2 relaxation rates in Figure 4(C), extracted from R2-CPMG dispersions, were monitored on the TROSY component of 2H,15Nlabeled CCHFV vOTU, which is in contrast to standard R2 relaxation rates shown in Figure 2 monitored using 15N-labeled CCHFV vOTU. Chemical exchange on this faster timescale of ms is preferentially consolidated within the active site of CCHFV vOTU both in the free state and upon Ub binding [Fig. 4(C), right]. Interestingly, there is a slight shift of a dynamic region from free to bound CCHFV vOTU that comprises Residues 95–101 [Fig. 4(C), red] to Residues 99–106 [Fig. 4(C), black], respectively, which appears to consolidate around the Cterminus of Ub. Thus, while dynamics on the ms timescale are predominantly quenched within the CCHFV vOTU/Ub complex, faster ms timescale motions persist near or adjacent to the active site. It should be noted that while R2-CPMG dispersions in the bound CCHFV vOTU/Ub complex failed to show measurable exchange near the active site residue C40, there are indications that chemical exchange here also persists on the ms timescale. For example, Residues 36–42 exhibit extreme line broadening within the complex and the resonance for C40 itself is not even observed in the complex, indicating that these residues do indeed exhibit exchange. Moreover, the R2 values at the mcpmg of 1000 Hz for this region surrounding the catalytic C40 remains high in both the free and bound forms [Fig. 4(C)].
Discussion and Conclusions NMR relaxation studies employed here indicate that despite the similarities in the static structural descriptions of CCHFV vOTU, both in the X-ray crystal and in solution (Fig. 1), CCHFV vOTU is a highly dynamic molecule on multiple timescales (Fig. 2). R2-CPMG dispersion experiments revealed multiple dynamic processes, which included slower processes likely involved in CCHFV vOTU substrate recognition and faster processes within the active site (Fig. 3). Despite the high noise of the relaxation data collected on the CCHFV vOTU/Ub complex, the R2-CPMG dispersion data collected using multiple concentrations of added Ub indicate that most of the exchange contributions on the slower timescales of milliseconds are diminished with increasing Ub,
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Figure 4. Probing dynamics within the CCHFV vOTU/Ub complex. (A) Left, overlay of 15N-HSQC spectra showing Q11 amide resonance of 0.7 mM CCHFV vOTU alone (red), with 0.8 mM Ub (blue), with 1.5 mM Ub (green), and with 4.3 mM Ub (black). Middle, blow up of the bound Q11 resonances. Right, R2-CPMG dispersions of the same bound concentrations of CCHFV vOTU and color-coded the same. (B) The same as (A) with A14. (C) R2 relaxation rates at the maximum applied field for R2-CPMG dispersion experiments are shown (mcpmg 5 1000 Hz) for both CCHFV vOTU alone (red) and CCHFV vOTU in complex with 4.3mM Ub (black). For CCHFV vOTU alone, the average R2 relaxation rate was 8.8 Hz with 0.5 SD of 1.5 Hz (red line indicates the sum of these) and for CCHFV vOTU in complex with Ub, the average R2 relaxation rate was 14.8 Hz with 0.5 SD of 2.4 Hz (black line indicates the sum of these). Elevated R2 values are mapped onto the complex for each, which are Residues 35–42, 54–58, 71–74, 95–101, and 148–151 for CCHFV vOTU alone (red balls), and Residues 34–44, 54–58, 78–79, 99–106, and 149–152 for CCHFV vOTU in complex (black balls). All data here were collected at 900 MHz at 20 C on 2H15N-labeled CCHFV vOTU measured using a TROSY-based and TROSY-CPMG sequence,30 which is in contrast to those data collected in Figure 2 on 15N-labeled CCHFV vOTU using standard BioPack non-TROSY sequences. CCHFV vOTU free spectra shown here were collected with 128 indirect points in 15N while bound spectra were collected with 420 indirect points in 15N for added resolution.
especially for residues implicated in binding Ub and ISG15 [Fig. 4(A)]. This is not surprising as the bound Ub likely represents the product complex after CCHFV vOTU cleavage of Ub from the respective Ub substrate or from another Ub linkage. In other words, if Ub is simply acting as an inhibitor or ligand then the sampled conformations may be significantly shifted to a more confined (i.e., similar)
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Figure 5. The inherent chemical exchange of CCHFV vOTU maps to the same region as Ub binding. (A) Chemical exchange for substrate-free CCHFV vOTU monitored through R2-CPMG dispersions at 900 MHz at 20 C (Rex, red) and the absolute change in amide chemical shifts upon CCHFV vOTU binding to Ub (|Ddfree-bound|, purple) are shown. The average |Ddfree-bound| was 0.82 ppm and 0.5 SD was 0.51 ppm (the purple line indicates the sum of these). (B) Residues exhibiting Rex values greater than 0.5 Hz (left, red) and |Ddfree-bound| greater than 0.5 SD of above the mean (right, purple) are shown as a surface representations. All data shown here were determined from R2-CPMG dispersions at both 600 MHz and 900 MHz collected at 20 C and simultaneously fit with the associated 900 MHz Rex values calculated from these fits shown in (A).
set of major conformers that would result in a quenching of the measured R2-CPMG dispersions. Similar quenching of dynamic exchange has been observed for other enzymes such as cyclophilin-A in complex with its inhibitor cyclosporine18 as well as proteins such as the Major Urinary Protein binding to a pheromone.22 The existence of inherent dynamics within the binding site of CCHFV vOTU as well as the subsequent shift in dynamic equilibria upon Ub binding that culminate in substantial quenching of chemical exchange are consistent with the theory of conformational selection and the Monod-Wyman-Changeux model of allostery.13 In fact, the remarkable correlation between inherent flexibility of CCHFV vOTU and substrate engagement is shown here by plotting the chemical exchange contributions extracted from R2-CPMG dispersion fits along with those regions that exhibit chemical shift differences upon binding Ub [Fig. 5(A)]. In other words, conformational selection is likely to play a significant role in substrate binding as inherent exchange maps very well to those regions exhibiting changes to their chemical environment [Fig. 5(B)]. In contrast, there are several regions that appear to exhibit an increase in dynamics within the CCHFV vOTU/Ub complex
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relative to CCHFV vOTU alone, at least within the faster regime of chemical exchange [Fig. 4(C)]. Substrate binding has been shown to increase measured ms-ms timescale dynamics within a subset of systems, such as that of glutaredoxin, which is consistent with an “induced fit” or Koshland-MemethyFilmer model.23 Thus, CCHFV vOTU exhibits a complicated set of dynamics as well as dynamic changes upon binding Ub. However, it is clear from these studies that dynamic excursions from the average structure do occur and CCHFV vOTU is flexible for function. Several recent findings have shown that protein dynamics are not necessarily a single cooperative process as previously reported.18,24 Instead, protein motions appear to be a collection of “dynamic segments” that are partially correlated to form dynamic networks as more recently proposed.14 For example, our group identified several conformational exchange processes around the active site of substrate-free cyclophilin-A that exhibit a 10-fold difference in their rates of movements within the ms–ms timescale.16 These findings of segmental dynamics are in contrast to previous studies that employed a global fit of exchange measured at only one static field,18,24 which was shown to be inaccurate by Loria and coworkers.20 In fact, the widespread application of global fitting to dynamics data forfeits the inherent atomic resolution provided by NMR experiments and, in many cases such as with cyclophilin-A, may be obscuring the true motions of the system. The presence of localized dynamics are further illustrated by recent findings from several groups that have led to the identification of segmental motions in the allosteric protein CheY that differ by approximately 10-fold15 and the HIV-1 protease that differ by approximately fourfold.12 For CCHFV vOTU probed here, there is also approximately an order of magnitude difference between the active site dynamics and dynamics within the N-terminal region (Fig. 3). In fact, the dynamic rates of exchange extracted from R2-CPMG dispersions of residues within the CCHFV vOTU active site are all orders of magnitude faster than the rate of turnover.4 Therefore, the notion that proteins, and in particular enzymes, may be dynamically fine-tuned machines intricately designed for function may be true, but this does not necessarily imply that enzymes undergo a single cooperative process that represents catalysis. Instead, enzyme function and allostery has been shown to comprise a plethora of underlying dynamic phenomena that include thermodynamic coupling.25 As new technologies emerge, it will likely become possible to utilize NMR to probe active enzyme systems that catalyze irreversible reactions such as those with CCHFV vOTU. Thus, applying NMR relaxation experiments during turnover would provide invaluable information to help
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further bridge our understanding between dynamics and function.
CCHF vOTU present. Data sets were analyzed with the NanoAnalyze software and fit to an independent model.
Material and Methods Protein expression and purification
NMR spectroscopy and data analysis
For CCHFV vOTU, the sequence encoding the protease Residues 1–169 was cloned into a pET15b vector (Novagen, Madison, WI) encoding an N-terminal 6xHis tag for subsequent expression in BL21/DE3 cells. These residues have been shown to be both necessary and sufficient for CCHFV vOTU structural integrity and activity.9 CCHFV vOTU was grown in LB or M9 minimal media supplemented with 15N-ammonium chloride for 15N-labeled samples. For 13C,15N,2H-labeled CCHFV vOTU in M9 minimal media, 13C-glucose with D2O was used instead of unlabeled glucose in H2O and spun cell pellets were refolded in their entirety as previously described for several proteins in order to exchange amide deuterons for protons.16,26–28 Cells were lysed via sonication directly in Ni Buffer (50 mM Na3PO4, 500 mM NaCl pH 7.5, and 10 mM imidazole) or refolded protein was dialyzed into Ni buffer and applied to a Ni-affinity column (GE Healthcare). Elutions were concentrated and cleaved with thrombin to remove the 6xHis tag. Lastly, size exclusion chromatography using a Superose 75 column (GE Healthcare) was used to further purify CCHFVp using NMR Buffer (50 mM Na3PO4, 150 mM NaCl pH 6.5, 1 mM DTT). All purifications were conducted on an AKTA FPLC system (GE Healthcare). For Ub, the sequence encoding the full-length protein of Residues 1–76 was cloned into a pET28b vector (Novagen), yet the 6xHis tag was removed as it was found unnecessary using the following purification that we developed based on the relative high stability of Ub. Specifically labeled Ub proteins were prepared as described above. Cells were lysed in Ub SP Buffer (50 mM NaOAc, pH 5.0) or refolded protein was dialyzed into Ub SP Buffer. Protein was applied to an SP-Sepharose column (GE Healthcare) and eluted with Ub SP Buffer plus 1 M NaCl. Eluted fractions containing Ub were concentrated and further purified over a Superose-75 in NMR Buffer.
For assignments, a 1-mM 13C,15N,2H-labeled CCHFV vOTU sample and 3 mM 13C,15N,2H-labeled Ub sample were used to assign each protein alone in the same NMR Buffer (50 mM Na3PO4, 150 mM NaCl pH 6.5, 1 mM DTT). For the complexes, samples of the bound conformation of each comprised 0.7 mM 13C,15N,2H-labeled protein with 0.8 mM unlabeled of the other. All assignment spectra were collected on either a Varian 600 MHz or Varian 800 MHz spectrometer equipped with a Cold Probe, except for 13C,15N,2H-labeled Ub that was collected on a Bruker 700 MHz equipped with a Cold Probe. Standard 3D HNCACB and HNcoCACB pulse sequences were used to assign the free proteins and a 15N-edited NOESY-HSQC was also used to help assign sequential amides for CCHFV vOTU. For the complexes, TROSY versions of HNCA, HNcoCA, HNCACB, and HNcoCACB were used with the latter two optimized to transfer magnitization to the CB using a tauCC of 6 ms. For relaxation experiments, standard R2, R1rho, and R1 relaxation experiments that comprise the BioPack software were used on a Varian 900 MHz spectrometer for a 1 mM 15N-labeled CCHFV vOTU. Relaxation times for R2 and R1rho were 0.01, 0.03, 0.05, 0.07, and 0.09 s while relaxation times for R1 were 0.01, 0.1, 0.3, 0.5, 0.7, 0.9, and 1.1 s. Relaxation rates were obtained for each data set using nmrPipe software fitting to a single exponential. For dispersion experiments, Transverse Relaxation Optimized SpectroscopY (TROSY)-based 15N-R2-CPMG pulse sequences were to either 1 mM 2H,15N-labeled proteins alone or complexes with concentrations described in the main text at both 600 MHz and 900 MHz29 and analyzed using the least squares fitting program CPMG_FIT, kindly provided by Dr. Dmitry Korzhnev (Departments of Molecular & Medical Genetics & Biochemistry, University of Toronto, Ontario, Canada). This program employs an analytical solution of the data to the full Carver-Richards equations as we have previously described.16,28
Isothermal titration calorimetry ITC experiments were performed in duplicate using a NanoITC system (TA Instruments, Lindon, UT). To assess the interaction between CCHF vOTU and Ub, a solution of 3.5 mM Ub was titrated into 0.65 mM CCHF vOTU. ITC runs were performed at 25 C and comprised of one injection of 1 lL followed by 24 injections of 2 lL for a total of 25 injections. Each injection was spaced 200 s apart. To account for the heat of dilution, 3.5 mM Ub was titrated in identical manner into the dialysis buffer with no
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