NMR observation of HIV-1 gp120 conformational flexibility resulting from V3 truncation Adi Moseri1,*, Einat Schnur1,*, Eran Noah1, Yuri Zherdev1, Naama Kessler1, Eshu Singhal Sinha1, Meital Abayev1, Fred Naider2, Tali Scherf3 and Jacob Anglister1 1 Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel 2 Department of Chemistry and Macromolecular Assembly Institute, College of Staten Island of the City University of New York, Staten Island, NY, USA 3 Department of Chemical Research Support, Weizmann Institute of Science, Rehovot, Israel

Keywords CD4M33; dynamics; gp120; NMR; V3 Correspondence J. Anglister, Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel Fax: +972 8 9343361 Tel: +972 8 9343394 E-mail: [email protected] *These authors contributed equally to this work. (Received 25 March 2014, revised 8 May 2014, accepted 9 May 2014) doi:10.1111/febs.12839

The envelope spike of HIV-1, which consists of three external gp120 and three transmembrane gp41 glycoproteins, recognizes its target cells by successively binding to its primary CD4 receptor and a coreceptor molecule. Until recently, atomic-resolution structures were available primarily for monomeric HIV-1 gp120, in which the V1, V2 and V3 variable loops were omitted (gp120core), in complex with soluble CD4 (sCD4). Differences between the structure of HIV gp120core in complex with sCD4 and the structure of unliganded simian immunodeficiency virus gp120core led to the hypothesis that gp120 undergoes a major conformational change upon sCD4 binding. To investigate the conformational flexibility of gp120, we generated two forms of mutated gp120 amenable for NMR studies: one with V1, V2 and V3 omitted (mutgp120core) and the other containing the V3 region [mutgp120core(+V3)]. The TROSY-1H-15NHSQC spectra of [2H,13C,15N]Arg-labeled and [2H,13C,15N]Ile-labeled unliganded mutgp120core showed many fewer crosspeaks than the expected number, and also many fewer crosspeaks in comparison with the labeled mut gp120core bound to the CD4-mimic peptide, CD4M33. This finding suggests that in the unliganded form, mutgp120core shows considerable flexibility and motions on the millisecond time scale. In contrast, most of the expected crosspeaks were observed for the unliganded mutgp120core(+V3), and only a few changes in chemical shift were observed upon CD4M33 binding. These results indicate that mutgp120core(+V3) does not show any significant conformational flexibility in its unliganded form and does not undergo any significant conformational change upon CD4M33 binding, underlining the importance of V3 in stabilizing the gp120core conformation.

Introduction The envelope spike of HIV-1 is responsible for the recognition of the virus’s target cells by successively

binding to the primary receptor CD4 and a coreceptor, which can be either the CCR5 or CXCR4 chemokine

Abbreviations DCN, 2H, 13C and 15N; gp120core, gp120 molecule containing residues 88–492 and with deletion of the variable regions V1, V2 and V3 [1]; gp120core(+V3), gp120 molecule containing residues 88–492 and with deletion of the variable regions V1, and V2; gp120core-e, gp120 molecule containing residues 44–492 and with deletion of the variable regions V1 and V2 and containing the opposing strands of the V3 base connected by a flexible linker (TRPNNGGSGSGGDIRQAH); gp120JR-FL, gp120 molecule of the HIV-1 JR-FL strain; HEK, human embryonic kidney; HSQC, Hetero Nuclear Single Quantum Coherence; MGN, Man5GlcNAc2 glycan; mutgp120core, gp120 molecule containing residues 88–492 and with deletion of the variable regions V1 V2 and V3; mutgp120core(+V3), gp120 molecule containing residues 88–492 and with deletion of the variable regions V1 and V2; sCD4, soluble extracellular form of human CD4; SIV, simian immunodeficiency virus; SPR, surface plasmon resonance; TROSY, transverse relaxation optimized spectroscopy.

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receptor. This spike consists of three gp120 and three gp41 molecules (originating from a gp160 precursor protein) held together by noncovalent interactions. Binding of CD4 causes a conformational change in the envelope spike, resulting in the formation and exposure of the coreceptor binding site. Binding to the coreceptor results in additional conformational changes in the envelope spike, leading to the fusion of the virus with its target cell, a process that is mediated by the transmembrane envelope protein gp41. The structure of the deglycosylated monomeric HIV gp120 core molecules, gp120core, in complex with a soluble extracellular form of human CD4 (sCD4) and different antibodies have been determined [1,2]. The gp120core construct studied lacked the V1/V2 and V3 variable domains that are presumed to be involved in the formation of the trimeric structure of the envelope spike. Truncation of these variable loops facilitated the crystallization of gp120core/sCD4 complexes. Both glycosylated and unglycosylated unliganded HIV-1 gp120core molecules have resisted crystallization. However, when glycosylated unliganded simian immunodeficiency virus (SIV) gp120core was crystallized and its structure was determined by X-ray crystallography [3], its structure was found to be very different from the structure of deglycosylated HIV-1 gp120core in complex with sCD4 [1,3]. The conformational change involved the formation of a b-sheet that created a bridge between the inner and the outer domains of gp120. Two strands of this bridging b-sheet, namely b20 and b21, have been implicated in coreceptor binding [4]. The large difference between the liganded HIV1 gp120core and unliganded SIV-1 gp120core structures led to the hypothesis that gp120 undergoes a major conformational change upon CD4 binding [5]. However, it has been suggested that the conformation of unliganded HIV-1 gp120 is dynamic and that it transiently populates the liganded state [3]. Thermodynamic studies of the binding of gp120core and full-length gp120 to sCD4 supported the hypothesis that a major conformational change is required for the formation of the coreceptor binding site [6,7]. Massspectroscopy studies revealed that unliganded deglycosylated gp120core is considerably more flexible than sCD4-bound deglycosylated gp120core, and that within this core molecule, the inner domain is more flexible than the outer domain [8]. The large unfavorable change in entropy upon sCD4 binding to gp120 and what seemed to be the creation of the CD4-binding site only upon complex formation led Kwong and coworkers to propose the ‘conformational masking’ mechanism (induced-fit) as one of the strategies that the virus has developed to evade the immune system [7]. 3020

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Recently, the structures of several unliganded deglycosylated extended gp120core molecules containing the base of V3 were solved by X-ray crystallography [9] and revealed a structure that was very similar to that of sCD4-bound gp120core and different from the structure of unliganded glycosylated SIV gp120core. These new structures have led Kwong and coworkers to suggest that gp120 exists in a dynamic equilibrium between the unliganded and sCD4-bound conformations and that, in gp120core constructs lacking V1/V2 but containing the base of V3 (gp120core-e), the equilibrium is shifted considerably towards the liganded conformation even for the free form of gp120 [9]. In the native unliganded trimeric spike, it has been assumed that the sCD4-bound conformation is considerably less populated [9,10]. Cryoelectron tomography revealed the architecture of native unliganded HIV-1 envelope spike on virions  resolution [11]. Whereas the structure of at ~ 20-A sCD4-bound HIV-1 gp120core could be fitted into this low-resolution structure of the trimer, the structure of unliganded SIV gp120core did not agree with the tomographic data [10,11]. Very recently, the structure of cleaved and stabilized HIV-1 unliganded envelope trimer was determined by X-ray crystallography [12] and by cryo-electron microscopy [13]. These two structures of the unliganded envelope trimer agree with earlier structures of sCD4-bound gp120core and sCD4-bound gp120core containing V3 [gp120core(+V3)] [14,15] and unliganded gp120core-e [9]. The structure of unliganded SIV gp120 molecule [3] was found to be very different from these two structures. Together, V3 and C4 form the binding site for the CCR5 and CXCR4 chemokine receptors, which are used by the virus as coreceptors for cell entry [16–19]. The amino acid sequence of V3 determines whether the HIV-1 strain will be an R5 or X4 virus and use the CCR5 or CXCR4 receptor, respectively [20]. On the basis of studies of gp120 with chimeric V3 or with deletions in V3, it has been suggested that the V3 stem (residues 296–305 and 321–330) is involved in binding to the N-terminal segment of CCR5, whereas the V3 crown (residues 306–320) is involved in binding to the CCR5 extracellular loops [18]. A model for the gp120 complex with a 27-residue N-terminal CCR5 peptide was derived on the basis of NMR and biochemical data [21]. In this model, the CCR5 N-terminal segment interacted with the base of V3 and with residues in C4. The crystal structure of a CXCR4 complex with a cyclic peptide antagonist suggests that the crown of V3 occupies a binding pocket formed by the transmembrane helices and ECL2 of the chemokine receptor [22]. In view of its crucial role in coreceptor FEBS Journal 281 (2014) 3019–3031 ª 2014 FEBS

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binding, it is important to design gp120 constructs that are suitable for detailed structural studies by either NMR or X-ray crystallography, and to investigate the dynamics of the gp120 envelope glycoprotein. It has been suggested that truncation of the whole of V3 leads to destabilization of gp120core in its unliganded form [9], explaining why HIV-1 gp120core could not be crystallized. NMR is an excellent technique with which to characterize, in a residue-specific manner, the flexibility and the dynamics of proteins [23]. Fast segmental mobility on the nanosecond timescale causes averaging of chemical shifts, and protein residues showing such motions are characterized by narrower line-widths of their NMR signals and longer transverse relaxation times than those of other residues that do not undergo such motions. On the other hand, resonances of protein residues undergoing slower motions, on the millisecond timescale, show considerable broadening that results in vanishing intensities of their peaks [24,25]. The 1H-15N-HSQC spectra of uniformly 15N-labeled proteins is best suited for studying the dynamics of proteins in a residue-specific manner, as each of the protein residues (except for prolines) is represented by one amide backbone crosspeak [26]. Comparison of the intensities and the line-widths of the 1H-15N-HSQC crosspeaks of a protein can provide qualitative information concerning the conformational flexibility of individual residues of the protein. More details can be obtained by quantitative studies of the transverse and longitudinal relaxation times of the backbone amides and by measurements of 1H-15N-NOEs [26]. NMR studies of the structure and dynamics of proteins larger than 25 kDa require deuteration of the nonexchangeable hydrogen atoms of the protein, in addition to 15N and 13C labeling [27], to increase the transverse relaxation times, narrow the signals, and in turn improve the signal-to-noise ratio of the NMR spectra. Such labeling can be easily achieved for proteins expressed in Escherichia coli. However, gp120 is heavily glycosylated, and its expression in E. coli gives rise to misfolded and nonfunctional molecules. This, together with its large size (~ 120 kDa), has hampered NMR studies of gp120. In a recent study, a 220-residue gp120 outer domain molecule was expressed in A549 adenocarcinoma human cells [28]. However, this gp120 molecule does not bind CD4. In the present study, we designed, expressed and labeled mutants of the 336-residue gp120core lacking or containing V3 [mutgp120core and mutgp120core(+V3), respectively]. These mostly monomeric mutants (~ 43 kDa with V3) retain high-affinity CD4 binding, enabling NMR studies of gp120 in the free and ligand-bound states. To FEBS Journal 281 (2014) 3019–3031 ª 2014 FEBS

HIV-1 gp120 conformational flexibility

simplify the HSQC spectra of mutgp120core and mut gp120core(+V3), we used residue-specific isotopic labeling. Here, we show that unliganded mutgp120core shows motions on the millisecond timescale and that, upon binding of the CD4-mimic peptide CD4M33, gp120 undergoes a considerable reduction in conformational flexibility. On the other hand, gp120core(+V3) showed considerably less flexibility, and did not show any significant conformational changes upon CD4M33 binding, in agreement with the crystallographic studies of Kwong et al. [9]. Our results show unequivocally that V3 has an important role (in addition to its role in coreceptor binding) in stabilizing the unliganded gp120 conformation and that its truncation results in considerable conformational flexibility. Therefore, the presence of V3, or at least its base, is essential for the structural integrity of unliganded gp120 constructs.

Results Design of gp120 for NMR studies To overcome the previously observed tendency of gp120core molecules to oligomerize [21], we designed a gp120core mutant derived from the HIV-1 JR-FL strain, mutgp120core, containing four mutations in the first a-helix (N99Q, E106Q, D107Q and D113Q). These residues are localized on the non-neutralizing face of the viral spike (Fig. 1), and were suggested to be buried within the gp120 trimer interface [29]. As shown below, mutgp120core is mostly monomeric under the NMR measurement conditions. Two mutgp120core molecules were expressed, one that contained V3 and lacked V1/V2 and one that lacked V1, V2, and V3. The mutgp120core molecules contained Man5GlcNAc2 glycans (MGN) at sites normally occupied by complex or hybrid glycans [30]. Deglycosylation with Endo-Hf left one GlcNac moiety at each glycosylation site. From here onwards, the glycosylated gp120 molecules are named MGN-mutgp120core and MGN-mut gp120core(+V3), and after deglycosylation (one remaining GlcNac moiety), mutgp120core and mut gp120core(+V3). The marked reduction of oligomerization in MGN-mutgp120core and MGN-mutgp120core(+V3) in comparison with the unmutated MGNgp120core(+V3) is evident from the preparative size exclusion chromatograms of these molecules (Fig. 2). It has been found previously that a high salt concentration (300 mM) alleviates somewhat the aggregation of both unliganded MGN gp120core(+V3) and gp120core(+V3), and purification of this unmutated construct [Fig. 2A; MGN gp120core(+V3)] was therefore performed at the higher salt concentration [21]. Nevertheless, even with a

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high salt concentration [which was not required for MGN-mut gp120core(+V3) or mutgp120core(+V3)], a considerable fraction of the unmutated MGNgp120core(+V3) population consisted of dimers and higher aggregates (Fig. 2A), whereas MGN-mutgp120core and MGN-mutgp120core(+V3) eluted mostly as monomers (Fig. 2B and 2C). MGN-mut gp120core(+V3) high-affinity binding to CD4M33 was verified with surface plasmon resonance (SPR) measurements and biotinylated CD4M33 immobilized on a streptavidin chip (data not shown). A KD of 67.0  2.7 nM was determined, indicating approximately five-fold weaker binding than for MGNgp120core [21]. Characterization of the oligomerization state of gp120 The oligomerization states of mutgp120core and gp120core(+V3) before and after CD4M33 binding were investigated by analytical size exclusion chromatog-

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Fig. 2. Preparative size exclusion chromatography profiles of MGN MGN MGNgp120 variants. (A) gp120core(+V3), (B) MGN-mut mut gp120core(+V3) and (C) gp120core on Superdex 200 16/ 60 pg in 50 mM Tris/HCl buffer (pH 8.0), with 300 mM NaCl for MGN gp120core(+V3), and 150 mM for MGN-mutgp120core(+V3) and MGNmut gp120core. The monomeric protein elutes at ~ 74 mL. Dimers and higher molecular mass aggregates can also be observed, and are significantly more abundant for MGNgp120core(+V3).

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Fig. 1. The location of the mutations on gp120core(+V3). Ribbon diagram of gp120core(+V3) (Reproduced from Protein Data Bank: 2QAD, [15]) showing, in red stick representation, residues mutated to glutamine to create the monomeric mut gp120core(+V3).

raphy and by sedimentation equilibrium. This analysis revealed that unliganded mutgp120core was ~ 85% monomeric and 12% dimeric, with minor populations of higher aggregates (Fig. 3A). Addition of CD4M33 in a 1 : 1 ratio resulted in a mutgp120core/CD4M33 complex with a slightly increased molecular mass, as expected, without any significant change in the populations of monomeric and dimeric forms (Fig. 3B). According to the elution time of specified protein markers, the estimated molecular mass of the monomeric form was ~ 40 kDa. Similarly, the chromatogram of unliganded mutgp120core(+V3) revealed a small amount of dimeric gp120 (12%), with the remaining protein being in the monomeric form (Fig. 3C). However, addition of CD4M33 in a 1 : 1 ratio shifted the monomer to dimer equilibrium to 60% dimer and 40% monomer, as shown in Fig. 3D. The fact that both gp120 constructs contained a mixture of monomers and dimers allowed only a rough estimate of the molecular mass by sedimentation equilibrium analysis. Moreover, the apparent molecular mass changed with the concentration of gp120 and the rotor angular velocity. In view of these complications, free mutgp120core showed an apparent molecular mass in the range of 39–43 kDa at a concentration of 21 lM and rotor angular velocities of 5152 and 11592g respectively (Fig. 4). Addition of CD4M33 increased the apparent molecular mass by ~ 8 kDa, which was more than the expected increase of ~ 4 kDa. Addition of CD4M33 to mutgp120core(+V3) doubled its apparent molecular mass from 49 to 57 kDa to 91–120 kDa, at 18 lM mutgp120core(+V3), providing a further indication that dimerization of this gp120 construct is favored by CD4M33 binding. The sedimentation equilibrium analysis is in very good agreement with the molecular mass estimates obtained from size exclusion chromatography. FEBS Journal 281 (2014) 3019–3031 ª 2014 FEBS

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Fig. 3. Analytical size exclusion chromatography profiles (Superdex 200 10/300) of unliganded and liganded mutgp120core and mutgp120core(+V3). (A) mutgp120core. (B) mutgp120core–CD4M33. (C) mutgp120core(+V3). (D) mutgp120core(+V3)–CD4M33. The protein solution was 20 mM Tris (pH 7), 150 mM NaCl, and 0.05% NaN3, and the flow rate was 0.5 mL
min 1. mAU, milli-Absorbance Units.

Unliganded mutgp120core shows conformational equilibrium on the millisecond timescale As mentioned above, the 1H-15N-HSQC spectra of proteins with a molecular mass lower than ~ 50 kDa can provide an indication of whether regions of the protein molecule undergo motions on the millisecond timescale [25,31,32]. Residues in segments that show such motions are characterized by broad crosspeaks,

Fig. 4. Sedimentation equilibrium of mutgp120core. Top panel: fitted sedimentation equilibrium of mutgp120core and mutgp120core– CD4M33 measured by analytical ultracentrifugation (280 nm, 11 591 g, 300 K) (top panel). The samples shown contained 21 lM envelope protein in the absence or presence of CD4M33 (black and gray, respectively) in Tris/HCl buffer (pH 7.0) with 150 mM NaCl. Bottom panel: residuals of free mutgp120core and mutgp120core– CD4M33 (black and gray, respectively).

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which are considerably weaker than the crosspeaks of the rest of the molecule. The extensive overlap expected in the 1H-15N-HSQC spectrum of the 336-residue uniformly 15N-labeled [U-15N]-mutgp120core(+V3) would most likely hamper detailed examination of the line-widths and intensities of the individual gp120 amide proton crosspeaks. Therefore, to simplify the spectrum, we used residue-specific 2H, 13C and 15N (DCN) labeling of gp120 [33]. The gp120 coreceptor-binding site, which is composed mostly of V3 and C4, is rich in arginines and isoleucines [4,15,21,34,35]. In view of the importance of these arginines and isoleucines in coreceptor binding, we focused our measurements on these two amino acids. Overall, there are 11 arginines in the mut gp120core construct used in the present study, and 15 in mutgp120core(+V3). Usually, the locations of isoleucines and arginines within the protein are different, with isoleucine side chains buried in inner hydrophobic regions, and the charged arginine side chains exposed to the solvent. Thus, the isotopic labeling of these two amino acids allowed us to probe different locations in the gp120 molecule. A section of the transverse relaxation optimized spectroscopy (TROSY) 1H-15N-HSQC spectrum of (DCN-Arg)-mutgp120core in its unliganded form, showing the arginine backbone amide crosspeaks, is shown in Fig. 5A (black crosspeaks). Only two backbone crosspeaks were clearly observed out of the 11 expected crosspeaks. In contrast, the TROSY-1H-15NHSQC spectrum of CD4M33-bound (DCN-Arg)-mutgp120 core (Fig. 5A, red crosspeaks) showed 11 intense crosspeaks corresponding to backbone amide protons, and two of them partially overlapped. The peaks labeled ‘sc’ (Fig. 5A) were identified as side chain crosspeaks (see Experimental procedures). Thus, all of the expected backbone amides of the arginines were clearly observed in the spectrum of mutgp120core/CD4M33. The size exclusion chromatography and sedimentation equilibrium analyses exclude the possibility that the vanishing of the crosspeaks is attributable to aggregation of unliganded mutgp120core. Thus, the absence of nine of the expected arginine backbone crosspeaks in the spectrum of unliganded mutgp120core is probably attributable to conformational equilibrium with exchange rates on the millisecond time scale manifested by a considerable part of the unliganded gp120 molecule, which leads to broadening of these crosspeaks beyond the limit of detection [25,31,32]. Isoleucines are more abundant than arginines in gp120. There are 27 isoleucines in mutgp120core. In the unliganded spectrum of (DCN-Ile)-mutgp120core (Fig. 5B, black crosspeaks), only a fraction of the 27

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Fig. 5. Overlay 1H/15N-HSQC NMR spectra of labeled mutgp120core before and after CD4M33 binding. (A) TROSY-1H/15N-HSQC spectra of (DCN-Arg)-mutgp120core; protein only (black) and in the presence of CD4M33 (1 : 1, red). (B) TROSY-1H/15N-HSQC spectra of (DCN-Ile)-mutgp120core; protein only (black) and in the presence of CD4M33 (1 : 1, red). Spectra were obtained at pH 7.0, 150 mM NaCl and 300 K on a Bruker AVIII800 NMR spectrometer equipped with a cryoprobe. sc denotes side chain peaks.

expected crosspeaks, three very strong crosspeaks and ~ 14 weaker crosspeaks, many of which were almost at the noise level, were observed. After the addition of CD4M33, the quality of the spectrum improved drastically and many more crosspeaks with a good signal-tonoise ratio were observed (Fig. 5B, red crosspeaks). This improvement in the quality of the HSQC spectrum after CD4M33 binding indicates that, whereas unliganded mut gp120core shows conformational dynamics on the millisecond timescale, mutgp120core/CD4M33 probably adopts one single major conformation. Thus, with the isotopic labeling of two amino acids that probe different locations in the protein structure, similar conclusions are obtained regarding the dynamics of mutgp120core. Unliganded and CD4M33-bound mut gp120core(+V3) adopt similar conformations Kwong and coworkers suggested that unliganded gp120core, which lacks the whole of V3, shows considerable mobility, owing to the abrogation of interactions and hydrogen bonds between the base of V3 and both the N-terminal and C-terminal segments of C4 [9]. This flexibility of unliganded gp120core was manifested by a large entropy change upon sCD4 binding [7]. A larger gp120 molecule, which, in addition to the core, contained the base of V3, i.e. mutgp120core-e, showed less than half of this entropy change in the same experiment [9]. To further investigate the role of V3 in stabilizing the gp120 conformation, we measured the TROSY-1H-15N-HSQC spectrum of unliganded (DCNArg)-mutgp120core(+V3). The HSQC spectrum of this gp120 molecule (Fig. 6A, black crosspeaks) revealed ~ 15 HSQC crosspeaks contributed by backbone amides, in comparison with the only two backbone 3024

crosspeaks that were clearly observed for unliganded mut gp120core (Fig. 5A, black crosspeaks). According to both sedimentation equilibrium analysis and size exclusion chromatography, both mutgp120core and mut gp120core(+V3) are mostly monomeric in their unliganded forms. Thus, this dramatic difference in the number of the observed HSQC crosspeaks between the two unliganded gp120 molecules is consistent with the conclusion that unliganded mutgp120core(+V3) does not show the same conformational flexibility as unliganded mutgp120core. Binding of CD4M33 to (DCNArg)-mutgp120core(+V3) resulted in only minor changes in the chemical shifts of arginines, and practically no changes in the number of observed HSQC crosspeaks or their intensity (Fig. 6A, red crosspeaks underneath black crosspeaks). Thus, it appears that mut gp120core(+V3) does not show significant changes in flexibility or a major change in its structure upon CD4M33 binding. To further examine the flexibility of mut gp120core(+V3) and possible conformational changes upon CD4M33 binding, TROSY-1H-15N-HSQC spectra were also obtained for (DCN-Ile)mut gp120core(+V3). The spectra were recorded for free (DCN-Ile)-mutgp120core(+V3) and for this protein in complex with CD4M33 (Fig. 6B). Approximately 26 of the 32 expected isoleucine crosspeaks were observed in the TROSY-1H-15N-HSQC spectrum of unliganded (DCN-Ile)-mutgp120core(+V3) (Fig. 6B, in black), many more than those observed in the spectrum of unliganded (DCN-Ile)-mutgp120core (black crosspeaks). This large difference in the observed number of isoleucine crosspeaks in the unliganded form of gp120 supports our conclusion that unliganded mut gp120core(+V3) does not show the same conformational flexibility as unliganded mutgp120core. FEBS Journal 281 (2014) 3019–3031 ª 2014 FEBS

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Fig. 6. Comparison of the TROSY-1H/15N-HSQC NMR spectra of labeled mutgp120core(+V3) before and after CD4M33 binding. (A) The TROSY-1H/15N-HSQC spectra of (DCN-Arg)-mutgp120core(+V3); protein only (black) and in the presence of CD4M33 (1 : 1, red). (B) The TROSY-1H/15N-HSQC spectra of (DCN-Ile)-mutgp120core(+V3); protein only (black) and in the presence of CD4M33 (1 : 1, red). Spectra were obtained at pH 7.0, 150 mM NaCl and 300 K on a Bruker AVIII800 NMR spectrometer equipped with a cryoprobe. c, complex; f, free.

The addition of CD4M33 to (DCN-Ile)gp120core(+V3) did not cause a drastic change in the HSQC spectrum, and most of the observed crosspeaks (Fig. 6B, red crosspeaks underneath black crosspeaks) showed minor or no changes in chemical shift upon CD4M33 binding. However, three crosspeaks, labeled ‘f’ (for free), disappeared upon the addition of CD4M33, and one new crosspeak, labeled ‘c’ (for complex), appeared. The disappearance of an NMR resonance or a change in its chemical shift upon ligand binding is usually attributed to protein residues involved in interactions with the ligand or to residues found in regions of the protein that experience conformational change upon ligand binding. Thus, the comparison of the two spectra supports the conclusion that, unlike mutgp120core, mut gp120core(+V3) does not undergo a major change in flexibility or in conformation upon CD4M33 binding.

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Assignment of the HSQC crosspeaks to V3 of gp120 – dynamic insights Comparison of the most intense crosspeaks in the HSQC spectrum of (DCN-Arg)-mutgp120core(+V3) in the unliganded form and in complex with CD4M33 (Fig. 6A black and red, respectively) with those of revealed four (DCN-Arg)-mutgp120core/CD4M33 strong crosspeaks (labeled V3 in Fig. 7A) that were not found in the protein lacking V3 (azure crosspeaks). It is very likely that the four new crosspeaks were contributed by the four arginines of the V3 region: Arg298, Arg304, Arg315, and Arg328. These four strong peaks of (DCN-Arg)-mutgp120core(+V3) in the unliganded and CD4M33-bound form (black on top of red crosspeaks and labeled V3 in Fig. 7A), FEBS Journal 281 (2014) 3019–3031 ª 2014 FEBS

respectively, did not show any significant change in chemical shift upon ligand binding, suggesting that they are not involved in CD4M33 binding. The unusually strong intensity of the four arginine crosspeaks attributed to V3 suggests that V3 is very flexible and undergoes motions on the nanosecond timescale, resulting in narrowing of its crosspeaks and stronger intensities. In the same manner, by comparing the most intense crosspeaks in the HSQC spectra of (DCN-Ile)-mutgp120 core(+V3) in the presence and absence of CD4M33 (red underneath black crosspeaks in Fig. 7B) with those of (DCN-Ile)-mutgp120core in the presence of CD4M33 (azure in Fig. 7B), we were able to identify the isoleucine crosspeaks of V3. Four strong crosspeaks in the spectrum of (DCNIle)-mutgp120core(+V3)/CD4M33 (red crosspeaks in Fig. 7B) that were not found in that of (DCNIle)-mutgp120core/CD4M33 are attributed to four of the five isoleucines of V3. The fifth crosspeak was missing, possibly because of overlap with the other crosspeaks. In the absence of CD4M33, the HSQC spectrum of (DCN-Ile)-mutgp120core(+V3) revealed five crosspeaks (black crosspeaks in Fig. 7B) corresponding to the five isoleucines of V3. Two of the crosspeaks, labeled ‘*V3’ in black, differ in their chemical shift from the isoleucine crosspeaks of CD4M33-bound mutgp120core(+V3), suggesting that two isoleucines of V3 interact with CD4M33 or are located in a region that underwent some conformational change upon CD4M33 binding. The strong intensities of the isoleucine crosspeaks attributed to V3 residues suggest that, like the V3 arginines, these isoleucines undergo fast motion in comparison with the considerably slower tumbling of the gp120 core, supporting the conclusion that V3 is very flexible.

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Fig. 7. Identification of V3 crosspeaks: overlay of the 1H/15N-HSQC NMR spectra of (DCN-Arg)-mutgp120core(+V3) and (DCN-Ile)-mutgp120core(+V3) before (black) and after CD4M33 binding (red), and of mutgp120core after CD4M33 binding (azure); crosspeaks attributed to V3 are the most intense. (A) TROSY-1H/15N-HSQC spectra of the three forms of (DCN-Arg)-mutgp120core(+V3). Only the most intense crosspeaks are shown. Those crosspeaks attributed to V3 arginines are labeled. (B) TROSY-1H/15N-HSQC spectra of (DCN-Ile)-mutgp120core(+V3). Spectra were obtained at pH 7.0, 150 mM NaCl and 300 K on a Bruker AVIII800 NMR spectrometer equipped with a cryoprobe.

Discussion

gp120 with the N-terminal segments of CCR5 and CXCR4 [21].

gp120 is suitable for NMR studies In this study, we designed, expressed and characterized mutated gp120 molecules, which proved to be suitable for NMR studies. Two gp120 forms were obtained: one that contained V3 and the other with deletion of the whole of V3, i.e. mutgp120core(+V3) and mut gp120core, respectively. Unlike the outer-domain gp120 molecule that was obtained by Kwong and coworkers, our larger gp120 molecule (molecular mass of ~ 40 kDa) contains both the outer and the inner domains, and is capable of binding sCD4 and the CD4-mimetic compound CD4M33. Residue-specific labeling of mutgp120core(+V3) and mutgp120core with DCN-Arg and DCN-Ile enabled us to assign HSQC crosspeaks to two classes of gp120 residue (arginine and isoleucine), and to obtain insights into the dynamics of regions of these gp120 molecules as discussed below. In the unliganded form, both mutgp120core(+V3) and mut gp120core were found to be predominantly (~ 85– 88%) monomeric. However, upon CD4M33 binding, mut gp120core(+V3)/CD4M33 showed an increased tendency to form a dimer (~ 60%), whereas mutgp120core/ CD4M33 showed a monomer/dimer ratio similar to that of unliganded mutgp120core. This finding suggests that V3 contributes to the formation of gp120 dimers and oligomers, and further manipulation of the mut gp120core(+V3) construct is required to alleviate its tendency to oligomerize upon CD4M33 binding. Such a construct is necessary to study the interactions of 3026

Unliganded mutgp120core shows conformational flexibility as a result of V3 truncation Examination of spectra of unliganded mutgp120core and mut gp120core(+V3) reveals that many of the expected crosspeaks are not observed for unliganded mut gp120core. The difference between the spectra of the unliganded and CD4M33-bound molecules was especially pronounced for (DCN-Arg)-mutgp120core. Whereas, for unliganded (DCN-Arg)-mutgp120core, only two backbone arginine crosspeaks were clearly observed (Fig. 5), all of the 11 expected signals were observed for unliganded (DCN-Arg)mut gp120core(+V3). The disappearance of the crosspeaks in the unliganded form of mutgp120core is most probably attributable to conformational flexibility, with motion on the microsecond time scale that results in broadening of the crosspeaks beyond the limit of detection [25,32]. The possibility that oligomerization of mutgp120core in the unliganded form caused the broadening of the crosspeaks beyond the limit of detection was eliminated by both size exclusion chromatography and sedimentation equilibrium, which indicated that this protein is predominantly in the monomeric form (Figs 2, 3A and 4A). Our findings regarding the flexibility of unliganded mutgp120core are in agreement with thermodynamic studies by Kwong and coworkers, which showed that CD4 binding to gp120core resulted

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HIV-1 gp120 conformational flexibility

in a large, unfavorable change in entropy [7], consistent with a reduction in conformational exchange. In contrast to the HSQC spectrum of unliganded (DCN-Arg)-mutgp120core, the spectrum of unliganded (DCN-Arg)-mutgp120core(+V3) reveals all of the expected arginine backbone crosspeaks, indicating that V3 stabilizes the mutgp120core(+V3) conformation and that, in the presence of intact V3, unliganded gp120core does not show conformational flexibility with motions on the millisecond timescale. This influence on the overall stability of gp120 can be explained by the extensive interactions of the base of V3 with C4 [9]. It has been suggested that complete truncation of V3 could destabilize the unliganded gp120 conformation by removing these interactions [9]. The conformation of unliganded mut gp120core(+V3) is similar to that of mut gp120core(+V3) when bound to CD4M33 Comparison of the HSQC spectra of (DCN-Arg)mut gp120core(+V3) in the presence and absence of CD4M33 reveals that the conformation of mutgp120 mut gp120core(+V3)/ core(+V3) is very similar to that of CD4M33. This finding is in agreement with the recent observation of Kwong and coworkers that the structure of an extended gp120core molecule containing the base of V3 is similar to that of gp120core molecules in complex with sCD4 [9]. Our finding is also in agreement with the recent X-ray and EM structures of unliganded trimeric gp140 molecules, which revealed only small differences between gp120core component of the trimeric complex and the structure of the core in monomeric gp120 complexes with sCD4 [12,13]. However, our results do not support the hypothesis of conformational masking of the CD4-binding site as suggested earlier by Kwong and coworkers on the basis of thermodynamic studies of gp120core binding to sCD4 [7], and by the very large difference between the structure of liganded HIV-1 gp120core and the crystal structure of unliganded SIV gp120 [3]. V3 in

mut

gp120core(+V3) undergoes fast motion

occur in the core domain. This motion on the nanosecond timescale causes conformational averaging and narrower NMR signals. As a result of these fast motions, the orientation of the V3-containing gp120 molecules is most probably not fixed relative to gp120core. Implications for coreceptor binding The destabilization of gp120core by truncation of V3 suggests that part of V3 is involved in extensive interactions with gp120core. In a published crystal structure, such interactions were observed between the base of V3 and the b19 and b21 strands in C4 [9]. Together, these regions form the surface interacting with the flexible N-terminal segment of CCR5 [21]. Molecular dynamics simulations show that three and six residues in the N-terminal and C-terminal segments, respectively, of V3 are not flexible [36]. Thus, the interaction of the V3 base with C4 not only stabilizes the structure of gp120core but also helps position the V3 in the correct orientation to form the binding site for the CCR5 N-terminal segment. Finally, Arg298, Pro299, Arg328 and Ala330 at the base of V3 are conserved in all HIV-1 clades, regardless of the phenotype. This suggests that, in most HIV-1 strains, common interactions occur between the V3 base and C4, a conclusion that is also supported by the molecular dynamic simulations of X4 and R5 gp120 containing V3 [36]. In addition to insights concerning the N-terminal and C-terminal segments of V3, molecular dynamics studies revealed that the stem and the crown of V3 are very flexible [36], in agreement with the flexibility of V3 arginines and isoleucines observed in the present study. The flexibility of the arginines and isoleucines is attributable to the lack of interactions between them and gp120core, and is enhanced by the presence of glycines at positions 321 and 325 on the C-terminal side of V3. It is reasonable to conclude that the flexibility of the V3 crown facilitates its insertion into the CCR5-binding and CXCR4-binding pockets formed by the transmembrane helices and ECL2 [22,37].

Conclusions

mut

We designed a truncated and mutated gp120 molecule that could be used in high-resolution NMR analyses of this HIV-1 envelope protein. The novel constructs that we developed were used to follow changes in its dynamics and in its conformation upon the binding of the CD4 mimic peptide CD4M33, and allowed us to identify arginines and isoleucines in V3. Our NMR results provide evidence for striking differences in the dynamics of unliganded gp120core and CD4M33-bound

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Comparison of the spectra of gp120core(+V3) and gp120core enabled us to identify the crosspeaks contributed by isoleucines and arginines of V3. Some of these residues were previously implicated in HIV-1 coreceptor binding [19,34]. The arginine and isoleucine crosspeaks attributed to V3 showed much stronger intensity than the crosspeaks of these residues in other regions of gp120. This strong intensity implies that the V3 arginines and isoleucines undergo much faster motions than

mut

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HIV-1 gp120 conformational flexibility

gp120core. Truncation of the whole of V3 leads to destabilization of gp120core, resulting in conformational motions on the millisecond timescale. In the presence of V3, the unliganded gp120 is stabilized and no significant conformational changes are observed upon CD4M33 binding. The stabilization of gp120core by interactions involving the V3 base and the flexibility of the V3 stem crown facilitate the interactions of the V3 base with N-terminal segment of CCR5 and the interactions of the V3 crown with the CCR5-binding pocket formed by the transmembrane helices and ECL2. We expect to apply the strategies used in this study to a ternary gp120/CD4M33/CCR5 peptide complex and to gain insights into the molecular pathway of HIV-1 infection.

Experimental procedures Peptide synthesis CD4M33 molecules with and without a biotin label were synthesized and purified as described previously [21].

Expression and purification of mut gp120core(+V3)

mut

gp120core and

A truncated gp120 molecule of the HIV-1 JR-FL strain (88–492gp120JR-FL) lacking the V1 and V2 variable loops was expressed in human embryonic kidney (HEK) 293 cells. To increase the affinity of the modified gp120JR-FL molecule for CD4, two glycosylation sites were mutated and removed, N301Q and T388A [88–492gp120DV1/V2 (N301Q;T388A)] [38]. Additionally, four residues in the first a-helix (Asn99, Glu106, Asp107, and Asp113) were mutated to glutamine in an attempt to create nonaggregating gp120, termed mutgp120core(+V3). A second construct, mut gp120core, in which Arg298–Ala330 of the V3 loop of mut gp120core(+V3) were replaced by a GAG sequence, was also designed. All of the mutations of the plasmid (described in Schnur et al. [21]) were generated with the QuikChange Site-Directed Mutagenesis Kit (Stratagene, CA, USA). MGN-mut gp120core(+V3) and MGN-mutgp120core were expressed in HEK293 cells lacking N-acetyl glucosaminyl transferase I enzymatic activity (GnTI-HEK293S cells [21,30]). Proteins expressed in cells lacking this enzyme are homogeneously glycosylated with MGN at sites normally occupied by complex or hybrid glycans. Treatment of this protein with Endo-Hf (New England Biolabs, MA, USA) removes the Man5-GlnNAc carbohydrates, leaving one GlcNac attached to an asparagine of the protein, resulting in a further reduction in molecular mass [~ 40 kDa; referred to herein as mutgp120core and mutgp120core(+V3)]. The cells were grown in DMEM supplemented with 10% fetal bovine serum (Biological Industries, Israel),

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50 lg
mL 1 puromycin, 20 000 units
L 1 penicillin, and 20 lg
mL 1 streptomycin. For the labeling of gp120, DMEM was prepared in-house with tissue culture-grade inorganic salts, vitamins, D-glucose, phenol red, sodium pyruvate, and unlabeled amino acids (except for the amino acid that was to be labeled), according to the DMEM recipe (https://www.lifetechnologies.com/il/en/home/technicalresources/media-formulation.9.html). Subsequently, the labelled amino acid (Cambridge Isotope Laboratories, Cambridge, MA, USA) was added to the medium. The DMEM was then supplemented with 10% dialyzed fetal bovine serum (Biological Industries, Israel), 25 mM Hepes (pH 7), 50 lg
mL 1 puromycin, 20 000 units
L 1 penicillin, and 20 lg
mL 1 streptomycin. MGN-mut gp120core(+V3) and MGN-mutgp120core were purified in several steps, the first of which was purification on a Cibacron column (Blue Sepharose 6 Fast Flow; GE Healthcare, Amersham UK). At the elution stage, a benzamidine column (HiTrap Benzamidine FF; GE Healthcare) followed the Cibacron column to remove serine proteases. The loading buffer was 20 mM Tris (pH 8), 100 mM NaCl, and 0.05% NaN3, and the elution buffer was 20 mM Tris (pH 8), 2 M NaCl, and 0.05% NaN3. The gp120 protein was then further purified with a nickel column (HisTrap FF; GE Healthcare), and the His-tag was then removed by tobacco etch virus protease cleavage. The tobacco etch virus protease containing a His-tag was removed with a second nickel column. The MGN-mutgp120MGN-mut gp120core yield at this stage was core(+V3) and 1 ~ 11–16 mg
L . For the preparation of mutgp120core(+V3) and mutgp120core, the gp120 protein was treated with EndoHf. The Endo-Hf (MBP-Endo-Hf) was removed with an amylose resin (New England Biolabs, Ipswich, MA, USA). This step resulted in gp120 protein with a yield of ~ 7– 12 mg
L 1. Finally, mutgp120core(+V3) and mutgp120core were purified by size exclusion chromatography, with a Superdex 200 column (HiLoad 16/600 Superdex 200, preparative grade; GE Healthcare). These purification steps resulted in a protein yield of ~ 4–6 mg
L 1 and > 95% purity. For the analytical size exclusion chromatography, the proteins were loaded onto a Superdex 200 column (Superdex 200 10/300 GL; GE Healthcare) in 20 mM Tris-HCl (pH 7.0), 150 mM NaCl and 0.05% NaN3 at 4 C, with a flow rate of 0.5 mL
min 1.

SPR measurements Binding affinities (KD) were determined by SPR with a ProteOn XPR36 Protein Interaction Array System (BioRad Haifa, Haifa, Israel) [39]. All experiments were carried out at 300 K with SPR buffer (20 mM Tris, pH 7.0, 0.05% NaN3, 0.005% Tween) containing either 150 mM or 50 mM NaCl. Biotinylated CD4M33 [40] was immobilized on a carboxymethylated dextran matrix chip, precoated with

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A. Moseri et al.

streptavidin (ProteOn NLC sensor chip). The immobilization procedure was allowed to continue until 200– 400 response units (RU) (1 RU = 1 pg protein
mm 2) of biotinylated CD4M33 were attached to the streptavidinprecoated surface of the chip. A blank sensor surface without an immobilized peptide served as a negative control and as a reference for the binding interaction. Binding was then measured with serial dilutions of unliganded mut MGN-mut gp120core and gp120core as well as mut gp120core(+V3) and MGN-mutgp120core(+V3) in SPR buffer (concentration ranging from nanomolar to micromolar), which were simultaneously injected over the peptide-immobilized surface at a flow rate of 30 lL
min 1. Data were analyzed with PROTEON MANAGER software after subtraction of a blank channel (negative control), by the use of equilibrium analysis or a Langmuir 1 : 1 kinetic model.

HIV-1 gp120 conformational flexibility

protein, dissolved in a 20 mM aqueous solution of D11-Tris buffer (pH 7.0), containing 95% H2O, 5% D2O, 150 mM NaCl, 1 mM D16-EDTA, 0.05% NaN3, 1 mM benzamidine, and P8340 protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). Concentrated solutions of CD4M33 (in H2O) were used for addition of the peptide to the gp120 samples. mutgp120core(+V3) and mutgp120core concentrations were determined from UV absorbance measurements at 280 nm. In order to identify peaks corresponding to the side chains of arginine, the TROSY-1H-15N-HSQC spectra were recorded with two different spectral widths. Folded peaks were attributed to arginine side chains. All NMR spectra were obtained on a Bruker AVIII800 spectrometer equipped with a 5 mm TCI cryoprobe. TROSY-1H-15N-HSQC [43] spectra were acquired at 300 K. Data were processed and analyzed with TOPSPIN (BrukerDe), NMRPIPE [44] and NMRVIEW [45,46].

Analytical ultracentrifugation Sedimentation equilibrium experiments were performed on a Beckman Optima XL-A analytical ultracentrifuge (Beckman, Coulter, CA, USA) at 300 K. Six-channel cells were used, with an An60 titanium rotor. The cells were scanned with a step size of 0.001 cm, and data points were averaged over 10 replicates. Solutions ranging from 12 lM to 21 lM mut mut of gp120core(+V3), gp120core(+V3)/CD4M33, mut mut gp120core and gp120core/CD4M33 were prepared in 20 mM Tris-HCl (pH 7.0), 150 mM NaCl, and 0.05% NaN3. Data were collected at 280 nm at different rotation speeds of 3401g, 5152g and 11591g. corresponding to the recommended speeds for trimeric, dimeric and monomeric gp120, respectively. Equilibrium was reached after 10 h, and verified by overlay and subtraction of at least two successive scans in 2 h intervals. Weight-averaged molecular masses were obtained by averaging the molecular masses from all data sets after equilibrium had been reached. The apparent molecular mass was calculated from the formula M = [2RT/(1 mq) x2][d(ln Abs)/dr2], where M is the solute molar mass (in grams per mole), R is the gas constant (g
cm 2
min 2), T is the temperature (K), 1 mq = 0.27 [m is the partial specific volume (mL
g 1); q is the density (g
mL 1)] [41], x is the angular velocity of the rotor (revolutions
min 1), and d (ln Abs)/dr2 is the first derivative of ln Abs with respect to r2, where Abs is the absorbance of the solute at r, which is the radial distance from the axis of rotation [42]. Residuals, which represent the difference between each experimental data point and the corresponding point on the curve calculated from the model equation, were calculated.

NMR measurements Samples of (DCN-Arg)-mutgp120core(+V3), (DCN-Ile)mut gp120core(+V3) or mutgp120core contained 0.07–0.14 mM

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Acknowledgements We thank D. Fass for help with the sedimentation equilibrium measurements. This study was supported by the Israel Science Foundation, the MINERVA foundation with funding from the Federal German Ministry for Education and Research, and the Kimmelman Center. J. Anglister is the Dr Joseph and Ruth Owades Professor of Chemistry. F. Naider is the Leonard and Esther Kurtz Term Professor at the College of Staten Island.

Author contributions A. Moseri and E. Schnur helped to design the experiments, carried out the experiments, analyzed the data, and helped to write the paper. E. Singhal Sinha prepared some of the gp120 samples, and carried out analytical size exclusion chromatography measurements and analysis. M. Abayev carried out and analyzed some of the NMR measurements, as well as some of the sedimentation equilibrium measurements and analysis. J. Anglister designed the experiments and wrote the paper. F. Naider helped to write the paper and contributed essential reagents. T. Scherf helped to perform the NMR measurements. E. Noah, N. Kessler and Y. Zherdev contributed essential reagents.

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