Structural conversion of the transformer protein RfaH: new insights derived from protein structure prediction and molecular dynamics simulations.

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Structural conversion of the transformer protein RfaH: new insights derived from protein structure prediction and molecular dynamics simulations ab

ab

Nicole Balasco , Daniela Barone a

a

& Luigi Vitagliano

Institute of Biostructures and Bioimaging, C.N.R., Naples I-80134, Italy

b

Dipartimento di Scienze e Tecnologie Ambientali Biologiche e Farmaceutiche, Seconda Università di Napoli, Caserta 81100, Italy Accepted author version posted online: 06 Dec 2014.

Click for updates To cite this article: Nicole Balasco, Daniela Barone & Luigi Vitagliano (2014): Structural conversion of the transformer protein RfaH: new insights derived from protein structure prediction and molecular dynamics simulations, Journal of Biomolecular Structure and Dynamics, DOI: 10.1080/07391102.2014.994188 To link to this article: http://dx.doi.org/10.1080/07391102.2014.994188

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Publisher: Taylor & Francis Journal: Journal of Biomolecular Structure and Dynamics DOI: http://dx.doi.org/10.1080/07391102.2014.994188

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Structural conversion of the transformer protein RfaH: new insights derived from protein structure prediction and molecular dynamics simulations

Nicole Balasco1,2*, Daniela Barone1,2, Luigi Vitagliano1*

1

Institute of Biostructures and Bioimaging, C.N.R., Naples I-80134, Italy

2

Dipartimento di Scienze e Tecnologie Ambientali Biologiche e Farmaceutiche, Seconda

Università di Napoli, Caserta 81100, Italy

*

Correspondence: [email protected] or [email protected], postal address Via

Mezzocannone 16, IBB-CNR, Naples I-80134, Italy; phone number ++39-0812534506

Keywords Secondary structure prediction; conformational conversion; molecular dynamics; transformer proteins; amino acid propensities

Abbreviations MD, Molecular dynamics; RMSF, Root Mean Square Fluctuation; RMSD, Root Mean Square Deviation; RMSIP, Root Mean Square Inner Product; PDB, Protein Data Bank; NTD, N-terminal domain; CTD, C-terminal domain; Rg, gyration radius

1


Abstract Recent structural investigations have shown that the C-terminal domain (CTD) of the transcription factor RfaH undergoes unique structural modifications that have a profound impact into its functional properties (Burmann, Knauer, Sevostyanova, Schweimer, Mooney, Landick, Artsimovitch, Roesch, 2012). These modifications cause a complete change in RfaHCTD topology that converts from an α-hairpin to a β-barrel fold. To gain insights into the determinants of this major structural conversion we here performed computational studies (protein structure prediction and molecular dynamics simulations) on RfaHCTD. Although these analyses, in line with literature data, suggest that the isolated RfaHCTD has a strong preference for the β-barrel fold, they also highlight that a specific region of the protein is endowed with a chameleon conformational behavior. In particular, the Leu-rich region (residues 141-145) has a good propensity to adopt both α-helical and β-structured states. Intriguingly, in the RfaH homolog NusG, whose C-terminal domain uniquely adopts the βbarrel fold, the corresponding region is rich in residues as Val or Ile that present a strong preference for the β-structure. On this basis, we suggest that the presence of this Leu-rich element in RfaHCTD may be responsible for the peculiar structural behavior of the domain. The analysis of the sequences of RfaH family (PfamA code PF02357) unraveled that other members potentially share the structural properties of RfaHCTD. These observations suggest that the unusual conformational behavior of RfaHCTD may be rare but not unique.

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Introduction


One of the basic paradigms of structural biology is that protein structure is dictated by the amino acid sequence (Anfinsen principle)(Anfinsen, 1973). The relation between sequence and structure is however not biunivocal. Indeed, similar sequences tend to assume rather similar structures whereas the same structure is frequently assumed by multitudes of unrelated sequences. In this context, the analysis of similar/identical sequences that adopt significantly different structures has attracted the attention of the scientific community (see for example the “Paracelsus Challenge”) (Dalal, Balasubramanian, & Regan, 1997). It is worth mentioning that short protein fragments in different contexts may easily assume different conformations (chameleon sequences) (Gendoo & Harrison, 2011). It has also been reported the case of specific protein regions that present significant differences between predicted and actual structures (discordant sequences) (Gendoo & Harrison, 2011).

A

special and rather widespread case of these structural ambiguities is represented by peptides and proteins that, in addition to their folded or unfolded physiological states, form in special conditions amyloid-like aggregates (Chiti & Dobson, 2006). The most striking example among structural/functional conversions between completely different folds is represented by the carboxy-terminal domain of the transcription factor RfaH (Burmann et al., 2012). This protein belongs to the NusG family which includes important regulators of transcription in all living organisms (Tomar & Artsimovitch, 2013; Werner & Grohmann, 2011). Escherichia coli (E. coli) RfaH is characterized by a modular organization consisting of an N-terminal RNP-like domain (NTD – residues 1-100) and a C-

3

terminal element with a KOW sequence motif (Belogurov et al., 2007; Kyrpides, Woese, & Ouzounis, 1996). The X-ray structure of the full length E. coli RfaH, showed that the Cterminal domain (CTD – residues 101-162) folds as an α-hairpin that is tightly bound to the NTD (Belogurov, et al., 2007). This interaction hides the RNAP binding site of RfaH. Indeed, only when RNAP pauses at ops-containing operons, RfaHCTD loses its contacts with


RfaHNTD allowing the interaction between RfaH and the polymerase (Belogurov, Mooney, Svetlov, Landick, & Artsimovitch, 2009). Very recently, Roesch and coworkers surprisingly observed that RfaHCTD undergoes a huge conformational switch from the all-α state to an allβ fold when it is detached from the rest of the protein (Burmann, et al., 2012; Svetlov & Nudler, 2012). By adopting this β-barrel fold, the domain acquires the ability to recruit ribosome, thereby activating translation (Tomar, Knauer, Nandymazumdar, Rosch, & Artsimovitch, 2013). Since RfaH simultaneously changes both topology and function, it has been considered a transformer protein (Knauer, Artsimovitch, & Rosch, 2012; Knauer, Rosch, & Artsimovitch, 2012). In order to gain further insights into this unique structural transformation we here preliminary performed some sequence analyses aimed at unraveling the intrinsic preferences of RfaHCTD. We then evaluated the stability of the isolated α-haipin (hereafter referred as αRfaHCTD) and β-barrel (βRfaHCTD) folds of the domain by performing molecular dynamics simulations in explicit solvent. Since these analyses provided some clues on the sequence determinants of RfaHCTD structural transition, we used our results to search KOW sequence motifs in order to identify other proteins that potentially share this peculiar conformational behavior with RfaH.

Materials and Methods Sequence analyses 4

RfaHCTD secondary structure predictions were performed using the servers PsiPred (http://bioinf.cs.ucl.ac.uk/psipred/) (McGuffin, Bryson, & Jones, 2000) and Net-CSSP which computes

the

contact-dependent

secondary

structure

propensities

(http://cssp2.sookmyung.ac.kr/) (Kim, Choi, Lee, Welsh, & Yoon, 2009). Molecular dynamics: models and protocols


Molecular dynamics simulations were performed on both αRfaHCTD and βRfaHCTD. αRfaHCTD consists of an extended helix-turn-helix motif in which the two α-helices span residues 118-130 (helix 1) and 134-156 (helix 2). On the other hand, βRfaHCTD is a compact five-stranded β-sheet. The starting model of the helical RfaHCTD was extracted from the crystal structure of the entire protein solved at 2.1 Å resolution (PDB: 2OUG) (Belogurov, et al., 2007) . Some regions (residues 109-114 and 158-162), that were missing in the crystal structure, were manually modeled as coil. In the simulation of βRfaHCTD the solution structure of the β-barrel CTD (PDB: 2LCL) (Burmann, et al., 2012) was used as starting model. Molecular dynamics simulations were performed by using the GROMACS software package 4.5.5 (Van Der Spoel et al., 2005) with Amber99sb as force field and TIP4P-EW as water model. The models were immersed cubic boxes filled with water molecules. The dimensions of the boxes were 7.59×7.19×7.25 nm3 (number of water molecules 12626) and 7.02×6.86×6.49 nm3 (number of water molecules 10067) for αRfaHCTD and βRfaHCTD, respectively. The simulations were run with periodic boundary conditions. Equilibration was conducted in two phases. In the first phase, conducted under an NVT ensemble, the temperature of the systems (300 K) was stabilized. Equilibration of pressure (1 atm) was then carried out under an NPT ensemble. Before starting the MD simulations, energies were minimized by fixing the protein atoms and then without restraints. The system temperature was brought to 300 K in a step-wise manner. In particular, 100 ps MD runs were carried out

5

at 50, 100, 150, 200, 250, and 300 K. The time step was 0.002 ps for both systems. The timescales were 250 ns and 160 ns for αRfaHCTD and βRfaHCTD, respectively. Bond lengths were constrained using the LINCS procedure. Lennard-Jones interactions were calculated with a cutoff of 10 Å. Electrostatic interactions were treated using the Particle Mesh Ewald (PME) method with a grid spacing of 0.12 nm.


In order to examine the collective motions of the proteins in the MD simulations, the essential degrees of freedom were extracted according to the ED method (Amadei, Linssen, & Berendsen, 1993). This method is based on the construction of the covariance matrix of the coordinate fluctuations. This matrix was diagonalized to obtain eigenvectors and eigenvalues. The first 10-20 eigenvectors usually represent most (80-90%) of the motion in the protein. To evaluate whether the simulation reached an adequate convergence in the essential space (Amadei, et al., 1993) the root mean square inner product (RMSIP) between two halves of the equilibrated trajectory was calculated as described by Di Nola and co-workers (Amadei, Ceruso, & Di Nola, 1999; Merlino, Vitagliano, Ceruso, Di Nola, & Mazzarella, 2002; Merlino, Vitagliano, Ceruso, & Mazzarella, 2003). The RMSIP between the first 10 eigenvectors is defined as

where

and

are the ith and jth eigenvectors from the first and second half of the

equilibrated trajectory, respectively. Trajectories were checked to assess the quality of the simulation using GROMACS routines and the program VMD (Humphrey, Dalke, & Schulten, 1996). In order to identify H-bond

6

interactions, both donor-acceptor distances (< 3.5 Å) and hydrogen-donor-acceptor angles (< 30°) were checked using GROMACS utilities (Van Der Spoel, et al., 2005).


Results Intrinsic structural preferences of RfaHCTD and NusGCTD: secondary structure prediction The servers Psipred and Net-CSSP were used to gain insights into the intrinsic structural preferences of RfaHCTD. For comparative purposes these analyses were also conducted on the C-terminal region of the homolog E. coli NusG (NusGCTD), which uniquely folds as a βbarrel and does not display any structural conversion. With the exception of the fragment 137-146 of RfaHCTD, Psipred predicts an overall preference for the β-structure for CTDs of both proteins (Figure S1A-B). PsiPred predicts a β-structure preference for the RfaH Cterminal region also when the sequence of the full-length protein was used (Figure S1C). Taking into account that this domain is helical in the experimental structure of the full-length protein, RfaHCTD represents a major discordant region. Therefore, the dual conformational behavior of this portion could have been highlighted prior the NMR characterization of the isolated RfaHCTD by simple comparisons between experimental structure and prediction. Net-CSSP provides similar predictive results showing that both RfaHCTD and NusGCTD have a significant preference for the β structure. However, a deeper inspection of Figure 1 indicates that the RfaHCTD region 137-146, which is a β-strand in the NMR structure of the isolated domain, has a significant preference for the helical state. This feature is not exhibited by the corresponding region of NusGCTD. Molecular dynamics simulation of RfaHCTD

7

In order to gain further insights into the huge structural transformation of RfaHCTD we performed all-atom molecular dynamics studies on both conformers of the domain, βRfaHCTD and αRfaHCTD. The β-barrel structure of βRfaHCTD is stable and rigid


The solution structure of βRfaHCTD (PDB: 2LCL) was employed as starting model (Figure 2A) in the MD simulation which was performed using GROMACS software (see Materials and Methods). Indicators commonly adopted to check system stability in MD analyses reveal that the β-motif is extremely stable within time. Furthermore, the very high value of the RMSIP, 0.92, calculated between the two halves of the equilibrated trajectory (20-160 ns), confirms that the simulation has reached an appropriate convergence in the simulation timescale. βRfaHCTD does not exhibit significant deviations from the starting model with average RMSD values, computed on the Cα atoms, of ~ 1.5 Å (Figure S2A). βRfaHCTD gyration radius (Rg) is stable (variations of about 1.5 Å) along the time (Figure S2C). The time evolution of the protein secondary structure elements (Figure 3A) is in good agreement with the very high stability observed. βRfaHCTD totally retains its secondary structure content throughout the simulation. The root mean square fluctuation (RMSF) values calculated on Cα atoms in the equilibrated region the trajectories are rather low for the structured elements (below 0.5 Å). Higher fluctuations (~ 1-2 Å) are exhibited by residues of loops (Figure S3). In order to gain further insights into the dynamics of this highly stable folding, we analyzed the collective motions of βRfaHCTD by essential dynamics. This analysis showed that most of the atomic displacements were contained in the first few eigenvectors. In particular, the essential subspace spanned by the first 10 eigenvectors covers about 72% of the total fluctuations. The regions involved in the motions described by the first eigenvector (26% of

8

the total fluctuations) can be visualized plotting the protein along this principal component in a film-like fashion (Figure 2B). In agreement with the RMSF profile, significant atomic displacements observed for the first eigenvector are mainly concentrated in loop regions.


The αRfaHCTD fold is highly unstable

The structure of αRfaHCTD, used as starting model in the MD simulation, corresponds to the residues 109-162 of the crystal structure of the full-length protein (see Materials and Methods) (Figure 2C). Since the early stages of the simulation, αRfaHCTD displayed a completely different dynamic behavior compared to βRfaHCTD. The trend of the RMSD values of the trajectory structures from the starting models shows that αRfaHCTD undergoes to significant variations in the interval 0-160 ns. Indeed, RMSD values, computed on Cα atoms, are in the range 3-13 Å (Figure S2B). In the 190-250 ns interval, RMSD values become less variable with an average of ~ 10 Å. An analogous trend can be observed in the evolution of αRfaHCTD gyration radius (Figure S2D). After significant fluctuations Rg assumes a lower value (about 11 Å) beyond 180 ns indicating a more compact structure. The instability of αRfaHCTD fold is confirmed by the time evolution of the secondary structure elements (Figure 3B) which clearly shows the unfolding of most of the helical regions of the protein. Interestingly, a short segment belonging to helix 2 (residues 135-145) retains the α-helical content in the simulation timescale.

Discussion In line with the experimental evidence, prediction programs and molecular dynamics simulations indicate that the isolated C-terminal region of RfaH has a strong preference for the β-structure. The MD simulation clearly indicates that αRfaHCTD α-hairpin is highly 9

unstable. Nevertheless, this analysis shows that the two helices of the α-hairpin motif display a rather different behavior. Indeed, helix 1 rapidly unfolds whereas a significant portion of helix 2 remains structured in the simulations. This finding is in line with previous MD studies carried out on this system by using rather different approaches (replica exchange in implicit solvent, targeted and high-temperature MD in implicit solvent) (Gc, Bhandari,


Gerstman, & Chapagain, 2014; Li et al., 2014). In both of these reports, which were published while the present work was in progress, a higher stability of helix 2 was observed. According to these authors, the rapid unfolding of helix 1 in the isolated RfaHCTD was ascribed to the loss of favorable interdomain contacts with RfaHNTD that stabilize this helix in the full length protein. However, a detailed analysis of the interfaces between RfaHNTD and the two helices clearly indicates that the interactions formed by helix 2 with the Nterminal domain are comparable or even larger than those involving helix 1. Indeed, the overall surface, computed using the server PDBePISA (http://www.ebi.ac.uk/pdbe/pisa/), of RfaHNTD/helix 2 and RfaHNTD/helix 1 interfaces is 590 Å2, and 320 Å2, respectively. RfaHNTD/helix 2 interface is characterized by stabilizing interactions such as a salt bridge (Glu 48 of the NTD and Arg138 of helix 2) and several hydrogen bonds. On the contrary, no electrostatic interaction is formed by RfaHNTD with helix 1. These observations indicate that helix 2 is able to maintain the helical structure despite the loss of important interactions. These considerations suggest that residues of helix 2 are intrinsically endowed with a higher propensity for helical states compared to residues of helix 1. The analysis of helix 2 sequence shows the presence of a Leu-rich segment (LLLNL, residues 141-145) which has a high intrinsic propensity for the helical conformation as shown by NET-CSSP (Figure 1). This reflects the good intrinsic propensity of Leu for α-helix (Fersht, 1999; Myers, Pace, & Scholtz, 1997). It is interesting to note that the corresponding region in NusGCTD, the RfaH homolog whose CTD forms a stable non-transient β-structure, shows an abundance of

10

valine/isoleucine (VSVSI, residues 160-164). These residues present a marked preference for the β-structure and a very poor propensity for the α-helix (Fersht, 1999; Myers, et al., 1997). This is consistent with the analysis of the PDB structures containing the LLLNL sequence motif present in RfaHCTD. Indeed, in addition to RfaH, we identified this motif in other eleven proteins. Notably, these fragments adopt a helical structure in nine cases (PDB codes:


1C8R, 1H8L, 2BL0, 2WZF, 3F7F, 3VE9, 4AN8, 4H3T, 4IMP). In the other two cases, the motif is either disordered (PDB code: 2E0Z) or it adopts a mixed β-/irregular structure (PDB code: 4O5A). On this basis, we can reasonably propose that the specific secondary structure propensity of the Leu-rich segment may be one of the determining factors for the peculiar, hitherto unique, conformational properties of RfaHCTD. In order to identify new proteins that potentially share this behavior with RfaHCTD we analyzed the sequence of this segment in proteins belonging to NusG family. The family (PfamA code PF02357) includes 3544 sequences with the NusG-KOW architecture. The vast majority of these proteins presents valine/isoleucine-rich sequences in the considered region. For example, as many as 1254 sequences exhibit the motif VSV.

On the other hand, very few members present residues with good helix

propensity. Indeed, only 4 sequences exhibit the Leu-rich segment LLL of RfaHCTD. This observation is confirmed by the analysis of the motifs XLL, LXL, LLX, where X is an αhelix preferrers (Ala, Arg or Met) (Fersht, 1999; Myers, et al., 1997). In particular, the motifs ALL, LML, LRL are observed only in 65, 29, and 1 sequences, respectively. By considering in the final ensemble only sequences with identities lower than 80%, we obtained

2, 17, 1, and 1 sequences containing LLL, ALL, LML, and LRL motifs,

respectively (Table S1). The analysis of the structural propensity of the selected sequences, carried out by using the server NET-CSSP, shows a close similarity with the trends observed for RfaHCTD. As shown in Figure 4, in which representative examples of the NET-CSSP

11

plots are reported, the chameleon behavior of these sequences is evident. It is important to note that the propensity of the Leu-rich region is higher for helical states compared to βstructure. This finding suggests that the identified sequences encode potential transformer proteins, which may undergo the structural transition observed in RfaH. In this context, it is interesting to note that these sequences present a relatively low sequence identity (range 17-


30 %) with RfaHCTD (Table S1). These observations are corroborated by the analysis of specific sequence features. With a single exception (uniprot code C2NS75), in all sequences the charged residues involved in the interdomain interaction in RfaH (Glu 48 and Arg 138) are functionally conserved. Currently, this sequence ensemble is essentially made by predicted or inferred from homology proteins. Only in one case the encoded protein has been experimentally detected (uniprot code Q5XE43). In conclusion, by combining prediction tools and MD simulation we highlighted the role of specific sequence regions that may play a role in the peculiar, but functional, conversion of RfaHCTD. Our data suggest that the conformational behavior of RfaHCTD is rare but not unique. Future characterizations of the proteins encoded by the sequences here identified may lead to the discovery of new transformer proteins. Present analyses also suggest that other examples of transformer proteins, unrelated to RfaH, may be unveiled by the analyses of discrepancies between secondary structure predictions and “actual” structures reported in the PDB. Finally, the identification of the structural determinants of this structural switch opens the possibility for the design and the development of new active biomolecules endowed with this intriguing property.

Acknowledgments

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This work was financially supported by MIUR (FIRB RBRN07BMCT). The authors thank


Luca De Luca for skilful technical assistance.

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Figure 1. Contact-dependent secondary structure propensities (CSSP) of RfaHCTD (A) and NusGCTD (B) computed using the Net-CSSP server.

16


Figure 2. (A) Structure of βRfaHCTD used as a starting model in the MD simulation. (B) βRfaHCTD Cα trace motion along the first eigenvector in a film-like fashion. A color scale (from violet to red) is used to represent the movement. (C) Crystallographic structure of the full-length RfaH. The isolated α-hairpin (starting model in the MD simulation) and the final unfolded RfaHCTD structures are shown.

17


Figure 3. Evolution of the secondary structure content of βRfaHCTD (A) and αRfaHCTD (B).

18


Figure 4. Representative examples of NET-CSSP plots of sequences containing the Leu-rich motif. The uniprot codes are C2NS75 (A), A8RDZ7 (B), Q5XE43 (C), and A0YKG2 (D).

19


Supplementary Material

Figure S1. Secondary structure prediction of the C-terminal domain of RfaH (A) and NusG (B) computed by using Psipred server. Prediction for the full-length sequences of RfaH and NusG are reported in panels C, and D, respectively.

1


Figure S2. Structure stability of βRfaHCTD and αRfaHCTD during the MD simulations. Panel A and B report the Cα RMSD values of trajectory structures against the starting models for βRfaHCTD and αRfaHCTD, respectively. In panel C and D, the gyration radii of βRfaHCTD and αRfaHCTD are shown, respectively.

2


Figure S3. Root mean square fluctuations of residues of βRfaHCTD calculated in the equilibrated region of the trajectory. The five strands of the CTD β-barrel are reported as red bars.

3

Table S1. Proteins or hypothetical proteins which present the Leu-rich motif that may be responsible


for the structural conversion of RfaHCTD. UNIPROT CODE

Motif

LLL LLL

Overall sequence identity with RfaHCTD 30.0% 29.1%

Overall sequence identity with NusGCTD 25.4% 26.9%

C2NS75 E7PZA3 A8RDZ7

LRL

31.2%

16.7%

Q5XE43

LML

27.0%

26.5%

A0YKG2

ALL

18.3%

46.2%

A0ZHA9

ALL

26.5%

49.2%

A2BP00

ALL

22.9%

38.7%

A3IVE9

ALL

17.3%

47.8%

A8YGJ0

ALL

18.6%

48.5%

A2C035

ALL

25.0%

44.9%

B0CAC8

ALL

23.4%

40.8%

B1X4D0

ALL

24.4%

46.2%

B4VRH0

ALL

27.7%

42.9%

B5IQN8

ALL

26.9%

46.2%

B4WIK1

ALL

26.2%

47.7%

B8HVL6

ALL

29.2%

47.7%

A4CY17

ALL

29.2%

46.4%

F7UN82

ALL

25.4%

50.7%

Q119S5

ALL

17.8%

47.7%

Q31QK2

ALL

24.6%

49.2%

Q8DM29

ALL

27.7%

46.2%

Status

Predicted Inferred from homology Predicted Experimental evidence at protein level Inferred from homology Inferred from homology Inferred from homology Inferred from homology Inferred from homology Inferred from homology Inferred from homology Inferred from homology Inferred from homology Inferred from homology Inferred from homology Inferred from homology Inferred from homology Inferred from homology Inferred from homology Inferred from homology Inferred from homology

Organism

Bacillus cereus Streptococcus dysgalactiae Eubacterium dolichum Streptococcus pyogenes Lyngbya sp Nodularia spumigena Prochlorococcus marinus Cyanothece sp. Microcystis aeruginosa Prochlorococcus marinus Synechococcus sp. Paulinella chromatophora Coleofasciculus chthonoplastes Cyanobium sp. Synechococcus sp. Cyanothece sp. Synechococcus sp. Synechocystis sp. Trichodesmium erythraeum Synechococcus elongatus Thermosynechococcus elongatus

4

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