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

<div><p>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. These modifications cause a complete change in RfaH^CTD topology that converts from an <i>α</i>-hairpin to a <i>β</i>-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 RfaH^CTD. Although these analyses, in line with literature data, suggest that the isolated RfaH^CTD has a strong preference for the <i>β</i>-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 <i>α</i>-helical and <i>β</i>-structured states. Intriguingly, in the RfaH homolog NusG, whose CTD uniquely adopts the <i>β</i>-barrel fold, the corresponding region is rich in residues as Val or Ile that present a strong preference for the <i>β</i>-structure. On this basis, we suggest that the presence of this Leu-rich element in RfaH^CTD 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 RfaH^CTD. These observations suggest that the unusual conformational behavior of RfaH^CTD may be rare but not unique.</p></div

KCTD5 is endowed with large, functionally relevant, interdomain motions

<p>The KCTD family is an emerging class of proteins that are involved in important biological processes whose biochemical and structural properties are rather poorly characterized or even completely undefined. We here used KCTD5, the only member of the family with a known three-dimensional structure, to gain insights into the intrinsic structural stability of the C-terminal domain (CTD) and into the mutual dynamic interplay between the two domains of the protein. Molecular dynamics (MD) simulations indicate that in the simulation timescale (120 ns), the pentameric assembly of the CTD is endowed with a significant intrinsic stability. Moreover, MD analyses also led to the identification of exposed β-strand residues. Being these regions intrinsically sticky, they could be involved in the substrate recognition. More importantly, simulations conducted on the full-length protein provide interesting information of the relative motions between the BTB domain and the CTD of the protein. Indeed, the dissection of the overall motion of the protein is indicative of a large interdomain twisting associated with limited bending movements. Notably, MD data indicate that the entire interdomain motion is pivoted by a single residue (Ser150) of the hinge region that connects the domains. The functional relevance of these motions was evaluated in the context of the functional macromolecular machinery in which KCTD5 is involved. This analysis indicates that the interdomain twisting motion here characterized may be important for the correct positioning of the substrate to be ubiquitinated with respect to the other factors of the ubiquitination machinery.</p

The dynamic properties of the Hepatitis C Virus E2 envelope protein unraveled by molecular dynamics

<p>Hepatitis C Virus (HCV) is one of the most persistent human viruses. Although effective therapeutic approaches have been recently discovered, their use is limited by the elevated costs. Therefore, the development of alternative/complementary strategies is an urgent need. The E2 glycoprotein, the most immunogenic HCV protein, and its variants represent natural candidates to achieve this goal. Here we report an extensive molecular dynamics (MD) analysis of the intrinsic properties of E2. Our data provide interesting clues on the global and local intrinsic dynamic features of the protein. Present MD data clearly indicate that E2 combines a flexible structure with a network of covalent bonds. Moreover, the analysis of the two most important antigenic regions of the protein provides some interesting insights into their intrinsic structural and dynamic properties. Our data indicate that a fluctuating β-hairpin represents a populated state by the region E2^412−423. Interestingly, the analysis of the epitope E2^427−446 conformation, that undergoes a remarkable rearrangement in the simulation, has significant similarities with the structure that the E2^430−442 fragment adopts in complex with a neutralizing antibody. Present data also suggest that the strict conservation of Gly436 in E2 protein of different HCV genotypes is likely dictated by structural restraints. Moreover, the analysis of the E2^412−423 flexibility provides insights into the mechanisms that some antibodies adopt to anchor Trp437 that is fully buried in E2. Finally, the present investigation suggests that MD simulations should systematically complement crystallographic studies on flexible proteins that are studied in combination with antibodies.</p

The intrinsic stability of the human prion β-sheet region investigated by molecular dynamics

<div><p>Human prion diseases are neurodegenerative disorders associated to the misfolding of the prion protein (PrP). Common features of prion disorders are the fibrillar amyloid deposits and the formation of prefibrillar oligomeric species also suggested as the origin of cytotoxicity associated with diseases. Although the process of PrP misfolding has been extensively investigated, many crucial aspects of this process remain unclear. We have here carried out a molecular dynamics study to evaluate the intrinsic dynamics of PrP β-sheet, a region that is believed to play a crucial role in prion aggregation. Moreover, as this region mediates protein association in dimeric assemblies frequently observed in prion crystallographic investigations, we also analyzed the dynamics of these intermolecular interactions. The extensive sampling of replica exchange shows that the native antiparallel β-structure of the prion is endowed with a remarkable stability. Therefore, upon unfolding, the persistence of a structured β-region may seed molecular association and influence the subsequent phases of the aggregation process. The analysis of the four-stranded β-sheet detected in the dimeric assemblies of PrP shows a tendency of this region to form dynamical structured states. The impact on the β-sheet structure and dynamics of disease associated point mutations has also been evaluated.</p> </div

A Loose Domain Swapping Organization Confers a Remarkable Stability to the Dimeric Structure of the Arginine Binding Protein from <i>Thermotoga maritima</i>

<div><p>The arginine binding protein from <i>Thermatoga maritima</i> (TmArgBP), a substrate binding protein (SBP) involved in the ABC system of solute transport, presents a number of remarkable properties. These include an extraordinary stability to temperature and chemical denaturants and the tendency to form multimeric structures, an uncommon feature among SBPs involved in solute transport. Here we report a biophysical and structural characterization of the TmArgBP dimer. Our data indicate that the dimer of the protein is endowed with a remarkable stability since its full dissociation requires high temperature as well as SDS and urea at high concentrations. In order to elucidate the atomic level structural properties of this intriguing protein, we determined the crystallographic structures of the apo and the arginine-bound forms of TmArgBP using MAD and SAD methods, respectively. The comparison of the liganded and unliganded models demonstrates that TmArgBP tertiary structure undergoes a very large structural re-organization upon arginine binding. This transition follows the Venus Fly-trap mechanism, although the entity of the re-organization observed in TmArgBP is larger than that observed in homologous proteins. Intriguingly, TmArgBP dimerizes through the swapping of the C-terminal helix. This dimer is stabilized exclusively by the interactions established by the swapping helix. Therefore, the TmArgBP dimer combines a high level of stability and conformational freedom. The structure of the TmArgBP dimer represents an uncommon example of large tertiary structure variations amplified at quaternary structure level by domain swapping. Although the biological relevance of the dimer needs further assessments, molecular modelling suggests that the two TmArgBP subunits may simultaneously interact with two distinct ABC transporters. Moreover, the present protein structures provide some clues about the determinants of the extraordinary stability of the biomolecule. The availability of an accurate 3D model represents a powerful tool for the design of new TmArgBP suited for biotechnological applications.</p></div

Domain-swapped dimer of ApoTmArgBP.

<p>(A) Cartoon representation of ApoTmArgBP swapping dimer. (B) Omit (Fo-Fc) map of the hinge region, contoured at 2σ. (C) Interactions mediated by the C-terminal helix. (D) Superposition of the chains A and B of ApoTmArgBP.</p

Analytical SEC-MALS of TmArgBP.

<p>The black and grey curves represent the Rayleigh ratio (left scale) of HoloTmArgBP and ApoTmArgBP, respectively; both are plotted against the retention time. Molecular masses are reported with the same colour code. In both experiments, average molecular masses values correspond to a dimeric state of the protein.</p

Structure and dynamics of the multi-domain resuscitation promoting factor RpfB from <i>Mycobacterium tuberculosis</i>

<p>RpfB is multidomain protein that is crucial for <i>Mycobacterium tuberculosis</i> resuscitation from dormancy. This protein cleaves cell wall peptidoglycan, an essential bacterial cell wall polymer formed by glycan chains of β-(1-4)-linked-N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) cross-linked by short peptide stems. RpfB is structurally complex being composed of five distinct domains, namely a catalytic, a G5 and three DUF348 domains. Here, we have undertaken a combined experimental and computation structural investigations on the entire protein to gain insights into its structure–function relationships. CD spectroscopy and light scattering experiments have provided insights into the protein fold stability and into its oligomeric state. Using the available structure information, we modeled the entire protein structure, which includes the two DUF348 domains whose structure is experimentally unknown, and we analyzed the dynamic behavior of RpfB using molecular dynamics simulations. Present results highlight an intricate mutual influence of the dynamics of the different protein domains. These data provide interesting clues on the functional role of non-catalytic domains of RpfB and on the mechanism of peptidoglycan degradation necessary to resuscitation of <i>M. tuberculosis</i>.</p

Multiple sequence alignment of TmArgBP with known homologues.

<p>For each protein the PDB code is reported in parenthesis. The signal peptide of TmArgBP is drawn in red. For the other proteins only the binding domain is reported.</p

Variation of tertiary (A) and quaternary (B) structures of ApoTmArgBP (orange) and HoloTmArgBP (magenta).

<p>In both panels, overlapped regions are 22–104 and 206–243 of lobe I. The arrows highlight the conformational changes occurring upon arginine binding.</p