Novel Intersubunit Interaction Critical for HIV-1 Core Assembly Defines a Potentially Targetable Inhibitor Binding Pocket

HIV-1 capsid protein (CA) plays critical roles in both early and late stages of the viral replication cycle. Mutagenesis and structural experiments have revealed that capsid core stability significantly affects uncoating and initiation of reverse transcription in host cells. This has led to efforts in developing antivirals targeting CA and its assembly, although none of the currently identified compounds are used in the clinic for treatment of HIV infection. A specific interaction that is primarily present in pentameric interfaces in the HIV-1 capsid core was identified and is reported to be important for CA assembly. This is shown by multidisciplinary characterization of CA site-directed mutants using biochemical analysis of virus-like particle formation, transmission electron microscopy of in vitro assembly, crystallographic studies, and molecular dynamic simulations. The data are consistent with a model where a hydrogen bond between CA residues E28 and K30′ from neighboring N-terminal domains (CA_(NTD)s) is important for CA pentamer interactions during core assembly. This pentamer-preferred interaction forms part of an N-terminal domain interface (NDI) pocket that is amenable to antiviral targeting

β-Bulges: extensive structural analyses of β-sheets irregularities.

International audienceβ-Sheets are quite frequent in protein structures and are stabilized by regular main-chain hydrogen bond patterns. Irregularities in β-sheets, named β-bulges, are distorted regions between two consecutive hydrogen bonds. They disrupt the classical alternation of side chain direction and can alter the directionality of β-strands. They are implicated in protein-protein interactions and are introduced to avoid β-strand aggregation. Five different types of β-bulges are defined. Previous studies on β-bulges were performed on a limited number of protein structures or one specific family. These studies evoked a potential conservation during evolution. In this work, we analyze the β-bulge distribution and conservation in terms of local backbone conformations and amino acid composition. Our dataset consists of 66 times more β-bulges than the last systematic study (Chan et al. Protein Science 1993, 2:1574-1590). Novel amino acid preferences are underlined and local structure conformations are highlighted by the use of a structural alphabet. We observed that β-bulges are preferably localized at the N- and C-termini of β-strands, but contrary to the earlier studies, no significant conservation of β-bulges was observed among structural homologues. Displacement of β-bulges along the sequence was also investigated by Molecular Dynamics simulations

Investigation of the impact of PTMs on the protein backbone conformation

International audiencePost-Translational Modifications (PTMs) are known to play a critical role in the regulation of the protein functions. Their impact on protein structures, and their link to disorder regions have already been spotted on the past decade. Nonetheless, the high diversity of PTMs types, and the multiple schemes of protein modifications (multiple PTMs, of different types, at different time, etc) make difficult the direct confrontation of PTM annotations and protein structures data.We so analyzed the impact of the residue modifications on the protein structures at local level. Thanks to a dedicated structure database, namely PTM-SD, a large screen of PTMs have been done and analyze at a local protein conformation levels using the structural alphabet Protein Blocks (PBs). We investigated the relation between PTMs and the backbone conformation of modified residues, of their local environment, and at the level of the complete protein structure. The two main PTM types (N-glycosylation and phosphorylation) have been studied in non-redundant datasets, and then, 4 different proteins were focused, covering 3 types of PTMs: N-glycosylation in renin endopeptidase and liver carboxylesterase, phosphorylation in cyclin-dependent kinase 2 (CDK2), and methylation in actin. We observed that PTMs could either stabilize or destabilize the backbone structure, at a local and global scale, and that these effects depend on the PTM types

Cis-trans isomerization of omega dihedrals in proteins.

International audiencePeptide bonds in protein structures are mainly found in trans conformation with a torsion angle ω close to 180°. Only a very low proportion is observed in cis conformation with ω angle around 0°. Cis-trans isomerization leads to local conformation changes which play an important role in many biological processes. In this paper, we reviewed the recent discoveries and research achievements in this field. First, we presented some interesting cases of biological processes in which cis-trans isomerization is directly implicated. It is involved in protein folding and various aspect of protein function like dimerization interfaces, autoinhibition control, channel gating, membrane binding. Then we reviewed conservation studies of cis peptide bonds which emphasized evolution constraints in term of sequence and local conformation. Finally we made an overview of the numerous molecular dynamics studies and prediction methodologies already developed to take into account this structural feature in the research area of protein modeling. Many cis peptide bonds have not been recognized as such due to the limited resolution of the data and to the refinement protocol used. Cis-trans proline isomerization reactions represents a vast and promising research area that still needs to be further explored for a better understanding of isomerization mechanism and improvement of cis peptide bond predictions

PredyFlexy: flexibility and local structure prediction from sequence.

International audienceProtein structures are necessary for understanding protein function at a molecular level. Dynamics and flexibility of protein structures are also key elements of protein function. So, we have proposed to look at protein flexibility using novel methods: (i) using a structural alphabet and (ii) combining classical X-ray B-factor data and molecular dynamics simulations. First, we established a library composed of structural prototypes (LSPs) to describe protein structure by a limited set of recurring local structures. We developed a prediction method that proposes structural candidates in terms of LSPs and predict protein flexibility along a given sequence. Second, we examine flexibility according to two different descriptors: X-ray B-factors considered as good indicators of flexibility and the root mean square fluctuations, based on molecular dynamics simulations. We then define three flexibility classes and propose a method based on the LSP prediction method for predicting flexibility along the sequence. This method does not resort to sophisticate learning of flexibility but predicts flexibility from average flexibility of predicted local structures. The method is implemented in PredyFlexy web server. Results are similar to those obtained with the most recent, cutting-edge methods based on direct learning of flexibility data conducted with sophisticated algorithms. PredyFlexy can be accessed at http://www.dsimb.inserm.fr/dsimb_tools/predyflexy/

Dynamics and deformability of α-, 310- and π-helices

National audienceProtein structures are often represented as seen in crystals: (i) rigid macromolecules (ii) with 3-states representations, namely helices, sheets and coils. Nonetheless, both information are incomplete because (i) proteins are highly dynamical macromolecules and (ii) the description of protein structures can be more precise. Regarding these two points, we have analysed and quantified the stability of helices by considering α-helices as well as the other two other helical local conformations, namely the 310-helices and the rare π-helices. Molecular dynamics simulations of 150 ns were performed on a large set of 169 representative protein domains with three independent simulations for each protein chain. The local protein conformations were studied during each simulation and analysed. Classical flexibility index (B-factor) was confronted to MD flexibility index (RMSf) and protein blocks (PB) assignation to characterize distorsions. Helical regions were classified according to their level of helicity from highly to none. For the first time, a precise quantification showed the percentage of rigid and flexible helices underlying unexpected behaviours. For instance, only 76.4% of the residues associated to α-helices keep this conformation, while this tendency drops to 40.5% for 310-helices and is never seen for π-helices. This analysis highlight the carefulness of considering protein structures as dynamic entity rather than static ones

Dynamics and deformability of α-, 310- and π-helices

Protein structures are often represented as seen in crystals as (i) rigid macromolecules (ii) with helices, sheets and coils. However, both definitions are partial because (i) proteins are highly dynamic macromolecules and (ii) the description of protein structures could be more precise. With regard to these two points, we analyzed and quantified the stability of helices by considering α-helices as well as 310- and π-helices. Molecular dynamic (MD) simulations were performed on a large set of 169 representative protein domains. The local protein conformations were followed during each simulation and analyzed. The classical flexibility index (B-factor) was confronted with the MD root mean square flexibility (RMSF) index. Helical regions were classified according to their level of helicity from high to none. For the first time, a precise quantification showed the percentage of rigid and flexible helices that underlie unexpected behaviors. Only 76.4% of the residues associated with α-helices retain the conformation, while this tendency drops to 40.5% for 310-helices and is never observed for π-helices. α-helix residues that do not remain as an α-helix have a higher tendency to assume β-turn conformations than 310- or π-helices. The 310-helices that switch to the α-helix conformation have a higher B-factor and RMSF values than the average 310-helix but are associated with a lower accessibility. Rare π-helices assume a β-turn, bend and coil conformations, but not α- or 310-helices. The view on π-helices drastically changes with the new DSSP (Dictionary of Secondary Structure of Proteins) assignment approach, leading to behavior similar to 310-helices, thus underlining the importance of secondary structure assignment methods

Identification of a small molecule splicing inhibitor targeting UHM domains

International audienceSplicing factor mutations are frequent in myeloid neoplasms, blood cancers,and solid tumors. Cancer cells harboring these mutations present aparticular vulnerability to drugs that target splicing factors such asSF3b155 or CAPERα. Still, the arsenal of chemical probes that targets thespliceosome is very limited. U2AF homology motifs (UHMs) are commonprotein interaction domains among splicing factors. They present ahydrophobic pocket ideally suited to anchor small molecules with the aimto inhibit protein–protein interaction. Here, we combined a virtual screeningof a small molecules database and an in vitro competition assay andidentified a small molecule, we named UHMCP1 that prevents theSF3b155/U2AF interaction. NMR analyses and molecular dynamics simulationsconfirmed the binding of this molecule in the hydrophobic pocketof the U2AF UHM domain. We further provide evidence that UHMCP1impacts splicing and cell viability and is therefore an interesting novel compoundtargeting an UHM domain with potential anticancer properties