Analysis of Integrin αIIb Subunit Dynamics Reveals Long-Range Effects of Missense Mutations on Calf Domains

Integrin αIIbβ3, a glycoprotein complex expressed at the platelet surface, is involved in platelet aggregation and contributes to primary haemostasis. Several integrin αIIbβ3 polymorphisms prevent the aggregation that causes haemorrhagic syndromes, such as Glanzmann thrombasthenia (GT). Access to 3D structure allows understanding the structural effects of polymorphisms related to GT. In a previous analysis using Molecular Dynamics (MD) simulations of αIIb Calf-1 domain structure, it was observed that GT associated with single amino acid variation affects distant loops, but not the mutated position. In this study, experiments are extended to Calf-1, Thigh, and Calf-2 domains. Two loops in Calf-2 are unstructured and therefore are modelled expertly using biophysical restraints. Surprisingly, MD revealed the presence of rigid zones in these loops. Detailed analysis with structural alphabet, the Proteins Blocks (PBs), allowed observing local changes in highly flexible regions. The variant P741R located at C-terminal of Calf-1 revealed that the Calf-2 presence did not affect the results obtained with isolated Calf-1 domain. Simulations for Calf- 1+ Calf-2, and Thigh + Calf-1 variant systems are designed to comprehend the impact of five single amino acid variations in these domains. Distant conformational changes are observed, thus highlighting the potential role of allostery in the structural basis of GT.

Insights into Comparative Modeling of VHH Domains

In the particular case of the Camelidae family, immunoglobulin proteins have evolved into a unique and more simplified architecture with only heavy chains. The variable domains of these chains, named VHHs, have a number of Complementary Determining Regions (CDRs) reduced by half, and can function as single domains making them good candidates for molecular tools. 3D structure prediction of these domains is a beneficial and advantageous step to advance their developability as molecular tools. Nonetheless, the conformations of CDRs loops in these domains remain difficult to predict due to their higher conformational diversity. In addition to CDRs loop diversity, our earlier study has established that Framework Regions (FRs) are also not entirely conformationally conserved which establishes a need for more rigorous analyses of these regions that could assist in template selection. In the current study, VHHs models using different template selection strategies for comparative modeling using Modeller have been extensively assessed. This study analyses the conformational changes in both CDRs and FRs using an original strategy of conformational discretization based on a structural alphabet. Conformational sampling in selected cases is precisely reported. Some interesting outcomes of the structural analyses of models also draw attention towards the distinct difficulty in 3D structure prediction of VHH domains.

PYTHIA: Deep Learning Approach for Local Protein Conformation Prediction

Protein Blocks (PBs) are a widely used structural alphabet describing local protein backbone conformation in terms of 16 possible conformational states, adopted by five consecutive amino acids. The representation of complex protein 3D structures as 1D PB sequences was previously successfully applied to protein structure alignment and protein structure prediction. In the current study, we present a new model, PYTHIA (predicting any conformation at high accuracy), for the prediction of the protein local conformations in terms of PBs directly from the amino acid sequence. PYTHIA is based on a deep residual inception-inside-inception neural network with convolutional block attention modules, predicting 1 of 16 PB classes from evolutionary information combined to physicochemical properties of individual amino acids. PYTHIA clearly outperforms the LOCUSTRA reference method for all PB classes and demonstrates great performance for PB prediction on particularly challenging proteins from the CASP14 free modelling category.

Structural alphabets for conformational analysis of nucleic acids available at dnatco.datmos.org

A detailed description of the dnatco.datmos.org web server implementing the universal structural alphabet of nucleic acids is presented. It is capable of processing any mmCIF- or PDB-formatted files containing DNA or RNA molecules; these can either be uploaded by the user or supplied as the wwPDB or PDB-REDO structural database access code. The web server performs an assignment of the nucleic acid conformations and presents the results for the intuitive annotation, validation, modeling and refinement of nucleic acids.

Analysis of Protein Disorder Predictions in the Light of a Protein Structural Alphabet

Intrinsically-disordered protein (IDP) characterization was an amazing change of paradigm in our classical sequence-structure-function theory. Moreover, IDPs are over-represented in major disease pathways and are now often targeted using small molecules for therapeutic purposes. This has had created a complex continuum from order-that encompasses rigid and flexible regions-to disorder regions; the latter being not accessible through classical crystallographic methodologies. In X-ray structures, the notion of order is dictated by access to resolved atom positions, providing rigidity and flexibility information with low and high experimental B-factors, while disorder is associated with the missing (non-resolved) residues. Nonetheless, some rigid regions can be found in disorder regions. Using ensembles of IDPs, their local conformations were analyzed in the light of a structural alphabet. An entropy index derived from this structural alphabet allowed us to propose a continuum of states from rigidity to flexibility and finally disorder. In this study, the analysis was extended to comparing these results to disorder predictions, underlying a limited correlation, and so opening new ideas to characterize and predict disorder.

A unified dinucleotide alphabet describing both RNA and DNA structures

By analyzing almost 120 000 dinucleotides in over 2000 nonredundant nucleic acid crystal structures, we define 96+1 diNucleotide Conformers, NtCs, which describe the geometry of RNA and DNA dinucleotides. NtC classes are grouped into 15 codes of the structural alphabet CANA (Conformational Alphabet of Nucleic Acids) to simplify symbolic annotation of the prominent structural features of NAs and their intuitive graphical display. The search for nontrivial patterns of NtCs resulted in the identification of several types of RNA loops, some of them observed for the first time. Over 30% of the nearly six million dinucleotides in the PDB cannot be assigned to any NtC class but we demonstrate that up to a half of them can be re-refined with the help of proper refinement targets. A statistical analysis of the preferences of NtCs and CANA codes for the 16 dinucleotide sequences showed that neither the NtC class AA00, which forms the scaffold of RNA structures, nor BB00, the DNA most populated class, are sequence neutral but their distributions are significantly biased. The reported automated assignment of the NtC classes and CANA codes available at dnatco.org provides a powerful tool for unbiased analysis of nucleic acid structures by structural and molecular biologists.

Discrete analysis of camelid variable domains: sequences, structures, and in-silico structure prediction

Antigen binding by antibodies requires precise orientation of the complementarity- determining region (CDR) loops in the variable domain to establish the correct contact surface. Members of the family Camelidae have a modified form of immunoglobulin gamma (IgG) with only heavy chains, called Heavy Chain only Antibodies (HCAb). Antigen binding in HCAbs is mediated by only three CDR loops from the single variable domain (VHH) at the N-terminus of each heavy chain. This feature of the VHH, along with their other important features, e.g., easy expression, small size, thermo-stability and hydrophilicity, made them promising candidates for therapeutics and diagnostics. Thus, to design better VHH domains, it is important to thoroughly understand their sequence and structure characteristics and relationship. In this study, sequence characteristics of VHH domains have been analysed in depth, along with their structural features using innovative approaches, namely a structural alphabet. An elaborate summary of various studies proposing structural models of VHH domains showed diversity in the algorithms used. Finally, a case study to elucidate the differences in structural models from single and multiple templates is presented. In this case study, along with the above-mentioned aspects of VHH, an exciting view of various factors in structure prediction of VHH, like template framework selection, is also discussed.

iPBAvizu: a PyMOL plugin for an efficient 3D protein structure superimposition approach

Background Protein 3D structure is the support of its function. Comparison of 3D protein structures provides insight on their evolution and their functional specificities and can be done efficiently via protein structure superimposition analysis. Multiple approaches have been developed to perform such task and are often based on structural superimposition deduced from sequence alignment, which does not take into account structural features. Our methodology is based on the use of a Structural Alphabet (SA), i.e. a library of 3D local protein prototypes able to approximate protein backbone. The interest of a SA is to translate into 1D sequences into the 3D structures. Results We used Protein blocks (PB), a widely used SA consisting of 16 prototypes, each representing a conformation of the pentapeptide skeleton defined in terms of dihedral angles. Proteins are described using PB from which we have previously developed a sequence alignment procedure based on dynamic programming with a dedicated PB Substitution Matrix. We improved the procedure with a specific two-step search: (i) very similar regions are selected using very high weights and aligned, and (ii) the alignment is completed (if possible) with less stringent parameters. Our approach, iPBA, has shown to perform better than other available tools in benchmark tests. To facilitate the usage of iPBA, we designed and implemented iPBAvizu, a plugin for PyMOL that allows users to run iPBA in an easy way and analyse protein superimpositions. Conclusions iPBAvizu is an implementation of iPBA within the well-known and widely used PyMOL software. iPBAvizu enables to generate iPBA alignments, create and interactively explore structural superimposition, and assess the quality of the protein alignments.

Conformational coupling by trans-phosphorylation in calcium calmodulin dependent kinase II

The calcium calmodulin dependent protein kinase II (CaMKII) is a dodecameric holoenzyme important for encoding memory. Its activation, triggered by binding of calcium calmodulin, persists autonomously after calmodulin dissociation. One (receiver) kinase captures and subsequently phos-phorylates the regulatory domain peptide of a donor kinase forming a chained dimer as a first stage of autonomous activation. Protein dynamics simulations examined the conformational changes triggered by dimer formation and phosphorylation, aimed to provide a molecular rationale for human mutations that result in learning disabilities. Ensembles generated from X-ray crystal structures were characterized by network centrality and community analysis. Mutual information related collective motions to local fragment dynamics encoded with a structural alphabet. Implicit solvent tCONCOORD conformational ensembles revealed the dynamic architecture of Inactive kinase domains was co-opted in the activated dimer but the network hub shifted from the nucleotide binding cleft to the captured peptide. Explicit solvent molecular dynamics (MD) showed nucleotide and substrate binding determinants formed coupled nodes in long-range signal relays between regulatory peptides in the dimer. Strain in the extended captured peptide was balanced by reduced flexibility of the receiver kinase C-lobe core. The relays were organized around a hydrophobic patch between the captured peptide and a key binding helix. The human mutations aligned along the relays. Thus, these mutations could disrupt the allosteric network alternatively, or in addition, to altered binding affinities. Non-binding protein sectors distant from the binding sites mediated the allosteric signalling; providing possible targets for inhibitor design. Phosphorylation of the peptide modulated the dielectric of its binding pocket to strengthen the patch, non-binding sectors, domain interface and temporal correlations between parallel relays. These results provide the molecular details underlying the reported positive kinase coop-erativity to enrich discussion on how autonomous activation by phosphorylation leads to long-term behavioural effects.Author SummaryProtein kinases play central roles in intracellular signalling. Auto-phosphorylation by bound nucleotide typically precedes phosphate transfer to multiple substrates. Protein conformational changes are central to kinase function, altering binding affinities to change cellular location and shunt from one signal pathway to another. In the brain, the multi-subunit kinase, CaMKII is activated by calcium calmodulin upon calcium jumps produced by synaptic stimulation. Auto-transphosphorylation of a regulatory peptide enables the kinase to remain activated and mediate long-term behavioural effects after return to basal calcium levels. A database of mutated residues responsible for these effects is difficult to reconcile solely with impaired nucleotide or substrate binding. Therefore, we have computationally generated interaction networks to map the conformational plasticity of the kinase domains where most mutations localize. The network generated from the atomic structure of a phosphorylated dimer resolves protein sectors based on their collective motions. The sectors link nucleotide and substrate binding sites in self-reinforcing relays between regulatory peptides. The self-reinforcement is strengthened by phosphorylation consistent with the reported positive cooperativity of kinase activity with calcium-calmodulin concentration. The network gives a better match with the mutations and, in addition, reveals target sites for drug development.