Leap-dynamics: efficient sampling of conformational space of proteins and peptides in solution

AbstractA molecular simulation scheme, called Leap-dynamics, that provides efficient sampling of protein conformational space in solution is presented. The scheme is a combined approach using a fast sampling method, imposing conformational ‘leaps’ to force the system over energy barriers, and molecular dynamics (MD) for refinement. The presence of solvent is approximated by a potential of mean force depending on the solvent accessible surface area. The method has been successfully applied to N-acetyl-L-alanine-N-methylamide (alanine dipeptide), sampling experimentally observed conformations inaccessible to MD alone under the chosen conditions. The method predicts correctly the increased partial flexibility of the mutant Y35G compared to native bovine pancreatic trypsin inhibitor. In particular, the improvement over MD consists of the detection of conformational flexibility that corresponds closely to slow motions identified by nuclear magnetic resonance techniques

BCR-ABL residues interacting with ponatinib are critical to preserve the tumorigenic potential of the oncoprotein

Patients with chronic myeloid leukemia in whom tyrosine kinase inhibitors (TKIs) fail often present mutations in the BCR-ABL catalytic domain. We noticed a lack of substitutions involving 4 amino acids (E286, M318, I360, and D381) that form hydrogen bonds with ponatinib. We therefore introduced mutations in each of these residues, either preserving or altering their physicochemical properties. We found that E286, M318, I360, and D381 are dispensable for ABL and BCR-ABL protein stability but are critical for preserving catalytic activity. Indeed, only a "conservative" I360T substitution retained kinase proficiency and transforming potential. Molecular dynamics simulations of BCR-ABLI360T revealed differences in both helix αC dynamics and protein-correlated motions, consistent with a modified ATP-binding pocket. Nevertheless, this mutant remained sensitive to ponatinib, imatinib, and dasatinib. These results suggest that changes in the 4 BCR-ABL residues described here would be selected against by a lack of kinase activity or by maintained responsiveness to TKIs. Notably, amino acids equivalent to those identified in BCR-ABL are conserved in 51% of human tyrosine kinases. Hence, these residues may represent an appealing target for the design of pharmacological compounds that would inhibit additional oncogenic tyrosine kinases while avoiding the emergence of resistance due to point mutations.This work was supported by an investigator grant to P.V. from Associazione Italiana per la Ricerca sul Cancro (AIRC) and by funding from the Biotechnology and Biological Sciences Research Council (BB/I023291/1 and BB/H018409/1 to AP and FF). P.B. is the recipient of an AIRC - Marie Curie fellowship

Homology-based modeling of universal stress protein from listeria innocua up-regulated under acid stress conditions

An Universal Stress Protein (USP) expressed under acid stress condition by Listeria innocua ATCC 33090 was investigated. The USP was up-regulated not only in the stationary phase but also during the exponential growth phase. The three dimensional (3D) structure of USP was predicted using a combined proteomic and bioinformatics approach. Phylogenetic analysis showed that the USP from Listeria detected in our study was distant from the USPs of other bacteria (such as Pseudomonas spp., Escherichia coli, Salmonella spp.) and clustered in a separate and heterogeneous class including several USPs from Listeria spp. and Lactobacillus spp. An important information on the studied USP was obtained from the 3D-structure established through the homology modelling procedure. In detail, the Model_USP-691 suggested that the investigated USP had a homo-tetrameric quaternary structure. Each monomer presented an architecture analogous to the Rossmann-like α/β fold with 5 parallel β-strands and 4 α-helices. The analysis of monomer-monomer interfaces and quality of the structure alignments confirmed the model reliability. In fact, the structurally and sequentially conserved hydrophobic residues of the β-strand 5 (in particular the residues V146 and V148) were involved in the inter-chains contact. Moreover, the highly conserved residues I139 and H141 in the region α 4 were involved in the dimer association and functioned as hot spots into monomer-monomer interface assembly. The hypothetical assembly of dimers was also supported by the large interface area and by the negative value of solvation free energy gain upon interface interaction. Finally, the structurally conserved ATP-binding motif G-2X-G-9X-G(S/T-N) suggested for a putative role of ATP in stabilizing the tetrameric assembly of the USP. Therefore, the results obtained from a multiple approach, consisting in the application of kinetic, proteomic, phylogenetic and modelling analyses, suggest that Listeria USP could be considered a new type of ATP-binding USP involved in the response to acid stress condition during the exponential growth phase

sciCSR infers B cell state transition and predicts class-switch recombination dynamics using single-cell transcriptomic data

Class-switch recombination (CSR) is an integral part of B cell maturation. Here we present sciCSR (pronounced 'scissor', single-cell inference of class-switch recombination), a computational pipeline that analyzes CSR events and dynamics of B cells from single-cell RNA sequencing (scRNA-seq) experiments. Validated on both simulated and real data, sciCSR re-analyzes scRNA-seq alignments to differentiate productive heavy-chain immunoglobulin transcripts from germline 'sterile' transcripts. From a snapshot of B cell scRNA-seq data, a Markov state model is built to infer the dynamics and direction of CSR. Applying sciCSR on severe acute respiratory syndrome coronavirus 2 vaccination time-course scRNA-seq data, we observe that sciCSR predicts, using data from an earlier time point in the collected time-course, the isotype distribution of B cell receptor repertoires of subsequent time points with high accuracy (cosine similarity ~0.9). Using processes specific to B cells, sciCSR identifies transitions that are often missed by conventional RNA velocity analyses and can reveal insights into the dynamics of B cell CSR during immune response

A Case-by-Case Evolutionary Analysis of Four Imprinted Retrogenes

Retroposition is a widespread phenomenon resulting in the generation of new genes that are initially related to a parent gene via very high coding sequence similarity. We examine the evolutionary fate of four retrogenes generated by such an event; mouse Inpp5f_v2, Mcts2, Nap1l5, and U2af1-rs1. These genes are all subject to the epigenetic phenomenon of parental imprinting. We first provide new data on the age of these retrogene insertions. Using codon-based models of sequence evolution, we show these retrogenes have diverse evolutionary trajectories, including divergence from the parent coding sequence under positive selection pressure, purifying selection pressure maintaining parent-retrogene similarity, and neutral evolution. Examination of the expression pattern of retrogenes shows an atypical, broad pattern across multiple tissues. Protein 3D structure modeling reveals that a positively selected residue in U2af1-rs1, not shared by its parent, may influence protein conformation. Our case-by-case analysis of the evolution of four imprinted retrogenes reveals that this interesting class of imprinted genes, while similar in regulation and sequence characteristics, follow very varied evolutionary paths

in-silico and in-vitro analysis of IL36RN mutations reveals critical residues for the function of the interleukin-36 receptor complex

Generalized pustular psoriasis (GPP) is a potentially life-threatening skin disease, associated with IL36RN mutations. IL36RN encodes the interleukin (IL)-36 receptor antagonist (IL-36Ra), a protein that downregulates the activity of IL-36 cytokines by blocking their receptor (IL-36R). While GPP can be treated with IL-36R inhibitors, the structural underpinnings of the IL-36Ra/IL-36R interaction remain poorly understood. Here, we sought to address this question by systematically investigating the effects of IL36RN mutations. We experimentally characterized the effects of 30 IL36RN variants on protein stability. In parallel, we used a machine-learning tool (Rhapsody) to analyse the IL-36Ra three-dimensional structure and predict the impact of all possible amino acid substitutions. This integrated approach identified 21 amino acids that are essential to IL-36Ra stability. We next investigated the effects of IL36RN changes on IL-36Ra/IL-36R binding and IL-36R signalling. By combining in-vitro assays and machine-learning with a second programme (mCSM), we identified 13 amino acids that are critical to IL-36Ra/IL36R engagement. Finally, we experimentally validated three representative predictions, further confirming the reliability of Rhapsody and mCSM. These findings shed light on the structural determinants of IL-36Ra activity, with potential to facilitate the design of new IL-36 inhibitors and aid the interpretation of IL36RN variants in diagnostic settings

Reordering Hierarchical Tree Based on Bilateral Symmetric Distance

BACKGROUND: In microarray data analysis, hierarchical clustering (HC) is often used to group samples or genes according to their gene expression profiles to study their associations. In a typical HC, nested clustering structures can be quickly identified in a tree. The relationship between objects is lost, however, because clusters rather than individual objects are compared. This results in a tree that is hard to interpret. METHODOLOGY/PRINCIPAL FINDINGS: This study proposes an ordering method, HC-SYM, which minimizes bilateral symmetric distance of two adjacent clusters in a tree so that similar objects in the clusters are located in the cluster boundaries. The performance of HC-SYM was evaluated by both supervised and unsupervised approaches and compared favourably with other ordering methods. CONCLUSIONS/SIGNIFICANCE: The intuitive relationship between objects and flexibility of the HC-SYM method can be very helpful in the exploratory analysis of not only microarray data but also similar high-dimensional data