Predicting allosteric pockets in protein biological assemblages.

MOTIVATION: Allostery enables changes to the dynamic behavior of a protein at distant positions induced by binding. Here, we present APOP, a new allosteric pocket prediction method, which perturbs the pockets formed in the structure by stiffening pairwise interactions in the elastic network across the pocket, to emulate ligand binding. Ranking the pockets based on the shifts in the global mode frequencies, as well as their mean local hydrophobicities, leads to high prediction success when tested on a dataset of allosteric proteins, composed of both monomers and multimeric assemblages. RESULTS: Out of the 104 test cases, APOP predicts known allosteric pockets for 92 within the top 3 rank out of multiple pockets available in the protein. In addition, we demonstrate that APOP can also find new alternative allosteric pockets in proteins. Particularly interesting findings are the discovery of previously overlooked large pockets located in the centers of many protein biological assemblages; binding of ligands at these sites would likely be particularly effective in changing the protein's global dynamics. AVAILABILITY AND IMPLEMENTATION: APOP is freely available as an open-source code (https://github.com/Ambuj-UF/APOP) and as a web server at https://apop.bb.iastate.edu/.

Cooperative mechanics of PR65 scaffold underlies the allosteric regulation of the phosphatase PP2A.

PR65, a horseshoe-shaped scaffold composed of 15 HEAT (observed in Huntingtin, elongation factor 3, protein phosphatase 2A, and the yeast kinase TOR1) repeats, forms, together with catalytic and regulatory subunits, the heterotrimeric protein phosphatase PP2A. We examined the role of PR65 in enabling PP2A enzymatic activity with computations at various levels of complexity, including hybrid approaches that combine full-atomic and elastic network models. Our study points to the high flexibility of this scaffold allowing for end-to-end distance fluctuations of 40-50 Å between compact and extended conformations. Notably, the intrinsic dynamics of PR65 facilitates complexation with the catalytic subunit and is retained in the PP2A complex enabling PR65 to engage the two domains of the catalytic subunit and provide the mechanical framework for enzymatic activity, with support from the regulatory subunit. In particular, the intra-repeat coils at the C-terminal arm play an important role in allosterically mediating the collective dynamics of PP2A, pointing to target sites for modulating PR65 function.

Sampling of Protein Conformational Space Using Hybrid Simulations: A Critical Assessment of Recent Methods.

Recent years have seen several hybrid simulation methods for exploring the conformational space of proteins and their complexes or assemblies. These methods often combine fast analytical approaches with computationally expensive full atomic molecular dynamics (MD) simulations with the goal of rapidly sampling large and cooperative conformational changes at full atomic resolution. We present here a systematic comparison of the utility and limits of four such hybrid methods that have been introduced in recent years: MD with excited normal modes (MDeNM), collective modes-driven MD (CoMD), and elastic network model (ENM)-based generation, clustering, and relaxation of conformations (ClustENM) as well as its updated version integrated with MD simulations (ClustENMD). We analyzed the predicted conformational spaces using each of these four hybrid methods, applied to four well-studied proteins, triosephosphate isomerase (TIM), 3-phosphoglycerate kinase (PGK), HIV-1 protease (PR) and HIV-1 reverse transcriptase (RT), which provide extensive ensembles of experimental structures for benchmarking and comparing the methods. We show that a rigorous multi-faceted comparison and multiple metrics are necessary to properly assess the differences between conformational ensembles and provide an optimal protocol for achieving good agreement with experimental data. While all four hybrid methods perform well in general, being especially useful as computationally efficient methods that retain atomic resolution, the systematic analysis of the same systems by these four hybrid methods highlights the strengths and limitations of the methods and provides guidance for parameters and protocols to be adopted in future studies.

Impact of new variants on SARS-CoV-2 infectivity and neutralization: A molecular assessment of the alterations in the spike-host protein interactions.

The emergence of SARS-CoV-2 variants necessitates rational assessment of their impact on the recognition and neutralization of the virus by the host cell. We present a comparative analysis of the interactions of Alpha, Beta, Gamma, and Delta variants with cognate molecules (ACE2 and/or furin), neutralizing nanobodies (Nbs), and monoclonal antibodies (mAbs) using in silico methods, in addition to Nb-binding assays. Our study elucidates the molecular origin of the ability of Beta and Delta variants to evade selected antibodies, such as REGN10933, LY-CoV555, B38, C105, or H11-H4, while being insensitive to others including REGN10987. Experiments confirm that nanobody Nb20 retains neutralizing activity against the Delta variant. The substitutions T478K and L452R in the Delta variant enhance associations with ACE2, whereas P681R promotes recognition by proteases, thus facilitating viral entry. The Ab-specific responses of variants highlight how full-atomic structure and dynamics analyses are required for assessing the response to newly emerging variants.

Precise druggability of the PTH type 1 receptor.

Class B G protein-coupled receptors (GPCRs) are notoriously difficult to target by small molecules because their large orthosteric peptide-binding pocket embedded deep within the transmembrane domain limits the identification and development of nonpeptide small molecule ligands. Using the parathyroid hormone type 1 receptor (PTHR) as a prototypic class B GPCR target, and a combination of molecular dynamics simulations and elastic network model-based methods, we demonstrate that PTHR druggability can be effectively addressed. Here we found a key mechanical site that modulates the collective dynamics of the receptor and used this ensemble of PTHR conformers to identify selective small molecules with strong negative allosteric and biased properties for PTHR signaling in cell and PTH actions in vivo. This study provides a computational pipeline to detect precise druggable sites and identify allosteric modulators of PTHR signaling that could be extended to GPCRs to expedite discoveries of small molecules as novel therapeutic candidates.

ClustENMD: efficient sampling of biomolecular conformational space at atomic resolution.

SUMMARY: Efficient sampling of conformational space is essential for elucidating functional/allosteric mechanisms of proteins and generating ensembles of conformers for docking applications. However, unbiased sampling is still a challenge especially for highly flexible and/or large systems. To address this challenge, we describe a new implementation of our computationally efficient algorithm ClustENMD that is integrated with ProDy and OpenMM softwares. This hybrid method performs iterative cycles of conformer generation using elastic network model for deformations along global modes, followed by clustering and short molecular dynamics simulations. ProDy framework enables full automation and analysis of generated conformers and visualization of their distributions in the essential subspace. AVAILABILITY AND IMPLEMENTATION: ClustENMD is open-source and freely available under MIT License from https://github.com/prody/ProDy. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

ProDy 2.0: increased scale and scope after 10 years of protein dynamics modelling with Python.

SUMMARY: ProDy, an integrated application programming interface developed for modelling and analysing protein dynamics, has significantly evolved in recent years in response to the growing data and needs of the computational biology community. We present major developments that led to ProDy 2.0: (i) improved interfacing with databases and parsing new file formats, (ii) SignDy for signature dynamics of protein families, (iii) CryoDy for collective dynamics of supramolecular systems using cryo-EM density maps and (iv) essential site scanning analysis for identifying sites essential to modulating global dynamics. AVAILABILITY AND IMPLEMENTATION: ProDy is open-source and freely available under MIT License from https://github.com/prody/ProDy. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

State-dependent sequential allostery exhibited by chaperonin TRiC/CCT revealed by network analysis of Cryo-EM maps.

The eukaryotic chaperonin TRiC/CCT plays a major role in assisting the folding of many proteins through an ATP-driven allosteric cycle. Recent structures elucidated by cryo-electron microscopy provide a broad view of the conformations visited at various stages of the chaperonin cycle, including a sequential activation of its subunits in response to nucleotide binding. But we lack a thorough mechanistic understanding of the structure-based dynamics and communication properties that underlie the TRiC/CCT machinery. In this study, we present a computational methodology based on elastic network models adapted to cryo-EM density maps to gain a deeper understanding of the structure-encoded allosteric dynamics of this hexadecameric machine. We have analysed several structures of the chaperonin resolved in different states toward mapping its conformational landscape. Our study indicates that the overall architecture intrinsically favours cooperative movements that comply with the structural variabilities observed in experiments. Furthermore, the individual subunits CCT1-CCT8 exhibit state-dependent sequential events at different states of the allosteric cycle. For example, in the ATP-bound state, subunits CCT5 and CCT4 selectively initiate the lid closure motions favoured by the overall architecture; whereas in the apo form of the heteromer, the subunit CCT7 exhibits the highest predisposition to structural change. The changes then propagate through parallel fluxes of allosteric signals to neighbours on both rings. The predicted state-dependent mechanisms of sequential activation provide new insights into TRiC/CCT intra- and inter-ring signal transduction events.

Essential site scanning analysis: A new approach for detecting sites that modulate the dispersion of protein global motions.

Despite the wealth of methods developed for exploring the molecular basis of allostery in biomolecular systems, there is still a need for structure-based predictive tools that can efficiently detect susceptible sites for triggering allosteric responses. Toward this goal, we introduce here an elastic network model (ENM)-based method, Essential Site Scanning Analysis (ESSA). Essential sites are here defined as residues that would significantly alter the protein's global dynamics if bound to a ligand. To mimic the crowding induced upon substrate binding, the heavy atoms of each residue are incorporated as additional network nodes into the α-carbon-based ENM, and the resulting shifts in soft mode frequencies are used as a metric for evaluating the essentiality of each residue. Results on a dataset of monomeric proteins indicate the enrichment of allosteric and orthosteric binding sites, as well as global hinge regions among essential residues, highlighting the significant role of these sites in controlling the overall structural dynamics. Further integration of ESSA with information on predicted pockets and their local hydrophobicity density enables successful predictions of allosteric pockets for both ligand-bound and -unbound structures. ESSA can be efficiently applied to large multimeric systems. Three case studies, namely (i) G-protein binding to a GPCR, (ii) heterotrimeric assembly of the Ser/Thr protein phosphatase PP2A, and (iii) allo-targeting of AMPA receptor, demonstrate the utility of ESSA for identifying essential sites and narrowing down target allosteric sites identified by druggability simulations.

Intrinsic dynamics is evolutionarily optimized to enable allosteric behavior.

Allosteric behavior is central to the function of many proteins, enabling molecular machinery, metabolism, signaling and regulation. Recent years have shown that the intrinsic dynamics of allosteric proteins defined by their 3-dimensional architecture or by the topology of inter-residue contacts favors cooperative motions that bear close similarity to structural changes they undergo during their allosteric actions. These conformational motions are usually driven by energetically favorable or soft modes at the low frequency end of the mode spectrum, and they are evolutionarily conserved among orthologs. These observations brought into light evolutionary adaptation mechanisms that help maintain, optimize or regulate allosteric behavior as the evolution from bacterial to higher organisms introduces sequential heterogeneities and structural complexities.

Protein-Ligand Complexes as Constrained Dynamical Systems.

This study focuses on how the low-frequency end of the vibrational spectrum related to the functional motions changes as a protein binds to a small ligand(s). Our recently proposed residue-specific (RESPEC) elastic network model provides a natural laboratory for this aim due to its systematic mixed coarse-graining approach and parametrization. Current analysis on a large data set of protein-ligand complexes reveals a universal curve enclosing the frequency distributions, which bears the features of previous computational and experimental studies. We mostly observe positive frequency shifts in the collective modes of the protein upon ligand binding. This observation, conforming to the Rayleigh-Courant-Fisher theorem, points to a constraining effect imposed by ligands on protein dynamics, which may be accompanied by a negative vibrational entropy difference. Positive frequency shifts in the global modes can thus be linked to the harmonic well getting steeper, because of interactions with the ligand(s).

RESPEC Incorporates Residue Specificity and the Ligand Effect into the Elastic Network Model.

RESPEC is a new framework that introduces residue specificity into elastic network modeling (ENM) to successfully render intact protein-ligand complexes as well as apo proteins. This framework establishes a broader application of coarse-graining idea via describing (i) a coarse-grained residue/node through its heavy atoms as virtual nodes, (ii) an effective B-factor for such a node, directly obtained from the experimental data, and (iii) a node-node interaction by a cumulative distance-dependent force constant. RESPEC improves the level of correlations with B-factors after optimizing the parameters of the model. In the absence of ligands, the mean correlations exceed 0.72, which is higher than the classical ENM results, based on a diverse set of proteins. Global modes satisfactorily describe the conformational transitions for apo structures. When the ligands are included at atomistic resolution in RESPEC calculations, mean correlation values exceed 0.9 over the same data set.

Moloney darn repair versus lichtenstein mesh hernioplasty for open inguinal hernia repair.

PURPOSE: To compare two tension-free techniques of inguinal hernia repair: the Moloney darn repair (MDR) and Lichtenstein mesh hernioplasty (LMH). METHODS: The subjects of this study were 651 patients from a total 732 who underwent open inguinal herniorrhaphy at our clinic between January 2000 and January 2006. We evaluated and compared analgesic requirement in the first 24 h, operative time, hospital stay, early postoperative complications, time until return to work, and recurrence, between patients who underwent MDR (group A) and patients who underwent LMH (group B). RESULTS: Group B patients required less analgesia in the first 24 h than group A patients. Conversely, the mean operative time and postoperative hospital stay were shorter in group A. Early postoperative complication rates and the time until return to work did not differ significantly between the two groups. During follow-up, recurrences developed in three patients from group A and four from group B. The cost of MDR was significantly less than that of LMH. CONCLUSIONS: Both MDR and LMH resulted in rapid recovery and low recurrence rates; however, the advantage of the MDR lies in the fact that it does not require mesh, so it is much less expensive.

[Infected urachal cyst]. / Enfekte urakus kisti.

The urachus is a vestigial remnant of the cloaca and allantois. It is usually obliterated at early postnatal life. When this obliteration is incomplete, in addition to congenital urachal anomalies such as patent urachus, umblical-urachal sinus, vesico-urachal diverticulum, and urachal cyst, acquired urachal pathologies as infections and neoplasms can emerge. In this case report we will evaluate an infected urachal cyst established in a 26 year-old female. She presented with complaints of abdominal pain and umblical discharge. suprapubic sensitivity, abdominal mass with an overlying hyperemic skin were detected. Patient whose clinical manifestations suggested the diagnosis of infected urachal cyst which was also supported by USG and CT findings was operated. Total cyst excision was performed.