Clustering Heterogeneous Conformational Ensembles of Intrinsically Disordered Proteins with t-Distributed Stochastic Neighbor Embedding.

Intrinsically disordered proteins (IDPs) populate a range of conformations that are best described by a heterogeneous ensemble. Grouping an IDP ensemble into "structurally similar" clusters for visualization, interpretation, and analysis purposes is a much-desired but formidable task, as the conformational space of IDPs is inherently high-dimensional and reduction techniques often result in ambiguous classifications. Here, we employ the t-distributed stochastic neighbor embedding (t-SNE) technique to generate homogeneous clusters of IDP conformations from the full heterogeneous ensemble. We illustrate the utility of t-SNE by clustering conformations of two disordered proteins, Aß42, and α-synuclein, in their APO states and when bound to small molecule ligands. Our results shed light on ordered substates within disordered ensembles and provide structural and mechanistic insights into binding modes that confer specificity and affinity in IDP ligand binding. t-SNE projections preserve the local neighborhood information, provide interpretable visualizations of the conformational heterogeneity within each ensemble, and enable the quantification of cluster populations and their relative shifts upon ligand binding. Our approach provides a new framework for detailed investigations of the thermodynamics and kinetics of IDP ligand binding and will aid rational drug design for IDPs.

Molecular dynamics simulations elucidate oligosaccharide recognition pathways by galectin-3 at atomic resolution.

The recognition of carbohydrates by lectins plays key roles in diverse cellular processes such as cellular adhesion, proliferation, and apoptosis, which makes it a therapeutic target of significance against cancers. One of the most functionally active lectins, galectin-3 is distinctively known for its specific binding affinity toward ß-galactoside. However, despite the prevalence of high-resolution crystallographic structures, the mechanistic basis and more significantly, the dynamic process underlying carbohydrate recognition by galectin-3 are currently elusive. To this end, we employed extensive Molecular Dynamics simulations to unravel the complete binding event of human galectin-3 with its native natural ligand N-acetyllactosamine (LacNAc) at atomic precision. The simulation trajectory demonstrates that the oligosaccharide diffuses around the protein and eventually identifies and binds to the biologically designated binding site of galectin-3 in real time. The simulated bound pose correlates with the crystallographic pose with atomic-level accuracy and recapitulates the signature stabilizing galectin-3/oligosaccharide interactions. The recognition pathway also reveals a set of transient non-native ligand poses in its course to the receptor. Interestingly, kinetic analysis in combination with a residue-level picture revealed that the key to the efficacy of a more active structural variant of the LacNAc lay in the ligand's resilience against disassociation from galectin-3. By catching the ligand in the act of finding its target, our investigations elucidate the detailed recognition mechanism of the carbohydrate-binding domain of galectin-3 and underscore the importance of ligand-target binary complex residence time in understanding the structure-activity relationship of cognate ligands.

Spontaneous transmembrane pore formation by short-chain synthetic peptide.

Amphiphilic ß-peptides, which are synthetically designed short-chain helical foldamers of ß-amino acids, are established potent biomimetic alternatives of natural antimicrobial peptides. An intriguing question is how the distinct molecular architecture of these short-chain and rigid synthetic peptides translates to its potent membrane-disruption ability. Here, we address this question via a combination of all-atom and coarse-grained molecular dynamics simulations of the interaction of mixed phospholipid bilayer with an antimicrobial 10-residue globally amphiphilic helical ß-peptide at a wide range of concentrations. The simulation demonstrates that multiple copies of this synthetic peptide, initially placed in aqueous solution, readily self-assemble and adsorb at membrane interface. Subsequently, beyond a threshold peptide/lipid ratio, the surface-adsorbed oligomeric aggregate moves inside the membrane and spontaneously forms stable water-filled transmembrane pores via a cooperative mechanism. The defects induced by these pores lead to the dislocation of interfacial lipid headgroups, membrane thinning, and substantial water leakage inside the hydrophobic core of the membrane. A molecular analysis reveals that despite having a short architecture, these synthetic peptides, once inside the membrane, would stretch themselves toward the distal leaflet in favor of potential contact with polar headgroups and interfacial water layer. The pore formed in coarse-grained simulation was found to be resilient upon structural refinement. Interestingly, the pore-inducing ability was found to be elusive in a non-globally amphiphilic sequence isomer of the same ß-peptide, indicating strong sequence dependence. Taken together, this work puts forward key perspectives of membrane activity of minimally designed synthetic biomimetic oligomers relative to the natural antimicrobial peptides.

Quantitative Assessment of the Conformational Heterogeneity in Amylose across Force Fields.

The inherent flexibility and conformational heterogeneity of a carbohydrate pose a challenge for its modeling and sampling by the existing classical force field. This work quantitatively assesses the quality of four popular carbohydrate force fields (CHARMM36, GLYCAM06, OPLS-AA, GROMOS53A6CARBO_R) against their ability to accurately model the conformational landscape of a dodecamer of single-stranded amylose, the key constituent of starch. While past NMR and X-ray studies have hinted at evidence of a helical structure of amylose and its spontaneous helix-coil transition, it remains to be seen how existing force fields fare against modeling its structural transition. Toward this end, we perform a multimicrosecond long extensive molecular dynamics simulation of dodecamer of a single-stranded amylose chain in explicit water in each of the four force fields and assess these force fields' ability to model relative structural transitions via analyzing the radius of gyration, glycosidic linkage orientation, and pyranose ring puckering of the amylose. In particular, the simulations show that while GLYCAM06 and CHARMM36 force fields predict a significant helix-coil transition in the amylose, GROMOS53A6CARBO_R and OPLS-AA majorly favor extended conformation. The Markov State Model (MSM), built using the simulation trajectories, for each force field, provides a comparative quantification of the population of key macrostates of amylose and elucidates an underlying network of pathways of their mutual interconversion. The macrostates obtained from MSM revealed that metastable helixlike and semicoil intermediate conformations are more probable for CHARMM36, whereas elongated or helixlike conformations are more probable in OPLS-AA and GROMOS53A6CARBO_R. GLYCAM06 showed significant probability for both helix and coil conformations along with intermediate conformations. We find that the differences in the conformations across force fields are governed by differences in the kinetics of glycosidic linkages and pyranose ring pucker conformers. All four force fields share one common point that the majority of α(1 → 4) glycosidic linkages preferred syn conformation, which is found to be energetically more favorable than anti. However, except for GROMOS53A6CARBO_R, all other force fields predicted non-negligible minor anti conformation. The multimicrosecond long simulations on the single-chain amylose, in combination with MSM, described here, suggest that sampling of α(1 → 4) linked oligosacharides on microsecond time scales enable quantitative predictions of helix-coil, glycosidic linkage, and pyranose ring exchange kinetics. These exchange kinetics have otherwise remained inaccessible to quantification by experiments or nanosecond time scale simulations which might have hindered the comparison of the possibility of helix-coil exchange across different force fields on equal footing.

In Silico Reoptimization of Binding Affinity and Drug-Resistance Circumvention Ability in Kinase Inhibitors: A Case Study with RL-45 and Src Kinase.

A major bottleneck in the development of kinase inhibitors has been the onset of drug resistance around the gatekeeper residues of Src kinase. Although recent times have seen the reports of certain second-generation kinase inhibitors which are capable of bypassing the drug resistance by circumventing kinase mutation, their kinase-binding efficacy has remained considerably weaker than that of the classical adenosine 5'-triphosphate-competitive kinase inhibitors. Using a recently synthesized second-generation kinase inhibitor RL-45 as a template, the current work integrates fragment-based drug discovery and quantitative structure-activity relationship study with enhanced molecular dynamics simulation approaches, namely, metadynamics and replica exchange free-energy perturbation, and demonstrates how one can optimally redesign and assess novel Src kinase inhibitors, by minimal introduction of new functional moieties around template kinase inhibitor. Interestingly, unlike many synthetic kinase inhibitors, these in silico optimized small-molecule derivatives of RL-45 are found to be potentially capable of serving dual purposes, crucial for efficacy of an ideal kinase inhibitor: (a) circumventing gatekeeper residue mutation-related drug resistance in Src kinase, unlike many commercial kinase inhibitors and (b) manifesting superior resilience against unbinding from the kinase active site. The computer simulation, boosted by enhanced sampling techniques, further reveals that these designed inhibitors bring about key interactions in the form of significantly long-standing hydrogen bonds and hydrophobic pocket otherwise weak in the template bioactive kinase inhibitor, which enhance the binding efficacy of these newly designed ligands in the kinase-binding pocket.