A coarse-grained methodology identifies intrinsic mechanisms that dissociate interacting protein pairs

We address the problem of triggering dissociation events between proteins that have formed a complex. We have collected a set of 25 non-redundant, functionally diverse protein complexes having high-resolution three-dimensional structures in both the unbound and bound forms. We unify elastic network models with perturbation response scanning (PRS) methodology as an efficient approach for predicting residues that have the propensity to trigger dissociation of an interacting protein pair, using the three-dimensional structures of the bound and unbound proteins as input. PRS reveals that while for a group of protein pairs, residues involved in the conformational shifts are confined to regions with large motions, there are others where they originate from parts of the protein unaffected structurally by binding. Strikingly, only a few of the complexes have interface residues responsible for dissociation. We find two main modes of response: In one mode, remote control of dissociation in which disruption of the electrostatic potential distribution along protein surfaces play the major role; in the alternative mode, mechanical control of dissociation by remote residues prevail. In the former, dissociation is triggered by changes in the local environment of the protein, e.g., pH or ionic strength, while in the latter, specific perturbations arriving at the controlling residues, e.g., via binding to a third interacting partner is required for decomplexation. We resolve the observations by relying on an electromechanical coupling model which reduces to the usual elastic network result in the limit of the lack of coupling. We validate the approach by illustrating the biological significance of top residues selected by PRS on select cases where we show that the residues whose perturbation leads to the observed conformational changes correspond to either functionally important or highly conserved residues in the complex

Inhibition of mutant RAS-RAF interaction by mimicking structural and dynamic properties of phosphorylated RAS

Undruggability of RAS proteins has necessitated alternative strategies for the development of effective inhibitors. In this respect, phosphorylation has recently come into prominence as this reversible post-translational modification attenuates sensitivity of RAS towards RAF. As such, in this study, we set out to unveil the impact of phosphorylation on dynamics of HRASWT and aim to invoke similar behavior in HRASG12D mutant by means of small therapeutic molecules. To this end, we performed molecular dynamics (MD) simulations using phosphorylated HRAS and showed that phosphorylation of Y32 distorted Switch I, hence the RAS/RAF interface. Consequently, we targeted Switch I in HRASG12D by means of approved therapeutic molecules and showed that the ligands enabled detachment of Switch I from the nucleotide-binding pocket. Moreover, we demonstrated that displacement of Switch I from the nucleotide-binding pocket was energetically more favorable in the presence of the ligand. Importantly, we verified computational findings in vitro where HRASG12D/RAF interaction was prevented by the ligand in HEK293T cells that expressed HRASG12D mutant protein. Therefore, these findings suggest that targeting Switch I, hence making Y32 accessible might open up new avenues in future drug discovery strategies that target mutant RAS proteins

Developing New Applications for Perturbation Response Scanning Method to Study Conformational Modulation of Globular Proteins

Conformational transitions in proteins facilitate precise physiological functions. Therefore, it is crucial to understand the mechanisms underlying these processes to modulate protein function. Yet, studying structural and dynamical properties of proteins are notoriously challenging due to the complexity of the underlying potential energy surfaces (PES). Perturbation Response Scanning (PRS) method has previously been developed to identify key residues that participate in the communication network responsible for specific conformational transitions. PRS is based on a residue-by-residue scan of the protein to determine the subset of residues/forces which provide the closest conformational change leading to a target conformational state, inasmuch as linear response theory applies to these motions. In this thesis, two novel methods are developed to further explore the dynamics of proteins. Perturb-Scan-Pull (PSP) method evaluates if conformational transitions may be triggered on the PES. It aims to study functionally relevant conformational transitions in proteins using results obtained by PRS and feeding them as inputs to steered molecular dynamics simulations. The success and the transferability of the method are evaluated on three protein systems having different complexity of motions on the PES: calmodulin, adenylate kinase, and bacterial ferric binding protein. Results indicate that PSP method captures the target conformation, while providing key residues and the optimum paths with relatively low free energy profiles. Unlike PSP method, which is developed to study conformational changes between two known states of a protein and considers the best force vector toward the target state, protein perturbation responses can be clustered with the hope of exploring the collective variables (CV) toward new conformations of a protein. The perturbation response clustering (PRC) technique is developed to study the alternative conformations available to proteins for which these have not yet been detected via experimental methods. Using collective variables predicted via clustering of the response vectors, new conformations are sampled, which capture low lying energy states that exist under specific circumstances in vivo. The methodologies developed in this thesis can be applied on a wide range of proteins having different functions and displaying various types of motions. More importantly, these methods can be extended to study nucleic acids (DNA, RNA) or membrane proteins by considering lipids molecules

Perturb-Scan-Pull: A novel method facilitating conformational transitions in proteins

Conformational transitions in proteins facilitate precise physiological functions. Therefor; it is crucial to understand the mechanisms underlying these processes to modulate protein function. Yet, studying structural and dynamical properties of proteins is notoriously challenging due to the complexity of the underlying potential energy surfaces (PES). We have previously developed the perturbation-response scanning (PRS) method to identify key residues that participate in the communication network responsible for specific conformational transitions. PRS is based on a residue-by-residue scan of the protein to determine the subset of residues/forces which provide the closest conformational change leading to a target conformational state, inasmuch as linear response theory applies to these motions. Here, we develop a novel method to further evaluate if conformational transitions may be triggered on the PES. We aim to study functionally relevant conformational transitions in proteins by using results obtained from PRS and feeding them as inputs to steered molecular dynamics simulations. The success and the transferability of the method are evaluated on three protein systems having different complexities of motion on the PES: calmodulin, adenylate kinase, and bacterial ferric binding protein. We find that the method captures the target conformation, while providing key residues and the optimum paths with relatively low free energy profiles

Compound heterozygous p. Arg949Trp and p. Gly970Ala mutations deteriorated the function of PEX1p: a study on PEX1 in a patient with Zellweger syndrome

The peroxisome is responsible for a variety of vital pathways in primary metabolism, including the very long-chain fatty-acid oxidation and plasmalogen lipid biosynthesis. Autosomal recessive disorder of the Zellweger spectrum (ZSD) is a major subset of peroxisome biogenesis disorders (PBDs) that can be caused by mutations in any of the 14 PEX genes. Zellweger syndrome (ZS) is the foremost common and severe phenotype within the heterogeneous ZSD. However, missense mutations encode proteins with residual functions, which are associated with phenotypes that are milder than ZS. Mutations in the PEX1 gene are among the most prevalent. PEX1 and PEX6 proteins, belonging to the AAA family of ATPases, form a hexameric complex, which is associated with peroxisome membranes and essential for peroxisome biology. In this study, a two-month-old Iranian boy with hypotonia, poor feeding, and difficulty in breathing was diagnosed with Zellweger syndrome. The parents of the patient were second cousins and healthy and no similar cases were observed in the parents' family. The PEX1 gene was sequenced in the patient and his parents. The compound heterozygous mutations, p. Arg949Trp and p. Gly970Ala, were identified in the patient, while the parents were heterozygous for these alleles. Sequence analysis of the mutant PEX1 D2 domain revealed that mutation p. Arg949Trp precisely occurred in a conserved arginine residue (P4 Arg), which hinders the substrate processing of the complex. Several database records have reported mutation p. Arg949Trp(R949W) but its clinical significance is given as uncertain. We report here a novel mutation, p. Gly970Ala, which is not recorded before and may prevent proper interaction of PEX1 and PEX6 proteins. In summary, the clinical findings and peroxisome profile of the patient suggested that compound heterozygosity for these two missense mutations resulted in a nonfunctional PEX1/PEX6 complex causing the severe ZS phenotype

Conformational multiplicity of bacterial ferric binding protein revealed by small angle x-ray scattering and molecular dynamics calculations

This study combines molecular dynamics (MD) simulations with small angle x-ray scattering (SAXS) measurements to investigate the range of conformations that can be adopted by a pH/ionic strength (IS) sensitive protein and to quantify its distinct populations in solution. To explore how the conformational distribution of proteins may be modified in the environmental niches of biological media, we focus on the periplasmic ferric binding protein A (FbpA) from Haemophilus influenzae involved in the mechanism by which bacteria capture iron from higher organisms. We examine iron-binding/release mechanisms of FbpA in varying conditions simulating its biological environment. While we show that these changes fall within the detectable range for SAXS as evidenced by differences observed in the theoretical scattering patterns calculated from the crystal structure models of apo and holo forms, detection of conformational changes due to the point mutation D52A and changes in ionic strength (IS) from SAXS scattering profiles have been challenging. Here, to reach conclusions, statistical analyses with SAXS profiles and results from different techniques were combined in a complementary fashion. The SAXS data complemented by size exclusion chromatography point to multiple and/or alternative conformations at physiological IS, whereas they are well-explained by single crystallographic structures in low IS buffers. By fitting the SAXS data with unique conformations sampled by a series of MD simulations under conditions mimicking the buffers, we quantify the populations of the occupied substates. We also find that the D52A mutant that we predicted by coarse-grained computational modeling to allosterically control the iron binding site in FbpA, responds to the environmental changes in our experiments with conformational selection scenarios that differ from those of the wild type

GROMACS 2023.3 Manual

<p>Full documentation for the GROMACS 2023.3 release version.</p&gt