Biology subjects

Orekhov, P.

Publications and source records attributed to Orekhov, P..

6 recordsLinked to original sources

bioRxiv2026

Influence of molecular representation and charge on protein-ligand structural predictions by popular co-folding methods

Recently developed deep learning-based tools can effectively generate structural models of complexes of proteins and non-proteinaceous compounds. While some of their predictive capabilities are truly exciting, others remain to be thoroughly tested. Here, we probe whether the ligand input format (Chemical Component Dictionary, CCD, or Simplified Molecular Input Line Entry System, SMILES) and charge (which depends on protonation) will affect the results of the predictions by four popular algorithms: AlphaFold 3, Boltz-2, Chai-1, and Protenix-v1. We chose methylamine and acetic acid as two of the simplest titratable chemicals that are omnipresent in proteins as amino and carboxy moieties, and are consequently ubiquitous in the Protein Data Bank models that are most commonly used for training. Unexpectedly, we found that for both molecules, in many cases the input format affected the prediction results, and did it much stronger compared to protonation, whereas changes in the formally specified charge of the molecules did not lead to changes in binding expected from experiments. We conclude that (i) ensuring identical results irrespective of input formats and (ii) inclusion of protonation-related steps into training and prediction pipelines are the two available paths for improvement of protein-ligand structure prediction algorithms.

bioinformatics↗

bioRxiv2023

Monitoring GPCR Conformation with GFP-Inspired Dyes

Solvatochromic compounds have emerged as valuable environment-sensitive probes for biological research, with the chromophore of the green fluorescent protein (GFP) being a well-studied example. In this study, we demonstrate that synthetic analogues of the GFP chromophore can be used to investigate ligand-induced conformational changes in proteins. We synthesized thiol-reactive derivatives of four analogues of the GFP chromophore that exhibit notable solvatochromism. We used these derivatives to label two proteins: the soluble calcium sensor recoverin (Rec) and the transmembrane G protein-coupled A2A adenosine receptor (A2AAR), via cysteines located or introduced in the regions that undergo structural changes upon ligand binding. Two of these dyes showed Ca2+-induced fluorescence changes when attached to Rec. Notably, our best-performing dye, DyeC, when attached to A2AAR, revealed agonist-induced changes in both fluorescence intensity and shape of the emission spectrum. Molecular dynamics (MD) simulations provided mechanistic insights into these changes showing the activation of A2AAR transfers DyeC to a more confined and more hydrophilic environment. Additionally, an allosteric modulator, HMA, induces changes in DyeC fluorescence spectra, indicating a distinct receptor conformation from apo, antagonist, or agonist-bound receptors. Our study demonstrates that GFP-inspired dyes are effective for detecting structural changes in GPCR (G protein-coupled receptors), with advantages such as the ability to perform both intensity-based and ratiometric tracking, red-shifted fluorescence spectra, high extinction coefficient, and sensitivity to allosteric modulation. These dyes expand the toolbox for tracking ligand-induced changes and facilitate new insights into conformational changes induced by allosteric modulators in GPCRs.

biophysics↗

bioRxiv2022

Structural basis for receptor selectivity and inverse agonism in S1P5 receptors

The bioactive lysophospholipid sphingosine-1-phosphate (S1P) acts via five different subtypes of S1P receptors (S1PR) - S1P1-5. S1P5 is predominantly expressed in nervous and immune systems, regulating the egress of natural killer cells from lymph nodes and playing a role in immune and neurodegenerative disorders, as well as carcinogenesis. Several S1PR therapeutic drugs have been developed to treat these diseases; however, they lack receptor subtype selectivity, which leads to side effects. In this article, we describe a 2.2 [A] resolution room temperature crystal structure of the human S1P5 receptor in complex with a selective inverse agonist determined by serial femtosecond crystallography (SFX) at the Pohang Accelerator Laboratory X-Ray Free Electron Laser (PAL-XFEL) and analyze its structure-activity relationship data. The structure demonstrates a unique ligand-binding mode, involving an allosteric subpocket, which clarifies the receptor subtype selectivity and provides a template for structure-based drug design. Together with previously published S1PR structures in complex with antagonists and agonists, the new S1P5-inverse agonist structure sheds light on the activation mechanism and reveals structural determinants of the inverse agonism in the S1PR-family.

biophysics↗

bioRxiv2021

Structure and dynamics of the SARS-CoV-2 envelope protein monomer

Coronaviruses, especially SARS-CoV-2, present an ongoing threat for human wellbeing. Consequently, elucidation of molecular determinants of their function and interaction with host is an important task. Whereas some of the coronaviral proteins are extensively characterized, others remain understudied. Here, we use molecular dynamics simulations to analyze the structure and dynamics of the SARS-CoV-2 envelope (E) protein (a viroporin) in the monomeric form. The protein consists of the hydrophobic -helical transmembrane domain (TMD) and amphiphilic -helices H2 and H3, connected by flexible linkers. We show that TMD has a preferable orientation in the membrane, while H2 and H3 reside at the membrane surface. Orientation of H2 is strongly influenced by palmitoylation of cysteines Cys40, Cys43 and Cys44. Glycosylation of Asn66 affects the orientation of H3. We also observe that the E protein both generates and senses the membrane curvature, preferably localizing with the C-terminus at the convex regions of the membrane. This may be favorable for assembly of the E protein oligomers, whereas induction of curvature may facilitate budding of the viral particles. The presented results may be helpful for better understanding of the function of coronaviral E protein and viroporins in general, and for overcoming the ongoing SARS-CoV-2 pandemic.

biophysics↗

bioRxiv2020

Sub-millisecond conformational dynamics of the A2A adenosine receptor revealed by single-molecule FRET

The complex pharmacology of G-protein-coupled receptors (GPCRs) is defined by their multi-state conformational dynamics. Single-molecule Forster Resonance Energy Transfer (smFRET) is well-suited to quantify dynamics for individual protein molecules, however, its application to GPCRs is challenging; therefore, smFRET has been limited to studies of interreceptor interactions in cellular membranes and receptors in detergent environments. Here, we performed smFRET experiments on functionally active human A2A adenosine receptor (A2AAR) molecules embedded in freely diffusing lipid nanodiscs to study their intramolecular conformational dynamics. We propose a dynamic model of A2AAR activation that involves a slow (>2 ms) exchange between the active-like and inactive-like conformations in both apo and antagonist-bound A2AAR, explaining the receptors constitutive activity. For the agonist-bound A2AAR, we detected faster (390{+/-}80 s) ligand efficacy-dependent dynamics. This work establishes a general smFRET platform for GPCR investigations that can potentially be used for drug screening and/or mechanism-of-action studies.

biophysics↗

bioRxiv2020

Molecular mechanism of light-driven sodium pumping

Microbial rhodopsins appeared to be the most abundant light-harvesting proteins on the Earth and are the major contributes to the solar energy captured in the sea. They possess highly diverse biological functions. Explosion of research on microbial rhodopsins led to breakthroughs in their applications, in particular, in neuroscience. An unexpected new discovery was a Na+-pumping KR2 rhodopsin from Krokinobacter eikastus, the first light-driven non-proton cation pump. A fundamental difference between proton and other cation pumps is that non-proton pumps cannot use tunneling or Grotthuss mechanism for the ion translocation and, therefore, Na+ pumping cannot be understood in the framework of classical proton pump, like bacteriorhodopsin. Extensive studies on the molecular mechanism of KR2 failed to reveal mechanism of pumping. The existing high-resolution structures relate only to the ground state of the protein and revealed no Na+ inside the protein, which is unusual for active ion transporters. KR2 is only known non proton cation transporter with demonstrated remarkable potential for optogenetic applications and, therefore, elucidation of the mechanism of cation transport is important. To understand conception of cation pumping we solved crystal structures of the functionally key O-intermediate state of physiologically relevant pentameric form of KR2 and its D116N and H30A key mutants at high resolution and performed additional functional studies. The structure of the O-state reveals a sodium ion near the retinal Schiff base coordinated by N112 and D116 residues of the characteristic (for the whole family) NDQ triad. The structural and functional data show that cation uptake and release are driven by a switching mechanism. Surprisely, Na+ pathway in KR2 is lined with the chain of polar pores/cavities, similarly to the channelrhodopsin-2. Using Parinello fast molecular dynamics approach we obtained a molecular movie of a probable ion release. Our data provides insight into the mechanism of a non-proton cation light-driven pumping, strongly suggest close relation of sodium pumps to channel rhodopsins and, we believe, expand the present knowledge of rhodopsin world. Certainly they might be used for engineering of cation pumps and ion channels for optogenetics.

biophysics↗