Biology subjects

Volkov, A. N.

Publications and source records attributed to Volkov, A. N..

13 recordsLinked to original sources

bioRxiv2026

Binding of long non-coding RNAs within the androgen receptor N-terminal tail modulates interdomain communication and liquid-liquid phase separation

The androgen receptor (AR) is a transcription factor whose overactivation is a primary driver of prostate cancer. Although AR interactions with several long non-coding RNAs (lncRNAs) have been implicated in castration-resistant prostate cancer, their underlying molecular mechanisms and functional consequences remain poorly understood. Here, we identify the N-terminal 37 residues of the intrinsically disordered AR N-terminal domain as the primary RNA-binding region that mediates selective interactions with the lncRNAs HOTAIR and SLNCR1. Residue-level mapping and mutational analysis define Y11, R13, and Q24 as key determinants of RNA recognition. This RNA-binding region partially overlaps with the F23QNLF27 motif, previously shown to mediate N/C interdomain communication with the ligand-binding domain through folding upon binding. We observe a partner-dependent binding mode in which this motif remains dynamically disordered upon RNA binding. LncRNA binding promotes phase separation of the N-terminal domain, indicating that lncRNAs upregulated in late-stage prostate cancer may lower the threshold for AR condensate formation and contribute to ligand-independent AR signaling. LncRNAs modulate communication between the N-terminal and ligand-binding domains within condensates, suggesting that lncRNA binding may tune hormone-dependent full-length AR signaling. These findings provide a mechanistic framework for AR-lncRNA regulation, laying the groundwork for future therapeutic strategies against advanced prostate cancer. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/728402v1_ufig1.gif" ALT="Figure 1000"> View larger version (22K): org.highwire.dtl.DTLVardef@bee76dorg.highwire.dtl.DTLVardef@14bb6edorg.highwire.dtl.DTLVardef@1d693b5org.highwire.dtl.DTLVardef@7b70d1_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

Interplay of stability and dynamics in the optimization of a highly proficient de novo enzyme

The development of highly active de novo enzymes that catalyze new-to-nature reactions is becoming increasingly possible. Here, we identify the features underlying the catalytic efficiency of a highly proficient de novo enzyme, from the original computational design to an optimized catalyst obtained through two rounds of directed evolution. Computational, spectroscopic, and biochemical studies reveal successfully designed features, including precise alignment of catalytic residues, transition state stabilization, and environmental tuning. In the most evolved enzyme, binding of a transition state analog also led to widespread increases in backbone conformational stability throughout the protein, except within a helix near the active site entrance, where the introduction of Gly and Pro increased dynamics and catalytic activity. Thus, the entire protein contributes to catalysis in the most optimized enzyme. Also, the initial design considered only the transition state but not substrate binding, leading to a dynamic Michaelis complex prior to optimization. These studies show the multiple features that need to be optimized to achieve high activity in a designed enzyme.

biochemistry↗

bioRxiv2026

Ionic strength modulates structural disorder and protein oligomerization in the marginally disordered Phd transcription factor

Some proteins combine sequence features that are typical both for folded proteins and intrinsically disordered proteins (IDPs). The borderline properties of these so-called "marginal" IDPs render their conformational ensembles highly sensitive to the environmental changes, which may be important for their function. Here, we investigate the prokaryotic transcription factor Phd, which regulates the phd-doc toxin-antitoxin module through an allosteric mechanism involving disorder-order transition. Using an ensemble of biophysical techniques, we show that the protein is completely disordered at low ionic strength, whereas increasing salt concentration promotes its collapse into a partially ordered monomeric state, followed by the formation of a structured dimer. Using a thermodynamic model, we decipher the linkage between ionic strength, protein stability, oligomer state and degree of disorder. Via small-angle X-ray scattering we derive the structural ensemble of dimeric Phd, revealing a gradation of disorder as a function of salt. The sequence and biophysical properties of Phd position it at the boundary between macroscopically distinct conformational ensembles, representing a large pool of states capable of engaging in functional disorder-to-order interactions, enabling Phd to act as conformational rheostat. Together with previous crystallographic data, this charts the full spectrum of disorder-to-order states in the bacterial transcription factor and underscores the structural plasticity of IDPs with the marginal sequence properties. SIGNIFICANCEWhile globular proteins adopt stable three-dimensional structures, intrinsically disordered proteins (IDPs) remain flexible and dynamically sample partially ordered states. This behavior is largely determined by amino acid composition; however, some proteins lie at the borderline between order and disorder. Here, we focus on such a protein, the Phd transcription, and show how its conformation changes from completely disordered to fully ordered. These transitions are modulated by ionic strength and binding to macromolecules, including homodimerization. Using a thermodynamic model, we map the Phd conformational space, revealing a broad ensemble of states with varying degrees of disorder. The borderline amino acids properties enable Phd to function as a conformational rheostat, coupling functional interactions to the series of graded order-disorder transitions.

biophysics↗

bioRxiv2025

Insights into the recruitment of the H3K4me3 reader Spp1 by the meiotic double-strand break protein Mer2

The formation of DNA double-strand breaks (DSBs) by Spo11 is tied to the loop-axis organization of meiotic chromosomes. Prior to DSB formation, chromatin loops marked by histone H3K4 trimethylation become tethered to the chromosome axis through interactions between Spp1 and Mer2. Mer2 is an essential partner of Spo11 thought to assemble the DSB machinery via biomolecular condensation, but these molecular assemblies remain poorly characterized. Here, using AlphaFold modeling, biochemical reconstitution, and biophysical validation, we explored the relationship between Mer2, Spp1 and their DNA-bound complexes. The tetrameric coiled-coil domain of Mer2 has four rotationally symmetrical sites that can bind a C-terminal -helix of Spp1. However, binding of one Spp1 subunit appears to allosterically modulate the affinity for the adjacent sites, leading to the assembly of 4x2 Mer2-Spp1 complexes. Mer2 also accommodates multiple DNA duplexes, allowing the assembly of tripartite Mer2-Spp1-DNA complexes with branched DNA substrates and effective recruitment of Spp1 within nucleoprotein condensates. However, because the Spp1- and DNA-binding sites of Mer2 partially overlap, Spp1 recruitment reduces DNA binding by Mer2, which is compensated for by a patch of positively charged residues within Spp1. These findings provide insights into the structural organization of Mer2 and Spp1 and their role in the assembly of the meiotic DSB machinery.

molecular biology↗

bioRxiv2025

Negative design enables cell-free expression and folding of designed transmembrane β-Barrels

De novo design of membrane proteins (MPs) is a rapidly growing field with transformative potential for synthetic biology. Yet, progress has lagged behind that of soluble proteins, largely due to limited understanding of the fundamental principles governing MP folding, stability, and solubility--and their integration into computational models. Here, we use a cell-free expression system to bypass the cytotoxicity of failed or insoluble designs and investigate how sequence features influence the folding of synthetic transmembrane {beta}-barrels (TMBs). We find that even small, idealized TMBs challenge classical protein design workflows: sequences optimized solely for thermodynamic stability misfold and aggregate, preventing membrane insertion. Instead of bulk hydrophobicity, aggregation and {beta}-sheet propensity emerge as key determinants of membrane association. By designing better folding variants of a synthetic TMB, we demonstrate that suppressing aggregation-prone intermediates through local destabilization of {beta}-strands ("negative design") significantly improves folding efficiency. Strikingly, even substitutions typically considered highly destabilizing, such as prolines or polar threonines exposed to the bilayer core, can improve folding when strategically positioned, without significantly compromising thermodynamic stability. Based on these findings, we propose a framework for joint optimization of native stability and folding pathways for future MP and nanopore design. Significance StatementMembrane proteins are essential to biology and biotechnology, yet designing them from scratch remains challenging. Using synthetic transmembrane {beta}-barrels and a cell-free expression system with lipid vesicles, we show that conventional design strategies focused solely on native state stabilization can lead to misfolding and aggregation. By incorporating "negative design" features--specific mutations that locally disrupt {beta}-strand structure--we improve folding efficiency without compromising stability. Remarkably, a protein language model outperformed traditional energy-based methods in predicting these beneficial mutations. Our findings highlight the critical role of folding kinetics in membrane protein design and introduce new principles for engineering synthetic membrane proteins and {beta}-barrel nanopores.

biophysics↗

bioRxiv2025

Recruitment of Mre11 to recombination sites during meiosis

The Mre11 nuclease, part of the conserved MRX complex involved in the repair of DNA double-strand breaks (DSBs), is also essential to initiate meiotic recombination in budding yeast by promoting Spo11-induced DSBs. Recruitment of Mre11 to meiotic DSB sites depends on Rec114-Mei4 and Mer2 (RMM) that organize the meiotic DSB machinery by a mechanism involving biomolecular condensation. Here, we explored the role of Mre11 during meiosis and its relationship to RMM condensation. We show that both Mre11 and MRX complexes form DNA-dependent, hexanediol sensitive condensates in vitro. In vivo, Mre11 assembles into DNA damage-dependent foci in vegetative cells and DSB-independent foci in meiotic cells. In vitro condensates and in vivo foci both depend on the C-terminal intrinsically-disordered region (IDR) of Mre11. Importantly, while the Mre11 IDR is dispensable for vegetative DNA repair it is essential during meiosis. The C-terminus of Mre11 forms a short -helix that binds a conserved region of Mer2, and mutating residues within this interface reduces Mre11 foci and DSB formation. Finally, we identified a SUMO-interacting motif within the Mre11 IDR that enhances recruitment of Mre11 during meiosis and facilitates DSB formation. This work identifies multiple mechanisms that collaborate to recruit Mre11 during meiosis to initiate recombination.

molecular biology↗

bioRxiv2024

De novo design of high-affinity single-domain antibodies

Antibody-based therapeutics have become indispensable in modern medicine, but traditional methods of antibody discovery often present with limitations in developability, cross-reactivity, and ethical concerns. While deep learning and generative approaches have shown promise in the design of high affinity protein binders, de novo antibody design remains challenging. Here, we present EvolveX, a structure-based computational pipeline for designing antibody fragments. EvolveX utilizes ModelX and empirical force field FoldX to optimize complementarity- determining regions (CDRs) and TANGO for aggregation analysis. We demonstrate the ability of EvolveX to redesign a single-domain VHH antibody fragment targeting mouse Vsig4 to address two challenges: enhancing stability and affinity for the original target and redesigning it for high affinity to the human ortholog. The redesigned variants of VHH fragments specific to mouse Vsig4 showed improved physicochemical properties, while retaining binding affinities comparable to the original version. Notably, EvolveX improved the binding affinity of VHHs to human Vsig4 by over 1000-fold, transforming low-affinity binders into nanomolar-affinity molecules. Structural analyses by X-ray crystallography and NMR confirmed the accuracy of the designs, which display optimized interactions with the antigen. NGS and re-modelling analysis further demonstrated the efficiency of FoldX-based design pipeline. Collectively, our study highlights EvolveXs potential to overcome current limitations in antibody design, offering a powerful tool for the development of next-generation therapeutics with enhanced specificity, stability, and efficacy.

bioinformatics↗

bioRxiv2023

Beyond the VSG Layer: Exploring the Role of Intrinsic Disorder in the Invariant Surface Glycoproteins of African Trypanosomes

In the bloodstream of mammalian hosts, African trypanosomes face the challenge of protecting their invariant surface receptors from immune detection. This crucial role is fulfilled by a dense, glycosylated protein layer composed of variant surface glycoproteins (VSGs), which undergo antigenic variation and provide a physical barrier that shields the underlying invariant surface glycoproteins (ISGs). The protective shields limited permeability comes at the cost of restricted access to the extracellular host environment, raising questions regarding the specific function of the ISG repertoire. In this study, we employ an integrative structural biology approach to show that intrinsically disordered membrane-proximal regions are a common feature of members of the ISG superfamily, conferring the ability to switch between compact and elongated conformers. While the folded, membrane-distal ectodomain is buried within the VSG layer for compact conformers, their elongated counterparts would enable the extension beyond it. This dynamic behavior enables ISGs to maintain a low immunogenic footprint while still allowing them to engage with the host environment when necessary. Our findings add further evidence to a dynamic molecular organization of trypanosome surface antigens wherein intrinsic disorder underpins the characteristics of a highly flexible ISG proteome to circumvent the constraints imposed by the VSG coat.

biochemistry↗

bioRxiv2023

Structural Insights into the Interaction Between Adenovirus C5 Hexon and Human Lactoferrin

Adenovirus (AdV) infection of the respiratory epithelium is common, but poorly understood. Human AdV species C types, such as HAdV-C5, utilize the Coxsackie-adenovirus receptor (CAR) for attachment and subsequently integrins for entry. CAR and integrins are however located deep within the tight junctions in the mucosa where they would not be easily accessible. Recently, a model for CAR-independent AdV entry was proposed. In this model, human lactoferrin (hLF), an innate immune protein, aids the viral uptake into epithelial cells by mediating interactions between the major capsid protein, hexon, and yet unknown host cellular receptor(s). However, a detailed understanding of the molecular interactions driving this mechanism is lacking. Here, we present a new cryo-EM structure of HAdV-5C hexon at high resolution alongside a hybrid structure of HAdV-5C hexon complexed with human lactoferrin (hLF). These structures reveal the molecular determinants of the interaction between hLF and HAdV-C5 hexon. hLF engages hexon primarily via its N-terminal lactoferricin (Lfcin) region, interacting with hexons hypervariable region 1 (HVR-1). Mutational analyses pinpoint critical Lfcin contacts and also identify additional regions within hLF that critically contribute to hexon binding. Our study sheds more light on the intricate mechanism by which HAdV-C5 utilizes soluble hLF/Lfcin for cellular entry. These findings hold promise for advancing gene therapy applications and inform vaccine development.

biochemistry↗

bioRxiv2022

Evolutionary conservation of the structure and function of meiotic Rec114-Mei4 and Mer2 complexes

Meiosis-specific Rec114-Mei4 and Mer2 complexes are thought to enable Spo11-mediated DNA double-strand-break (DSB) formation through a mechanism that involves DNA-dependent condensation. However, the structure, molecular properties, and evolutionary conservation of Rec114-Mei4 and Mer2 are unclear. Here, we present AlphaFold structures of Rec114-Mei4 and Mer2 complexes, supported by nuclear magnetic resonance (NMR) spectroscopy, small-angle X-ray scattering (SAXS), and mutagenesis. We show that dimers composed of the Rec114 C-terminus form -helical chains that cup an N-terminal Mei4 -helix, and that Mer2 forms a parallel homotetrameric coiled coil. Both Rec114-Mei4 and Mer2 bind preferentially to branched DNA substrates, indicative of multivalent protein-DNA interactions. Indeed, the Rec114-Mei4 interaction domain contains two DNA-binding sites that point in opposite directions and drive condensation. The Mer2 coiled-coil domain bridges co-aligned DNA duplexes, likely through extensive electrostatic interactions along the length of the coiled coil. Finally, we show that the structure of Rec114-Mei4 and Mer2 are conserved across eukaryotes, while DNA-binding properties vary significantly. This work provides insights into the mechanism whereby Rec114-Mei4 and Mer2 complexes promote the assembly of the meiotic DSB machinery, and suggests a model where Mer2 condensation is the essential driver of assembly, with the DNA-binding activity of Rec114-Mei4 playing a supportive role.

molecular biology↗

bioRxiv2021

Vibrio cholerae ParE2 toxin modulates its operon transcription by stabilization of an antitoxin DNA ruler

The parDE2 operon of Vibrio cholerae encodes a type II TA system, which is one of three loci in the superintegron of small chromosome II that show modest similarity to the parDE operon of plasmid RK2. ParE2, like plasmid RK2-encoded ParE, inhibits DNA gyrase, an essential topoisomerase that is also the target of quinolone antibacterial agents. Mechanistic understanding on ParE2 toxin inhibition by direct interaction with its cognate antitoxin and transcriptional autoregulation of the TA system are currently lacking. ParD2, the ribbon-helix-helix (RHH) antitoxin, auto-represses the parDE2 promoter. This repression is enhanced by ParE2, which therefore functions as a transcriptional co-repressor. Here we present protein-DNA interaction studies and high-resolution X-ray structures of the ParD2:ParE2 complex and isolated ParD2 antitoxin, revealing the basis of toxin inhibition and autoregulation of the TA operon by conditional cooperativity. Native mass spectrometry, SAXS and MALS studies confirm the presence of different oligomerization states of ParD2 in solution and the role of the DNA-binding hexameric ParD26:ParE22 assembly in transcriptional repression.

microbiology↗

bioRxiv2021

Entropic pressure controls oligomerization of Vibrio cholerae ParD2 antitoxin

ParD2 is the antitoxin component of the parDE2 toxin-antitoxin module from Vibrio cholerae and consists of an ordered DNA binding domain followed by an intrinsically disordered ParE-neutralizing domain. In absence of the C-terminal IDP domain, VcParD2 crystallizes as a doughnut-shaped hexadecamer formed by the association of eight dimers. This assembly is stabilized via hydrogen bonds and salt bridges rather than hydrophobic contacts. In solution, oligomerization of the full-length protein is restricted to a stable, open 10-mer or 12-mer, likely as a consequence of entropic pressure from the IDP tails. The relative positioning of successive VcParD2 dimers mimics the arrangement of Streptococcus agalactiae CopG dimers on their operator and allows for an extended operator to wrap around the VcParD2 oligomer.

biophysics↗

bioRxiv2021

Novel anti-repression mechanism of H-NS proteins by a phage "early protein".

H-NS family proteins, bacterial xenogeneic silencers, play central roles in genome organization and in the regulation of foreign genes. It is thought that gene repression is directly dependent on the DNA binding modes of H-NS family proteins. These proteins form lateral protofilaments along DNA. Under specific environmental conditions they switch to bridging two DNA duplexes. This switching is a direct effect of environmental conditions on electrostatic interactions between the oppositely charged DNA binding and N-terminal domains of H-NS proteins. The Pseudomonas lytic phage LUZ24 encodes the early protein gp4, which modulates the DNA binding and function of the H-NS family protein MvaT of Pseudomonas aeruginosa. However, the mechanism by which gp4 modulates MvaT activity remains elusive. In this study, we show that gp4 specifically interferes with the formation and stability of the bridged MvaT-DNA complex. Structural investigations suggest that gp4 acts as an "electrostatic zipper" between the oppositely charged domains of MvaT protomers, and stabilizes a structure resembling their "half-open" conformation, resulting in relief of gene silencing and adverse effects on P. aeruginosa growth. The ability to control H-NS conformation and thereby its impact on global gene regulation and growth might open new avenues to fight Pseudomonas multidrug resistance.

biochemistry↗