Biology subjects

Czub, J.

Publications and source records attributed to Czub, J..

9 recordsLinked to original sources

bioRxiv2025

Origin of Class B J-domain proteins involved in amyloid transactions

J-domain protein (JDP) chaperones function widely in proteostasis. Notably, eukaryotic class B JDPs of the cytosol/nucleus prevent assembly or drive disassembly of amyloid aggregates known to cause neurodegenerative diseases, yet their evolutionary origin is not known. Members of the most ubiquitous class B subgroup, canonical B (BC) JDPs, lack the signature zinc finger region (ZnF) of the more prevalent class A JDPs, while having other key features in common. Our phylogenetic analysis revealed that BC JDPs evolved more than once from class A duplicates, losing their ZnF. The cytonuclear BCs emerged at the base of eukaryotes. Cytonuclear class B (i.e. B(ST)) JDPs that have a substrate binding domain of unknown origin, distinct from that of As and BCs, emerged from a BC duplication at the base of metazoans and subsequently multiplied by duplications. Origin of B(ST)s, which are capable of suppressing formation of amyloid aggregates, predated the emergence of disease-causing amyloidogenic proteins. Using ancestral sequence resurrection, we tested when cytonuclear Bs evolved their amyloid related functions. We found that their common ancestor with As, AncAB that has a ZnF does not facilitate disassembly of amyloid fibrils, while AncB, which lacks a ZnF, is active. Overall, our findings are consistent with the idea that, though the ZnF of class A JDPs is important for some roles, its loss allowed evolution of novel functions, as illustrated by the ability of BC and B(ST) JDPs to control amyloid aggregate levels. Significance statementAcross procaryotes and eukaryotes J-domain proteins (JDPs) are key players in Hsp70 chaperone systems that maintain cellular protein homeostasis. The abundant class A and B JDPs have structural similarities, yet their origin has remained unresolved. Here we show that B JDPs independently evolved from As more than once. In each case A lost its zinc finger (ZnF) domain, suggesting that such loss has allowed evolution of new functions. Supporting this idea, biochemical resurrection of an ancestral eukaryotic B revealed that its ability to disassemble amyloid aggregates, differentiating it from As, evolved after ZnF loss. Later, the subset of Bs implicated in suppression of disease-causing amyloid aggregate formation originated from a duplicate of this B in the common ancestor of animals.

evolutionary biology↗

bioRxiv2025

Lipid Dynamics and Organization Around Voltage-Gated Sodium Channels: A Coarse-Grained Perspective

Lipid-ion channel interactions play a critical role in channel function and membrane structural organization. Despite this importance, the mechanisms behind the rearrangement of lipids around ion channels are still unclear. To investigate this, we conducted coarse grained (CG) molecular dynamics simulations of voltage gated sodium ion channels (NavAB) embedded in a ternary lipid bilayer composed of 1,2-dilinoleoyl-sn- glycero-3-phosphocholine (DIPC), 1,2-dipalmitoyl-sn-glycero- 3-phosphocholine (DPPC), and cholesterol (CHOL) at varying CHOL concentrations (6.62%, 17.62% and 30.00%). By analyzing lipid organization and membrane structure, we examined how membrane composition and channel state (activated and resting) influence lipid redistribution near the channel interface. Our key finding is a pronounced preference for DIPC for the channel vicinity, observed consistently for all CHOL concentrations and channel states. Our simulations reveal that hydrophobic mismatch dictates lipid sorting near NavABs. The hydrphobic thickness of the channel favors flexible DIPC lipids, which are packed efficiently around it, while excluding thicker DPPC lipids. This exclusion drives DPPC and cholesterol to form ordered domains farther from the channel interface. Mixing entropy analysis supports local lipid de-mixing near the channel, aligning with the emergence of phase-separated domains. Notably, the hydrophobic thickness of NavAB remained stable and in close agreement with the experimental values, indicating that lipid-specific properties drive reorganization near the channel. Overall, our findings demonstrate that hydrophobic mismatch is a key driver of lipid reorganization and domain formation around ion channels, regardless of CHOL concentration or channel conformational state.

biophysics↗

bioRxiv2025

Mechanism and energetics of JDP induced Hsp70's conformational transition towards catalytically active state

Hsp70 chaperones are crucial for maintaining protein homeostasis by regulating the stability and conformational states of client polypeptides through cycles of their binding and release. These cycles require conformational transitions of Hsp70 driven by ATP binding and hydrolysis. The ATPase activity of Hsp70 is controlled by J-domain protein (JDP) cochaperones, which allosterically stimulate ATP hydrolysis via interactions between their J-domains and Hsp70. The J-domain binds at the interface between the nucleotide (NBD) and substrate (SBD) binding domains of ATP bound Hsp70. Although, it was established that the JD interaction involves residues of helices II and III, and the interhelical loop critical for ATPase stimulation, the mechanism by which the allosteric signal induced by J-domain binding is transmitted to the distal nucleotide-binding pocket of Hsp70 remains unclear, as do the conformational changes leading to the ATP hydrolysis. Here, we addressed these questions by means of all-atom free energy simulations and dynamic network analysis, starting from the crystal structures of ATP-bound Hsp70 DnaK alone and in complex with the J-domain of DnaJ. We demonstrated that the presence of the J-domain results in the rearrangement of the nucleotide-binding pocket into a hydrolysis competent state, characterized by close contact between universally conserved T199 of NBD and {gamma}-phosphate of ATP. With network analysis we revealed that the allosteric signal for this rearrangement is transmitted along the {beta}-strand containing T199. Finally, we provide rationale for the signal transmission, where steric repulsion between the J-domains helix III and SBD induces a push of the T199 containing {beta}-strand. Overall, our study provides mechanistic insights into allosteric signal transmission within Hsp70, bridging the gap between J-domain binding and ATPase stimulation. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/655504v2_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@1ff0f30org.highwire.dtl.DTLVardef@3e4157org.highwire.dtl.DTLVardef@133f159org.highwire.dtl.DTLVardef@12a35c2_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

Large-scale conformational analysis explains G-quadruplex topological landscape

G-quadruplexes (G4) are four-stranded nucleic acid structures formed within sequences containing repeated guanine tracts separated by loop regions. Abundant in the human genome, they play crucial roles in transcription regulation and genome maintenance. Although theoretically capable to adopt 26 different folding topologies--primarily differing in loop arrangements--only 14 of these have been observed experimentally. This raises fundamental questions about whether the remaining topologies are energetically inaccessible and what molecular factors shape the conformational landscape of G-quadruplexes. To address these questions, we systematically explored the conformational space of G-quadruplexes using a set of 128 G4-forming DNA sequences with varying loop lengths. Evaluation of nearly 20,000 unique G4 conformations revealed significant foldability differences across the 26 theoretical topologies. Crucially, we demonstrated that the presence of long-distance propeller loops in 12 of these topologies imposes strict loop length constraints, hindering their formation, especially in sequences with shorter loops. Additionally, we found that the occurrence of long-distance propeller loops is governed by G4 helicity, resulting in opposite folding preferences in right-handed and left-handed G4s. By providing geometric explanation for G4 folding patterns, our study advances the understanding of the G-quadruplex conformational landscape and offers valuable insights for the rational design of G4 structures. Author summaryDNA sequences enriched in guanines have the remarkable ability to form helical, four-stranded structures called G-quadruplexes (G4s). These structures have been found across the human genome, where they play a vital role in the regulation of various cellular processes, such as gene expression, replication, or genome maintenance. Moreover, designed G4 structures can be utilized as versatile building blocks in a variety of nanodevices. G4s are characterized by extensive structural diversity, arising from the multiple ways of arranging a DNA strand into a G4. Among the 26 geometrically valid arrangements--called looping topologies--only 14 have been proven experimentally, posing the question of whether the remaining topologies are energetically restricted and, if so, what molecular factors shape the topological landscape of G-quadruplexes. To address this question, in this work, we systematically searched the G4 conformational space for a set of 128 DNA sequences. We used an MD-based de novo folding procedure to evaluate foldability on nearly 20,000 unique G4 conformations. An analysis of the obtained data revealed that the topological landscape of G4s is predominantly restricted by long-distance propeller loops, whose occurrence among topologies is governed by G4s helicity.

bioinformatics↗

bioRxiv2024

Mechanism of expanded DNA recognition in xCas9

xCas9 is an evolved variant of the CRISPR-Cas9 genome editing system, engineered to improve specificity and reduce undesired off-target effects. How xCas9 expands the DNA targeting capability of Cas9 by recognizing a series of alternative Protospacer Adjacent Motif (PAM) sequences while ignoring others is unknown. Here, we elucidate the molecular mechanism underlying xCas9s expanded PAM recognition and provide critical insights for expanding DNA targeting. We demonstrate that while wild-type Cas9 enforces stringent guanine selection through the rigidity of its interacting arginine dyad, xCas9 introduces flexibility in R1335, enabling selective recognition of specific PAM sequences. This increased flexibility confers a pronounced entropic preference, which also improves recognition of the canonical TGG PAM. Furthermore, xCas9 enhances DNA binding to alternative PAM sequences during the early evolution cycles, while favouring binding to the canonical PAM in the final evolution cycle. This dual functionality highlights how xCas9 broadens PAM recognition and underscores the importance of fine-tuning the flexibility of the PAM-interacting cleft as a key strategy for expanding the DNA targeting potential of CRISPR-Cas systems. These findings deepen our understanding of DNA recognition in xCas9 and may apply to other CRISPR-Cas systems with similar PAM recognition requirements.

biophysics↗

bioRxiv2023

Low-barrier hydrogen bond determines target-binding affinity and specificity of the antitubercular drug bedaquiline

The role of short strong hydrogen bonds (SSHB) in ligand-target binding remains largely unexplored, thereby hindering a potentially important avenue in the rational drug design. Here, we investigate the interaction between bedaquiline (Bq), a potent anti-tuberculosis drug, and the mycobacterial ATP synthase, to unravel the role of a specific hydrogen bond to a conserved acidic residue in the target affinity and specificity. Our ab initio molecular dynamics simulations reveal that this bond belongs to the SSHB category and accounts for a substantial fraction of the target binding energy. We also demonstrate that the presence of an extra acidic residue (D32), found exclusively in mycobacteria, cooperatively enhances the HB strength ensuring the specificity for the mycobacterial target. Consistently, we show that the removal of D32 markedly weakens the affinity, leading to Bq resistance associated with mutations of D32 to non-acidic residues. By designing simple Bq analogs, we then explore the possibility to overcome the resistance and potentially broaden the Bq antimicrobial spectrum by making the SSHB independent on the presence of the extra acidic residue.

biophysics↗

bioRxiv2021

Determinants of directionality and efficiency of the ATP synthase Fo motor at atomic resolution

Fo subcomplex of ATP synthase is an membraneembedded rotary motor that converts proton motive force into mechanical energy. Despite a rapid increase in the number of high-resolution structures, the mechanism of tight coupling between proton transport and motion of the rotary c-ring remains elusive. Here, using extensive all-atom free energy simulations, we show how the motors directionality naturally arises from the interplay between intra-protein interactions and energetics of protonation of the c-ring. Notably, our calculations reveal that the strictly conserved arginine in the a-subunit (R176) serves as a jack-of-all-trades: it dictates the direction of rotation, controls the protonation state of the proton-release site and separates the two proton-access half-channels. Therefore, arginine is necessary to avoid slippage between the proton flux and the mechanical output and guarantees highly efficient energy conversion. We also provide mechanistic explanations for the reported defective mutations of R176, reconciling the structural information on the Fo motor with previous functional and single-molecule data.

biophysics↗

bioRxiv2021

Molecular mechanism of proton-coupled ligand translocation by the bacterial efflux pump EmrE

AO_SCPLOWBSTRACTC_SCPLOWThe current surge in bacterial multi-drug resistance (MDR) is one of the largest challenges to public health, threatening to render ineffective many therapies we rely on for treatment of serious infections. Understanding different factors that contribute to MDR is hence crucial from the global "one health" perspective. In this contribution, we focus on the prototypical broad-selectivity proton-coupled antiporter EmrE, one of the smallest known ligand transporters that confers resistance to aromatic cations in a number of clinically relevant species. As an asymmetric homodimer undergoing an "alternating access" protomer-swap conformational change, it serves as a model for the mechanistic understanding of more complex drug transporters. Here, we present a free energy and solvent accessibility analysis that indicates the presence of two complementary ligand translocation pathways that remain operative in a broad range of conditions. Our simulations show a previously undescribed desolvated apo state and anticorrelated accessibility in the ligand-bound state, explaining on a structural level why EmrE does not disrupt the pH gradient through futile proton transfer. By comparing the behavior of a number of model charged and/or aromatic ligands, we also explain the origin of selectivity of EmrE towards a broad class of aromatic cations. Finally, we explore unbiased pathways of ligand entry and exit to identify correlated structural changes implicated in ligand binding and release, as well as characterize key intermediates of occupancy changes.

biophysics↗

bioRxiv2020

Two-step mechanism of J-domain action in driving Hsp70 function

J-domain proteins (JDPs), obligatory Hsp70 cochaperones, play critical roles in protein homeostasis. They promote key allosteric transitions that stabilize Hsp70 interaction with substrate polypeptides upon hydrolysis of its bound ATP. Although a recent crystal structure revealed the physical mode of interaction between a J-domain and an Hsp70, the structural and dynamic consequences of J-domain action once bound and how Hsp70s discriminate among its multiple JDP partners remain enigmatic. We combined free energy simulations, biochemical assays and evolutionary analyses to address these issues. Our results indicate that the invariant aspartate of the J-domain perturbs a conserved intramolecular Hsp70 network of contacts that crosses domains. This perturbation leads to destabilization of the domain-domain interface - thereby promoting the allosteric transition that triggers ATP hydrolysis. While this mechanistic step is driven by conserved residues, evolutionarily variable residues are key to initial JDP/Hsp70 recognition - via electrostatic interactions between oppositely charged surfaces. We speculate that these variable residues allow an Hsp70 to discriminate amongst JDP partners, as many of them have coevolved. Together, our data points to a two-step mode of J-domain action, a recognition stage followed by a mechanistic stage. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/901538v2_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@ba0d3aorg.highwire.dtl.DTLVardef@ba9193org.highwire.dtl.DTLVardef@ab7974org.highwire.dtl.DTLVardef@1b2de33_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗