Biology subjects

Golcuk, M.

Publications and source records attributed to Golcuk, M..

13 recordsLinked to original sources

bioRxiv2026

The Molecular Interplay of Tau and Motor Proteins on Microtubules

Tau is a microtubule (MT)-associated protein essential for neuronal cytoskeletal organization and intracellular transport, yet how tau physically regulates motor proteins remains elusive. Here, we integrate single-molecule imaging, cryo-electron microscopy, and molecular dynamics (MD) simulations to investigate the molecular mechanisms underlying tau-mediated inhibition of kinesin-1 and cytoplasmic dynein motility. We show that tau suppresses MT landing and triggers frequent pausing and premature detachment when motors encounter tau-enriched patches. Unexpectedly, tau impairs each motor using different mechanisms. Tau and kinesin-1 compete for the same surface on tubulin, precluding simultaneous binding to the same MT site. On the other hand, while dynein and tau can co-occupy the same tubulin, tau inhibits the transition of dynein into its high-affinity MT-binding state. Thus, tau can act as a physical or a conformational roadblock in a concentration-dependent manner, exerting distinct structural constraints on kinesin and dynein transport. These findings establish a structural framework for tau-mediated regulation of axonal transport.

biophysics↗

bioRxiv2026

Coupling of tubulin acetylation to microtubule stabilization through molecular mimicry

Microtubules support diverse cellular functions through regulation by microtubule-associated proteins and tubulin post-translational modification, yet how these two layers are mechanistically integrated remains unclear. -tubulin acetylation marks mechanically resilient microtubules, and its incorporation in defined microtubule sub-populations is not well understood. Here, we identify MTCL1 as a molecular link between microtubule stabilization and post-translational modification installation. We find that MTCL1 stabilizes microtubules and alters the luminal surface when copolymerized with tubulin, remodeling -tubulin and enhancing TAT-mediated tubulin acetylation through molecular mimicry. This effect depends on assembly history and is not observed in pre-assembled microtubules. Targeted deletion of MTCL1 in zebrafish impacts axonal organization, leading to motor defects and increased seizure susceptibility. These findings establish MTCL1 as a licensing factor that couples microtubule stabilization with acetylation to regulate neuronal function.

biophysics↗

bioRxiv2025

Structure and mechanism of microtubule stabilization and motor regulation by MAP9

Microtubule-associated proteins (MAPs) regulate the organization of microtubules and control intracellular transport, but their individual contributions to microtubule dynamics and motor regulation remain poorly understood. Here, we identify MAP9 as a critical factor that stabilizes microtubules and facilitates neuronal morphogenesis. MAP9 knockdown abolishes the outgrowth of neurites, a phenotype not observed through the loss of other neuronal MAPs. Cryo-electron microscopy revealed that, unlike other MAPs that bind along protofilaments, MAP9 binds around the microtubule as a long alpha helix using five consecutive repeats. This unique binding mode enables MAP9 to staple adjacent protofilaments, thereby preventing microtubule depolymerization. We also showed that MAP9 selectively permits kinesin-3 and dynein motility while hindering kinesin-1 through interactions with a divergent loop-8 of the kinesin motor domain. Our results establish MAP9 as a key MAP required for neuronal growth and uncover how it differentially regulates intracellular transport driven by kinesin motors.

biophysics↗

bioRxiv2025

Covariance-Based MD Simulation Analysis Pinpoints Nanobody Attraction and Repulsion Sites on SARS-CoV-2 Omicron Spike Protein

Mutations in the SARS-CoV-2 spike receptor-binding domain (RBD) of the Omicron variant enable broad escape from neutralizing antibodies (Abs) and nanobodies (Nbs), yet the atomistic basis of epitope-dependent loss of Nb binding remains unclear. We performed all-atom molecular dynamics (MD) simulations of 13 Nbs bound to the Omicron RBD to characterize their binding modes, binding-pose stability, and interfacial dynamics. Analysis of the trajectories and corresponding free-energy landscapes revealed that most Nbs occupied a single dominant basin but showed varying degrees of within-basin heterogeneity and often exhibited orientation shifts, whereas two Nbs (Nb21 and Sb14) showed pronounced pose plasticity and dissociated from the RBD. For the remaining 11 Nbs, covariance-matrix analysis mapped stabilizing and destabilizing residue couplings, revealing recurrent hydrophobic anchor patches within the receptor-binding motif and Nb-specific interaction patterns reflecting differences in the sequences and chemistries of their complementarity-determining regions. Omicron substitutions rewired interaction networks, shifted Nb binding orientations relative to the corresponding experimentally determined reference structures (obtained with the ancestral WT RBD for all Nbs except H3), and introduced unfavorable repulsion, either directly at mutation sites or indirectly via mutation-driven reorientation, thereby reducing interfacial stability. Low-speed steered MD unbinding simulations of the 11 Nb-RBD complexes and the ACE2-RBD complex, performed at a pulling velocity within the range used in high-speed AFM-based force spectroscopy, yielded lower mean unbinding work for all 11 Nbs than for ACE2. These simulations also revealed heterogeneous, stepwise Nb unbinding pathways in which receptor-binding ridge (RBR)-mediated anchoring maintained partial Nb attachment through newly formed interactions and stabilized transient intermediate binding poses. Together, these Nb-RBD interaction fingerprints and pathway-resolved unbinding simulations pinpoint epitope-specific determinants and mutation-induced clash sites, providing a mechanistic basis for diminished Nb binding.

biophysics↗

bioRxiv2025

A Practical Covariance-Based Method for Efficient Detection of Protein-Protein Attractive and Repulsive Interactions in Molecular Dynamics Simulations

Molecular dynamics simulations of large protein-protein complexes require scalable analysis. We present a correlation-based workflow that systematically identifies both attractive (stabilizing) interactions, such as salt bridges, hydrogen bonds, and hydrophobic contacts, as well as repulsive (destabilizing) interactions across extensive interfaces. By constructing an inter-protein covariance matrix, filtering residue pairs based on distance, and identifying interactions underlying these correlations, our method focuses computational resources on the most relevant regions of the interface while preserving a high level of detail.

biophysics↗

bioRxiv2025

Structural Basis of Pseudomonas FapC Biofilm-Forming Functional Amyloid Formation

Biofilm-protected Pseudomonas aeruginosa causes chronic infections that are difficult to treat. FapC, the major biofilm forming functional-amyloid in Pseudomonas, is essential for biofilm integrity, yet its structural details remain unresolved. Using an integrative structural biology approach, we combine solution NMR-based structural ensemble of unfolded monomeric FapC, a [~]3.3 [A] resolution CryoEM density map of FapC fibril, and all-atom MD simulations to capture transition from unfolded to folded monomer to fibrillar fold, providing a complete structural view of FapC biogenesis. CryoEM reveals a unique triple-layer {beta}-solenoid cross-{beta} fibril composed of a single protofilament. MD simulations initiated from monomeric and fibrillar FapC mapped structural transitions, offering mechanistic insights into amyloid assembly and disassembly. Understanding FapC reveals how Pseudomonas exploits functional amyloids for biofilm formation and establishes a structural and mechanistic foundation for developing therapeutics targeting biofilm-related infection and antimicrobial resistance.

microbiology↗

bioRxiv2023

A force-sensitive mutation reveals a spindle assembly checkpoint-independent role for dynein in anaphase progression

The cytoplasmic dynein-1 (dynein) motor organizes cells by shaping microtubule networks and moving a large variety of cargoes along them. However, dyneins diverse roles complicate in vivo studies of its functions significantly. To address this issue, we have used gene editing to generate a series of missense mutations in Drosophila Dynein heavy chain (Dhc). We find that mutations associated with human neurological disease cause a range of defects in larval and adult flies, including impaired cargo trafficking in neurons. We also describe a novel mutation in the microtubule-binding domain (MTBD) of Dhc that, remarkably, causes metaphase arrest of mitotic spindles in the embryo but does not impair other dynein-dependent processes. We demonstrate that the mitotic arrest is independent of dyneins well-established roles in silencing the spindle assembly checkpoint. In vitro reconstitution and optical trapping assays reveal that the mutation only impairs the performance of dynein under load. In silico all-atom molecular dynamics simulations show that this effect correlates with increased flexibility of the MTBD, as well as an altered orientation of the stalk domain, with respect to the microtubule. Collectively, our data point to a novel role of dynein in anaphase progression that depends on the motor operating in a specific load regime. More broadly, our work illustrates how cytoskeletal transport processes can be dissected in vivo by manipulating mechanical properties of motors.

cell biology↗

bioRxiv2023

The Mechanism and Energetics of the Dynein Priming Stroke

Dyneins is an AAA+ motor responsible for motility and force generation towards the minus end of microtubules. Dynein motility is powered by nucleotide-dependent transitions of its linker domain, which transitions between straight (post-powerstroke) and bent (pre-powerstroke) conformations. To understand the dynamics and energetics of the linker, we per-formed all-atom molecular dynamics (MD) simulations of human dynein-2 primed for its power stroke. Simulations re-vealed that the linker can adopt either a bent conformation or a semi-bent conformation, separated by a 5.7 kT energy bar-rier. The linker cannot switch back to its straight conformation in the pre-powerstroke state due to a steric clash with the AAA+ ring. Simulations also showed that an isolated linker has a free energy minimum near the semi-bent conformation in the absence of the AAA+ ring, indicating that the linker stores mechanical energy as it bends and releases this energy during the powerstroke.

biophysics↗

bioRxiv2021

The Omicron Variant Increases the Interactions of SARS-CoV-2 Spike Glycoprotein with ACE2

SARS-CoV-2 infection is initiated by binding of the receptor-binding domain (RBD) of its spike glycoprotein to the peptidase domain (PD) of angiotensin-converting enzyme 2 (ACE2) receptors in host cells. Recently detected Omicron variant of SARS-CoV-2 (B.1.1.529) is heavily mutated on RBD. Currently, the most common Omicron variants are the original BA.1 Omicron strain and the BA.2 variant, which became more prevalent since it first appeared. To investigate how these mutations affect RBD-PD interactions, we performed all-atom molecular dynamics simulations of the BA.1 and BA.2 RBD-PD in the presence of full-length glycans, explicit water and ions. Simulations revealed that RBDs of BA.1 and BA.2 variants exhibit a more dispersed interaction network and make an increased number of salt bridges and hydrophobic interactions with PD compared to wild-type RBD. Although BA.1 and BA.2 differ in two residues at the RBD-ACE2 interface, no major difference in RBD-PD interactions and binding strengths were observed between these variants. Using the conformations sampled in each trajectory, the Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) method estimated ~34% and ~51% stronger binding free energies for BA.1 and BA.2 RBD, respectively, than wild-type RBD, which may result in higher binding efficiency of the Omicron variant to infect host cells.

immunology↗

bioRxiv2021

Binding mechanism of neutralizing Nanobodies targeting SARS-CoV-2 Spike Glycoprotein

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters human cells upon binding of its spike (S) glycoproteins to ACE2 receptors. Several nanobodies neutralize SARS-CoV-2 infection by binding to the receptor-binding domain (RBD) of S protein, but the underlying mechanism is not well understood. Here, we identified an extended network of pairwise interactions between RBD and nanobodies H11-H4, H11-D4, and Ty1 by performing all-atom molecular dynamics (MD) simulations. Simulations of the nanobody-RBD-ACE2 complex revealed that H11-H4 more strongly binds to RBD without overlapping with ACE2 and triggers dissociation of ACE2 due to electrostatic repulsion. In comparison, Ty1 binding results in dissociation of ACE2 from RBD due to an overlap with the ACE2 binding site, whereas H11-D4 binding does not trigger ACE2 dissociation. Mutations in SARS-CoV-2 501Y.V1 and 501.V2 variants resulted in a negligible effect on RBD-ACE2 binding. However, the 501.V2 variant weakened H11-H4 and H11-D4 binding while strengthening Ty1 binding to RBD. Our simulations indicate that all three nanobodies can neutralize 501Y.V1 while Ty1 is more effective against the 501.V2 variant.

immunology↗

bioRxiv2020

The mechanism of motor inhibition by microtubule-associated proteins

Microtubule (MT)-associated proteins (MAPs) regulate intracellular transport by selectively recruiting or excluding kinesin and dynein motors from MTs. We used single-molecule and cryo-electron imaging to determine the mechanism of MAP-motor interactions in vitro. Unexpectedly, we found that the regulatory role of a MAP cannot be predicted based on whether it overlaps with the motor binding site or forms liquid condensates on the MT. Although the MT binding domain (MTBD) of MAP7 overlaps with the kinesin-1 binding site, tethering of kinesin-1 by the MAP7 projection domain supersedes this inhibition and results in biphasic regulation of kinesin-1 motility. Conversely, the MTBD of tau inhibits dynein motility without overlapping with the dynein binding site or by forming tau islands on the MT. Our results indicate that MAPs sort intracellular cargos moving in both directions, as neither dynein nor kinesin can walk on a MAP-coated MT without favorably interacting with that MAP. HIGHLIGHTSO_LIMAP7 binds to a novel site and can coexist with tau on the MT. C_LIO_LIKinesin-1 motility is biphasically regulated by MAP7 accumulation on the microtubule. C_LIO_LIMT decoration of MAPs inhibits motors even when they do not block the motor binding site. C_LIO_LIMotors need to interact with a MAP to walk on MAP-decorated MTs C_LI

biophysics↗

bioRxiv2020

Critical Interactions Between the SARS-CoV-2 Spike Glycoprotein and the Human ACE2 Receptor

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects human cells upon binding of its spike (S) glycoproteins to ACE2 receptors and causes the coronavirus disease 2019 (COVID-19). Therapeutic approaches to prevent SARS-CoV-2 infection are mostly focused on blocking S-ACE2 binding, but critical residues that stabilize this interaction are not well understood. By performing all-atom molecular dynamics (MD) simulations, we identified an extended network of salt bridges, hydrophobic and electrostatic interactions, and hydrogen bonding between the receptor-binding domain (RBD) of the S protein and ACE2. Mutagenesis of these residues on the RBD was not sufficient to destabilize binding but reduced the average work to unbind the S protein from ACE2. In particular, the hydrophobic end of RBD serves as the main anchor site and unbinds last from ACE2 under force. We propose that blocking the hydrophobic surface of RBD via neutralizing antibodies could prove an effective strategy to inhibit S-ACE2 interactions.

immunology↗

bioRxiv2020

Exploring Conformational Transition of 2019 Novel Coronavirus Spike Glycoprotein Between Its Closed and Open States Using Molecular Dynamics Simulations

Since its first recorded appearance in December 2019, a novel coronavirus (SARS-CoV-2) causing the disease COVID-19 has resulted in more than 2,000,000 infections and 128,000 deaths. Currently there is no proven treatment for COVID-19 and there is an urgent need for the development of vaccines and therapeutics. Coronavirus spike glycoproteins play a critical role in coronavirus entry into the host cells, as they provide host cell recognition and membrane fusion between virus and host cell. Thus, they emerged as popular and promising drug targets. Crystal structures of spike protein in its closed and open states were resolved very recently in March 2020. These structures comprise 77% of the sequence and provide almost the complete protein structure. Based on down and up positions of receptor binding domain (RBD), spike protein can be in a receptor inaccessible closed or receptor accessible open state, respectively. Starting from closed and open state crystal structures, and also 16 intermediate conformations, an extensive set of all-atom molecular dynamics (MD) simulations in the presence of explicit water and ions were performed. Simulations show that in its down position, RBD has significantly lower mobility compared to its up position; probably caused by the 6 interdomain salt bridges of RBD in down position compared to 3 in up position. Free energy landscapes based on MD simulations revealed a semi-open state located between closed and open states. Minimum energy pathway between down and up positions comprised a gradual salt bridge switching mechanism. Furthermore, although significantly lower than open state, ACE2 binding surface of RBD contained a partial solvent accessibility in its closed state.

biophysics↗