Chong, S.-H. | bioRxiv Science

bioRxiv2025

Dynamic Shielding and Allosteric Modulation of Erythropoietin by Glycosylation

Glycosylation is a ubiquitous and essential post-translational modification that regulates protein structure, solubility, and function. Yet, the mechanisms by which glycans modulate the physicochemical properties of protein surfaces remain incompletely understood. Erythropoietin (EPO), a therapeutic glycoprotein with three N-glycosylation sites, provides a tractable model for dissecting site-specific glycan effects on protein functions. Here, we employ glycan-focused enhanced molecular dynamics simulations, specifically generalized replica exchange with solute tempering (gREST) to overcome the limited sampling of conventional MD and capture the extensive conformational heterogeneity of glycans. Our results demonstrate that glycan effects are highly non-additive: specific combinations of glycosylation sites yield emergent structural outcomes through spatial and dynamical cooperativity. Among them, the N83-linked glycan plays a dominant role in shielding a hydrophobic surface helix, thereby reducing local solvent-accessible hydropathy. Strikingly, the extent of this glycan-mediated surface masking quantitatively correlates with experimentally measured retention times in hydrophobic interaction chromatography, establishing a functional link between molecular-scale shielding and macroscopic behavior. These findings reveal that N-glycans modulate protein surfaces not only through local steric occlusion but also via long-range allosteric effects, providing a new framework for understanding and engineering glycoprotein properties.

biophysics↗

bioRxiv2025

Neck-region-microtubule interactions direct counterclockwise stepping of kinesin-1

The "neck" region of kinesin is a structurally conserved element critical for force generation, stepping directionality, and cargo transport along microtubules, yet its atomic-scale structure in a functional context remains unresolved. Here, we employ all-atom replica exchange molecular dynamics simulations to resolve a high-confidence conformation of the neck region in dimeric human kinesin-1 bound to a realistic microtubule lattice, and use this structure to simulate kinesins initial stepping motion. Our simulations reveal that the neck coiled-coil is oriented perpendicular to the microtubules axis and positioned near its surface--a conformation consistent with earlier proposals but lacking high-resolution validation. Importantly, simulations indicate that neck-microtubule interactions bias the stepping trajectory, directing the rear kinesin head to overtake the front head from the right (counterclockwise stepping). These findings establish a mechanism by which neck-microtubule interactions govern directional bias in kinesins initial step, offering new insight into the molecular basis of its motility.

biophysics↗