O'Neill, S. E. | bioRxiv Science

bioRxiv2025

Calcium induced N-terminal gating and pore collapse in connexin-46/50 gap junctions

Gap junctions facilitate electrical and metabolic coupling essential for tissue function. Under ischemic conditions (e.g., heart attack or stroke), elevated intracellular calcium (Ca2+) levels uncouple these cell-to-cell communication pathways to protect healthy cells from cytotoxic signals. Using single-particle cryo-EM, we elucidate details of the Ca2+-induced gating mechanism of native connexin-46/50 (Cx46/50) gap junctions. The resolved structures reveal Ca2+ binding sites within the channel pore that alter the chemical environment of the permeation pathway and induce diverse occluded and gated states through N-terminal domain remodeling. Moreover, subunit rearrangements lead to pore collapse, enabling steric blockade by the N-terminal domains, reminiscent of the "iris model" of gating proposed over four decades ago. These findings unify and expand key elements of previous gating models, providing mechanistic insights into how Ca2+ signaling regulates gap junction uncoupling and broader implications for understanding cell stress responses and tissue protection.

biophysics↗

bioRxiv2023

The α-crystallin chaperones undergo a quasi-ordered co-aggregation process in response to saturating client interaction

Small heat shock proteins (sHSPs) are ATP-independent chaperones vital to cellular proteostasis, preventing protein aggregation events linked to various human diseases including cataract. The -crystallins, A-crystallin (Ac) and B-crystallin (Bc), represent archetypal sHSPs that exhibit complex polydispersed oligomeric assemblies and rapid subunit exchange dynamics. Yet, our understanding of how this plasticity contributes to chaperone function remains poorly understood. This study investigates structural changes in Ac and Bc during client sequestration under varying degree of chaperone saturation. Using biochemical and biophysical analyses combined with single-particle electron microscopy (EM), we examined Ac and Bc in their apo-states and at various stages of client-induced co-aggregation, using lysozyme as a model client. Quantitative single-particle analysis unveiled a continuous spectrum of oligomeric states formed during the co-aggregation process, marked by significant client-triggered expansion and quasi-ordered elongation of the sHSP scaffold. These structural modifications culminated in an apparent amorphous collapse of chaperone-client complexes, resulting in the creation of co-aggregates capable of scattering visible light. Intriguingly, these co-aggregates maintain internal morphological features of highly elongated sHSP scaffolding with striking resemblance to polymeric -crystallin species isolated from aged lens tissue. This mechanism appears consistent across both Ac and Bc, albeit with varying degrees of susceptibility to client-induced co-aggregation. Importantly, our findings suggest that client-induced co-aggregation follows a distinctive mechanistic and quasi-ordered trajectory, distinct from a purely amorphous process. These insights reshape our understanding of the physiological and pathophysiological co-aggregation processes of sHSPs, carrying potential implications for a pathway toward cataract formation.

biophysics↗