Crystal Structure Of Full-length Kcsa Trapped In Open Conformation Reveals That C-terminal Domain Fine Tunes Activation And Coupled Inactivation

Abstract

Using chaperone-assisted crystallography, we have recently determined the crystal structure of full-length (FL) KcsA in its closed conformation. The FL structure reveals that the C-terminal domain (CTD) extends ~70 A towards the cytoplasm as a canonical four helix bundle stabilizing the closed state. Electrophysiological analysis of KcsA/Fab4 complex demonstrates that CTD remains as a bundle during activation gating. This motivated us to crystallize open conformation (OC) of FL KcsA using the same antibody fragments and a recently developed constitutively open mutant, which was crystallized in its truncated form. We solved the FL KcsA/Fab2 complex structure in the open state at 3.9 A resolution. FL open-structure bends at G104 and exhibits ~10 A displacement at the V115 creating four side windows large enough to accommodate hydrated K+ ions right below the gate. The CTD remains as four helix bundle, exerting strain on the bulge helices which connects the bundle and the transmembrane domain.Based on the current structure and an earlier set of truncated KcsA structures displaying different degree of openings, we suggest that the cytoplasmic domain not only stabilizes the closed state but also fine tunes the level of opening at the activation gate and thus determine the level of inactivation occurring at the selectivity filter. Brownian Dynamics, simulation, electrophysiological studies and electrostatic calculation are ongoing efforts to dissect the ion permeation pathway.