Partial closure of the γ-tubulin ring complex by CDK5RAP2 activates microtubule nucleation.

Microtubule nucleation is templated by the γ-tubulin ring complex (γ-TuRC), but its structure deviates from the geometry of α-/ß-tubulin in the microtubule, explaining the complex's poor nucleating activity. Several proteins may activate the γ-TuRC, but the mechanisms underlying activation are not known. Here, we determined the structure of the porcine γ-TuRC purified using CDK5RAP2's centrosomin motif 1 (CM1). We identified an unexpected conformation of the γ-TuRC bound to multiple protein modules containing MZT2, GCP2, and CDK5RAP2, resulting in a long-range constriction of the γ-tubulin ring that brings it in closer agreement with the 13-protofilament microtubule. Additional CDK5RAP2 promoted γ-TuRC decoration and stimulated the microtubule-nucleating activities of the porcine γ-TuRC and a reconstituted, CM1-free human complex in single-molecule assays. Our results provide a structural mechanism for the control of microtubule nucleation by CM1 proteins and identify conformational transitions in the γ-TuRC that prime it for microtubule nucleation.

Proton Release Reactions in the Inward H+ Pump NsXeR.

Directional ion transport across biological membranes plays a central role in many cellular processes. Elucidating the molecular determinants for vectorial ion transport is key to understanding the functional mechanism of membrane-bound ion pumps. The extensive investigation of the light-driven proton pump bacteriorhodopsin from Halobacterium salinarum(HsBR) enabled a detailed description of outward proton transport. Although the structure of inward-directed proton pumping rhodopsins is very similar to HsBR, little is known about their protonation pathway, and hence, the molecular reasons for the vectoriality of proton translocation remain unclear. Here, we employ a combined experimental and theoretical approach to tracking protonation steps in the light-driven inward proton pump xenorhodopsin from Nanosalina sp. (NsXeR). Time-resolved infrared spectroscopy reveals the transient deprotonation of D220 concomitantly with deprotonation of the retinal Schiff base. Our molecular dynamics simulations support a proton release pathway from the retinal Schiff base via a hydrogen-bonded water wire leading to D220 that could provide a putative gating point for the proton release and with allosteric interactions to the retinal Schiff base. Our findings support the key role of D220 in mediating proton release to the cytoplasmic side and provide evidence that this residue is not the primary proton acceptor of the proton transiently released by the retinal Schiff base.