Structure-Based Functional Modification Study of a Cyanobacterial Chloride Pump for Transporting Multiple Anions.

Understanding the structure and functional mechanisms of cyanobacterial halorhodopsin has become increasingly important, given the report that Synechocystis halorhodopsin (Sy HR), a homolog of the cyanobacterial halorhodopsin from Mastigocladopsis repens (Mr HR), can take up divalent ions, such as SO 4 2−, as well as chloride ions. Here, the crystal structure of Mr HR, containing a unique "TSD" chloride ion conduction motif, was determined as a homotrimer at a resolution of 1.9 Å. The detailed structure of Mr HR revealed a unique trimeric topology of the light-driven chloride pump, with peculiar coordination of two water molecules and hydrogen-mediated bonds near the TSD motif, as well as a short B–C loop. Structural and functional analyses of Mr HR revealed key residues responsible for the anion selectivity of cyanobacterial halorhodopsin and the involvement of two chloride ion-binding sites in the ion conduction pathway. Alanine mutant of Asn63, Pro118, and Glu182 locating in the anion inlet induce multifunctional uptake of chloride, nitrate, and sulfate ions. Moreover, the structure of N63A/P118A provides information on how Sy HR promotes divalent ion transport. Our findings significantly advance the structural understanding of microbial rhodopsins with different motifs. They also provide insight into the general structural framework underlying the molecular mechanisms of the cyanobacterial chloride pump containing Sy HR, the only molecule known to transport both sulfate and chloride ions. Unlabelled Image • The structure of the cyanobacterial chloride pump containing a unique "TSD" motif was solved at 1.9 Å resolution. • The homotrimeric topology and key ion-conducting residues of MrHR differ with general halorhodopsins. • Mutations in the chloride inlet induce multifunctional uptake of Cl−, NO 3 −, SO 4 2− ions. • The mutant structure of chloride inlet provides structural evidence on different anion accessibilities. [ABSTRACT FROM AUTHOR]