Structural insights into light-driven anion pumping in cyanobacteria

Abstract

Transmembrane ion transport is a key process in living cells. Active transport of ions is carried out by various ion transporters including microbial rhodopsins (MRs). MRs perform diverse functions such as active and passive ion transport, photo-sensing, and others. In particular, MRs can pump various monovalent ions like Na+, K+, Cl−, I−, NO3−. The only characterized MR proposed to pump sulfate in addition to halides belongs to the cyanobacterium Synechocystis sp. PCC 7509 and is named Synechocystis halorhodopsin (SyHR). The structural study of SyHR may help to understand what makes an MR pump divalent ions. Here we present the crystal structure of SyHR in the ground state, the structure of its sulfate-bound form as well as two photoreaction intermediates, the K and O states. These data reveal the molecular origin of the unique properties of the protein (exceptionally strong chloride binding and proposed pumping of divalent anions) and sheds light on the mechanism of anion release and uptake in cyanobacterial halorhodopsins. The unique properties of SyHR highlight its potential as an optogenetics tool and may help engineer different types of anion pumps with applications in optogenetics.

Light-Driven Chloride and Sulfate Pump with Two Different Transport Modes

“…This is generally the case for all anion pumps if examined by pH electrodes, including SyHR. 19 To verify the active nature of the inward anion transport, voltage and current across the membrane should be controlled. 20 For this purpose, we expressed AaClR in ND7/23 cells and performed electrophysiological measurements.…”

“…18 In contrast, the crystal structure of SyHR in the presence of sulfate ions did not visualize such sulfate ions near the Schiff base and thus concluded no binding of sulfate as the Schiff base counterion. 19 Here, we measured the absorption spectra of AaClR under various salt conditions (NaCl and Na 2 SO 4 ). C-terminal Histagged AaClR was overexpressed in E. coli (C43(DE3) strain), solubilized by DDM (2%), purified by HisTrap Co 2+ -affinity column chromatography, and after dialysis, purified protein was reconstituted into lipids (POPE:POPG; 3:1) at a 1:20 ratio.…”

“…Sulfate transport by SyHR may follow the general principle with the initial binding. 18 Or even without initial binding, 19 similar mechanism of the sulfate pump by AaClR may work for SyHR. AaClR and SyHR share 26% amino acid identity, which is lower than those between AaClR and FR (31%) and between AaClR and NMR-3 (38%).…”

“…Therefore, substrate binding to the resting state follows the canonical scheme of active transport. In fact, recent crystal structure of the chloride-bound form of SyHR observed a chloride ion near the retinal Schiff base . In remarkable contrast, crystal structure of the sulfate-bound form of SyHR monitored a sulfate ion at the cytoplasmic surface but not at the Schiff base region.…”

“…This property of AaClR is similar to that of KR2 5 and consistent with the crystal structure of SyHR. 19 Photocycle dynamics of AaClR resemble those of a non-functional cycle in the absence of anions. Nevertheless, flash photolysis and difference FTIR spectroscopy provide an insight into the sulfate ion transport in AaClR.…”