Research ArticleStructural Biology

Design of a light-gated proton channel based on the crystal structure of Coccomyxa rhodopsin

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Sci. Signal.  19 Mar 2019:
Vol. 12, Issue 573, eaav4203
DOI: 10.1126/scisignal.aav4203

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The light between pumps and channels

Whereas pumps move ions actively, channels move ions passively. Fudim et al. generated a high-resolution crystal structure for Coccomyxa subellipsoidea rhodopsin, a light-activated proton pump. These data enabled the authors to identify a critical interaction between Arg83 and Tyr14 in a transmembrane domain and generate a point mutant of this rhodopsin that behaved as a light-gated proton channel. These results provide greater insight into the molecular determinants that distinguish proton pumps from channels.

Abstract

The light-driven proton pump Coccomyxa subellipsoidea rhodopsin (CsR) provides—because of its high expression in heterologous host cells—an opportunity to study active proton transport under controlled electrochemical conditions. In this study, solving crystal structure of CsR at 2.0-Å resolution enabled us to identify distinct features of the membrane protein that determine ion transport directivity and voltage sensitivity. A specific hydrogen bond between the highly conserved Arg83 and the nearby nonconserved tyrosine (Tyr14) guided our structure-based transformation of CsR into an operational light-gated proton channel (CySeR) that could potentially be used in optogenetic assays. Time-resolved electrophysiological and spectroscopic measurements distinguished pump currents from channel currents in a single protein and emphasized the necessity of Arg83 mobility in CsR as a dynamic extracellular barrier to prevent passive conductance. Our findings reveal that molecular constraints that distinguish pump from channel currents are structurally more confined than was generally expected. This knowledge might enable the structure-based design of novel optogenetic tools, which derive from microbial pumps and are therefore ion specific.

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