Structure of an Inward Proton-Transporting Anabaena Sensory Rhodopsin Mutant: Mechanistic Insights

Abstract

Microbial rhodopsins are light-activated, seven-a-helical, retinylidene transmembrane proteins that have been identified in thousands of organisms across archaea, bacteria, fungi, and algae. Although they share a high degree of sequence identity and thus similarity in structure, many unique functions have been discovered and characterized among them. Some function as outward proton pumps, some as inward chloride pumps, whereas others function as light sensors or ion channels. Unique among the microbial rhodopsins characterized thus far, Anabaena sensory rhodopsin (ASR) is a photochromic sensor that interacts with a soluble 14-kDa cytoplasmic transducer that is encoded on the same operon. The sensor itself stably interconverts between all-trans-15-anti and 13-cis-15-syn retinal forms depending on the wavelength of illumination, although only the former participates in a photocycle with a signaling M intermediate. A mutation in the cytoplasmic half-channel of the protein, replacing Asp217 with Glu (D217E), results in the creation of a light-driven, single-photon, inward proton transporter. We present the 2.3 Å structure of dark-adapted D217E ASR, which reveals significant changes in the water network surrounding Glu217, as well as a shift in the carbon backbone near retinal-binding Lys210, illustrating a possible pathway leading to the protonation of Glu217 in the cytoplasmic half-channel, located 15 Å from the Schiff base. Crystallographic evidence for the protonation of nearby Glu36 is also discussed, which was described previously by Fourier transform infrared spectroscopy analysis. Finally, two histidine residues near the extracellular surface and their possible role in proton uptake are discussed.

Oligomeric states of microbial rhodopsins determined by high-speed atomic force microscopy and circular dichroic spectroscopy

“…1 ). X-ray crystal analysis of ASR suggested a dimeric oligomer 12 , 13 , while CD spectroscopy, SSNMR spectroscopy, and EPR spectroscopy suggested trimers in lipids 15 . In addition, a double electron-electron resonance EPR study using site-directed spin labeling of two ASR cysteine mutants confirmed the trimeric nature of ASR in lipids 16 .…”

“…For example, crystal structures of sodium pump rhodopsin, Krokinobacter eikastus rhodopsin 2 (KR2) 8 , exhibited different oligomer forms depending on the pH conditions: monomers at pH 4.0 and 4.3, and pentamers at pH 4.9 and 5.6 9 , 10 . X-ray crystallography resolved Anabaena sensory rhodopsin (ASR) 11 as a dimer 12 , 13 , whereas CD spectroscopy, solid-state nuclear magnetic resonance (SSNMR) spectroscopy and electron paramagnetic resonance (EPR) spectroscopy suggested trimers in lipids 14 – 16 . The situation for proteorhodopsin (PR) is more complex.…”

Protonation–state‐Coupled Conformational Dynamics in Reaction Mechanisms of Channel and Pump Rhodopsins

“…Similar principles may be at work in other rhodopsins. Indeed, recent crystal structures of microbial rhodopsins such as C1C2 , Anabaena sensory rhodopsin or the sodium pump KR2 indicate the presence of charged and polar groups that may contribute to the stability of zwitterionic states (Figs and ). In the case of bovine rhodopsin, a threonine group important for the protonation state of the retinal and its counterion (T94, see ref.…”

“…An intriguing feature of some of these proteins is that mutation of specific protein groups can change the functioning of the protein. For example, bacteriorhodopsin pumps chloride ions when D85 is mutated to Thr , or in certain acidic conditions , Natronobacterium pharaonis halorhodopsin can pump protons when azide is present , the E90K/T159C mutant of C. reinhardtii channelrhodopsin‐2 is a chloride channel and replacement of a single Asp group into Glu suffices for Anabaena sensory rhodopsin to become an inward proton pump . A common feature of these functional inter‐conversions among microbial rhodopsins is that mutations target sidechains that can hydrogen bond.…”

Mechanism of Inward Proton Transport in an Antarctic Microbial Rhodopsin

“…Inversion of proton transport vectoriality has been observed for some outward H + pumps, such as bacteriorhodopsin (BR), green-absorbing proteorhodopsin (GPR), and Gloeobacter rhodopsin (GR), and was hypothesized to originate either from combination of mutations and double-photon processes, or from channel activities under strong proton gradients, among other factors. − More recently, new groups of microbial rhodopsins have emerged, which transport protons exclusively in the cytoplasmic direction, but the mechanism and biological relevance of inward proton pumping remain unclear. Until now, only one group of inward pumping microbial rhodopsins, named xenorhodopsins (XeRs), has been described; the first discovered member of this group, Anabaena sensory rhodopsin (ASR), functions as a sensor and lacks transport activity, but mutating its cytoplasmic proton acceptor causes robust inward proton transport. , Other XeRs, from Parvularcula oceani (PoXeR), Nanosalina (NsXeR) and its two other nanohaloarchaeal homologues, and Rubricoccus marinus (RmXeR), showed inward proton transport by their wild-types (WTs). − …”