High Resolution Structural Insights into Heliorhodopsin Family

Abstract

Rhodopsins are the most abundant light-harvesting proteins. A new family of rhodopsins, heliorhodopsins (HeRs), was recently discovered. In opposite to the known rhodopsins their N-termini face the cytoplasm. HeRs structure and function remain unknown. We present structures of two HeR-48C12 states at 1.5 Å showing its remarkable difference from all known rhodopsins. Its internal extracellular part is completely hydrophobic, while the cytoplasmic part comprises a cavity ('active site'), surrounded by charged amino acids and containing a cluster of water molecules, presumably being a primary proton acceptor from the Schiff base. At acidic pH a planar triangle molecule (acetate) is present in the 'active site' which demonstrated its ability to maintain such anions as carbonate or nitrate. Structure-based bioinformatic analysis identified 10 subfamilies of HeRs suggesting their diverse biological functions. The structures and available data suggest an enzymatic activity of HeR-48C12 subfamily and their possible involvement into fundamental redox biological processes.

High-resolution structural insights into the heliorhodopsin family

“…While the biological function of HeRs remains unknown, thorough study of different 48C12 states is of great potential benefit for elucidating it. To investigate A B the conformational rearrangements in the HeR associated with pH decrease, we also solved the crystal structure of 48C12 at 1.5 Å using the crystals grown at pH 4.3 (36). Indeed, pH of the surrounding solution affects the functionality and the structure of microbial rhodopsins due to protonation or deprotonation of the key residues (37-39).…”

Characterization of heliorhodopsins detected via functional metagenomics in freshwater Actinobacteria, Chloroflexi and Archaea

“…S5). HeRs from the monophyletic group A (subfamily 1 in (Kovalev et al 2020)) are represented mostly by proteins from Actinobacteria, including HeR-48C12 and several of the clones reported here, while group B is more diverse and includes Bc HeR (Chloroflexi) and…”

“…Notice however that reminiscently of the DegV proteins, the crystal structures of HeRs revealed that they bind such lipids from the membranes used for crystallization as the FA monooleate and the alkane eicosane and that the corresponding binding site is localized to the fenestration that exposes the β-ionone ring of the retinal moiety (Shihoya et al 2019, Kovalev et al 2020. Taken together, these observations are suggestive of HeRs participating in FA modification in a light-dependent manner.…”

“…Sequences for the phylogenetic analysis of HeRs were collected from genomic and metagenomic assemblies (see below) and Uniprot. Initial experiments confirmed the disproportionately high rates of evolution in the putative superclade of viral and eukaryotic HeRs (Kovalev et al 2020), and they were removed from the analysis, focusing thus on the prokaryotic HeRs only. Sequences were clustered in two steps:…”

Heliorhodopsin evolution is driven by photosensory promiscuity in monoderms

“…Identified during a functional metagenomics screen and characterised by low sequence similarity when compared to type-1 rhodopsins, HeRs attracted increasing research interest due to their peculiar membrane orientation (i.e. N-terminus in the cytoplasm and the C-terminus in the extracellular space) (Pushkarev et al 2018), unusual protein structure (Kovalev et al 2020) and controversial taxonomic distribution (Flores-Uribe, Hevroni, and Ghai 2019). While electrophysiological (Pushkarev et al 2018), physicochemical (Tanaka et al 2020) and structural (Shihoya et al 2019;Kovalev et al 2020) studies achieved great progress in elucidating a series of characteristics ranging from photocycle length (indicating no pumping activity) to detailed protein organization, they provide no data regarding the biological function of HeRs.…”