Crystal Structure of Escherichia coli-Expressed Haloarcula marismortui Bacteriorhodopsin I in the Trimeric Form

December Crystal Structure of Escherichia coli- Expressed Haloarcula marismortui Bacteriorhodopsin I in the Trimeric Form Vitaly Shevchenko 0 1 6 7 Ivan Gushchin 1 2 3 4 6 7 Vitaly Polovinkin 1 2 3 4 6 7 Ekaterina 6 7 Round 0 6 7 Valentin Borshchevskiy 0 1 6 7 Petr Utrobin 1 2 3 6 7 Alexander Popov 5 6 7 Taras 6 7 Balandin 0 6 7 Georg B uldt 1 6 7 Valentin Gordeliy 0 1 2 3 4 6 7 0 Institute of Complex Systems (ICS-6) Structural Biochemistry, Research Centre Ju lich GmbH , Ju lich, Germany, 1 Laboratory for advanced studies of membrane proteins, Moscow institute of physics and technology , Dolgoprudniy , Russia, 2 Univ. Grenoble Alpes, IBS , Grenoble , France, 3 CNRS, IBS , Grenoble , France, 4 CEA, IBS , Grenoble , France, 5 European Synchrotron Radiation Facility , Grenoble , France 6 Funding: The work was supported by the program "Chaires d'excellence" edition 2008 of ANR France and CEA(IBS) - HGF(FZJ) STC 5.1 specific agreement. Part of this work was supported by BMBF (PhoNa - Photonic Nanomaterials). The work was supported by RFBR (research projects 13-04-91320 and 12-04-31290) and the Russian state Program for enhancing the competitiveness of MIPT among the world's leading research and education centers of the Ministry of education and science. The work used the platforms of the Grenoble Instruct centre (ISBG; UMS 3518 CNRS- CEA-UJF-EMBL) with support from FRISBI (ANR- 10-INSB-05-02) and GRAL (ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology (PSB). VP is deeply thankful to Fondation Nanosciences for financial support. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript 7 Editor: Oleg Y. Dmitriev, University of Saskatchewan , Canada Bacteriorhodopsins are a large family of seven-helical transmembrane proteins that function as light-driven proton pumps. Here, we present the crystal structure of a new member of the family, Haloarcula marismortui bacteriorhodopsin I (HmBRI) D94N mutant, at the resolution of 2.5 A. While the HmBRI retinal-binding pocket and proton donor site are similar to those of other archaeal proton pumps, its proton release region is extended and contains additional water molecules. The protein's fold is reinforced by three novel inter-helical hydrogen bonds, two of which result from double substitutions relative to Halobacterium salinarum bacteriorhodopsin and other similar proteins. Despite the expression in Escherichia coli and consequent absence of native lipids, the protein assembles as a trimer in crystals. The unique extended loop between the helices D and E of HmBRI makes contacts with the adjacent protomer and appears to stabilize the interface. Many lipidic hydrophobic tail groups are discernible in the membrane region, and their positions are similar to those of archaeal isoprenoid lipids in the crystals of other proton pumps, isolated from native or native-like sources. All these features might explain the HmBRI properties and establish the protein as a novel model for the microbial rhodopsin proton pumping studies. - Microbial rhodopsins are a large family of seven-helical transmembrane proteins that contain the covalently attached cofactor retinal [1]. Upon absorption of a photon, the retinal isomerizes and starts a series of structural transformations, correlated with spectral changes and called photocycle [1, 2]. Among the members of the family are light-driven proton, anion or cation pumps, light-gated ion channels and photoreceptors [1]. The most studied class of microbial rhodopsins are the proton pumps. It has been established that there are three distinct regions involved in the proton translocation: the proton donor site, the retinal binding pocket, and finally, the proton release region. In Halobacterium salinarum bacteriorhodopsin (HsBR), identified in 1971 by Oesterhelt and Stoeckenius [3], the proton translocation cycle starts with isomerization of the retinal and transfer of the proton from the retinal Schiff base to the side-chain of the proton acceptor D85 [2]. In the next stage, the proton is transferred to the proton release group, consisting of two closely situated glutamates E194 and E204 and several water molecules. Then, the Schiff base is reprotonated from the proton donor D96, which itself is reprotonated from the cytoplasm. Although the general details of this proton pumping mechanism are well known, there are some discrepancies between the published structures of the intermediate states [4, 5], some of which might be explained by high radiation susceptibility of bacteriorhodopsin [6] or twinning of crystals [7]. Consequently, different details of the bacteriorhodopsin proton pumping still continue to be investigated, related to both the proton release [8] and the proton uptake [9]. In the recent years, structures of many microbial rhodopsins have been determined. Structures of four different archaeal proton pumps are known, HsBR [10, 11], archaerhodopsin-1 and -2 (ar-1 and ar-2) [12], and deltarhodopsin-3 (dr-3) [13], as well as the structures of bacterial pumps xanthorhodopsin [14], various proteorhodopsins [15, 16] and eukaryotic Acetabularia rhodopsin [17] and Chlamydomonas reinhardtii channelrhodopsin [18]. Here, we present the structure of bacteriorhodopsin I from Haloarcula marismortui (henceforth HmBRI). HmBRI is one of the six Haloarcula marismortui retinylidene proteins and one of its two bacteriorhodopsins, whose sequences are 50% identical [19]. The protein can be expressed in Escherichia coli in large quantities and in fact can be used as a tag for production of other membrane proteins [20]. The structure reveals the conserved proton donor and retinal-binding pocket sites, and expanded proton release region. There are three additional inter-helical hydrogen bonds in the HmBRI transmembrane region. Despite heterologous expression, the protein assembles as a trimer in crystals. The loop between the helices D and E of HmBRI is extended, makes contacts with the adjacent protomer and appears to stabilize the interface. These features might result in a higher stability of HmBRI and explain its high expression level in E. coli cells. E. coli production allows for easy genetic manipulation and rapid production of mutants, which will be helpful for studies of the proton transport mechanism as well as for protein engineering for optogenetics needs [1, 21]. Results and Discussion HmBRI expression and spectroscopic characterization HmBRI was heterologously expressed in E. coli and purified by nickel-affinity and size-exclusion (SEC) chromatography. The SEC elution profile revealed that the protein exists in two forms in the detergent solution (Figure 1A), presumably monomeric and oligomeric (most probably trimeric). Both forms behave identically on the sodium dodecyl sulfate polyacrylamide electrophoresis. Although the approximate molecular weights of the two species can be calculated as 130 kDa (the li (...truncated)