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Crystal structure of Escherichia coli-expressed Haloarcula marismortui bacteriorhodopsin I in the trimeric form.

Shevchenko V, Gushchin I, Polovinkin V, Round E, Borshchevskiy V, Utrobin P, Popov A, Balandin T, Büldt G, Gordeliy V - PLoS ONE (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Complex Systems (ICS-6) Structural Biochemistry, Research Centre Jülich GmbH, Jülich, Germany; Laboratory for advanced studies of membrane proteins, Moscow institute of physics and technology, Dolgoprudniy, Russia.

ABSTRACT
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 Å. 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.

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Comparison of the ordered lipidic tails observed in HmBRI structure with those observed in the structures of trimeric HsBR [23], [34]–[36], ar-2 [29] and dr-3 [13].The HmBRI surface is shown in green, parts of the adjacent protomers are in beige, HmBRI lipids are in magenta, the other lipids are in yellow and bacterioruberin molecules observed in aR-2 and dR-3 structures are in orange. (A) Lipids inside the trimer. (B) Lipids close to the helices A, B and G. (C) Lipids close to the helices E and F. To obtain the positions of the lipidic moieties observed in the structures of the other proteins, their trimeric assemblies were aligned first to HmBRI trimer.
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pone-0112873-g008: Comparison of the ordered lipidic tails observed in HmBRI structure with those observed in the structures of trimeric HsBR [23], [34]–[36], ar-2 [29] and dr-3 [13].The HmBRI surface is shown in green, parts of the adjacent protomers are in beige, HmBRI lipids are in magenta, the other lipids are in yellow and bacterioruberin molecules observed in aR-2 and dR-3 structures are in orange. (A) Lipids inside the trimer. (B) Lipids close to the helices A, B and G. (C) Lipids close to the helices E and F. To obtain the positions of the lipidic moieties observed in the structures of the other proteins, their trimeric assemblies were aligned first to HmBRI trimer.

Mentions: Interestingly, all the retinylidene proteins, for which the trimeric assembly was observed in crystal, were purified from natural or natural-like sources [12], [13], [23], [34]–[38]. The HmBRI crystals, presented here, were grown with the protein expressed in Escherichia coli, a bacterium, although the protein itself originates from the archaeon Haloarcula marismortui. The ordered lipid tails, belonging either to the E. coli lipids or the in meso crystallization matrix lipid monooleoyl, are observed at the same positions as the native lipids in other structures (Figure 8). First, there are three paired hydrophobic tails at the extracellular side of the intra-trimer cavity of HmBRI (Figure 8A), where binding of highly specific sulfated triglycoside lipid S-TGA-1 is observed in HsBR crystals [34]–[36]. Second, there are ordered hydrophobic tails on the outer surface of the trimer (Figures 8B and 8C), whose position is also very similar to that in the crystals of HsBR and other proteins. It is remarkable that the lipid binding mode is conserved not only across the different proteins of the family but even across the different kinds of the hydrophobic tail moieties of the lipid molecules (branched isoprenoid chains in archaea and straight fatty acid chains in bacteria). Thus, stronger ability to assemble into a trimer and bind the host lipids might be another reason for the efficient expression of HmBRI in E. coli cells.


Crystal structure of Escherichia coli-expressed Haloarcula marismortui bacteriorhodopsin I in the trimeric form.

Shevchenko V, Gushchin I, Polovinkin V, Round E, Borshchevskiy V, Utrobin P, Popov A, Balandin T, Büldt G, Gordeliy V - PLoS ONE (2014)

Comparison of the ordered lipidic tails observed in HmBRI structure with those observed in the structures of trimeric HsBR [23], [34]–[36], ar-2 [29] and dr-3 [13].The HmBRI surface is shown in green, parts of the adjacent protomers are in beige, HmBRI lipids are in magenta, the other lipids are in yellow and bacterioruberin molecules observed in aR-2 and dR-3 structures are in orange. (A) Lipids inside the trimer. (B) Lipids close to the helices A, B and G. (C) Lipids close to the helices E and F. To obtain the positions of the lipidic moieties observed in the structures of the other proteins, their trimeric assemblies were aligned first to HmBRI trimer.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4257550&req=5

pone-0112873-g008: Comparison of the ordered lipidic tails observed in HmBRI structure with those observed in the structures of trimeric HsBR [23], [34]–[36], ar-2 [29] and dr-3 [13].The HmBRI surface is shown in green, parts of the adjacent protomers are in beige, HmBRI lipids are in magenta, the other lipids are in yellow and bacterioruberin molecules observed in aR-2 and dR-3 structures are in orange. (A) Lipids inside the trimer. (B) Lipids close to the helices A, B and G. (C) Lipids close to the helices E and F. To obtain the positions of the lipidic moieties observed in the structures of the other proteins, their trimeric assemblies were aligned first to HmBRI trimer.
Mentions: Interestingly, all the retinylidene proteins, for which the trimeric assembly was observed in crystal, were purified from natural or natural-like sources [12], [13], [23], [34]–[38]. The HmBRI crystals, presented here, were grown with the protein expressed in Escherichia coli, a bacterium, although the protein itself originates from the archaeon Haloarcula marismortui. The ordered lipid tails, belonging either to the E. coli lipids or the in meso crystallization matrix lipid monooleoyl, are observed at the same positions as the native lipids in other structures (Figure 8). First, there are three paired hydrophobic tails at the extracellular side of the intra-trimer cavity of HmBRI (Figure 8A), where binding of highly specific sulfated triglycoside lipid S-TGA-1 is observed in HsBR crystals [34]–[36]. Second, there are ordered hydrophobic tails on the outer surface of the trimer (Figures 8B and 8C), whose position is also very similar to that in the crystals of HsBR and other proteins. It is remarkable that the lipid binding mode is conserved not only across the different proteins of the family but even across the different kinds of the hydrophobic tail moieties of the lipid molecules (branched isoprenoid chains in archaea and straight fatty acid chains in bacteria). Thus, stronger ability to assemble into a trimer and bind the host lipids might be another reason for the efficient expression of HmBRI in E. coli cells.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Complex Systems (ICS-6) Structural Biochemistry, Research Centre Jülich GmbH, Jülich, Germany; Laboratory for advanced studies of membrane proteins, Moscow institute of physics and technology, Dolgoprudniy, Russia.

ABSTRACT
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 Å. 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.

Show MeSH
Related in: MedlinePlus