Figure 1.
HmBRI size-exclusion chromatography (SEC) elution profile and visual light absorbance spectra in different environments.
(A) SEC elution profile of HmBRI. Two distinct peaks are observed, that correspond supposedly to monomeric HmBRI (at 73.4 ml, peak ratio is ∼1.95) and oligomeric HmBRI (at 67.8 ml, peak ratio is ∼1.85). Five 2 ml fractions (1–5) were collected for subsequent crystallization. (B) Spectra of the light-adapted HmBRI in the hexagonal crystals, the first and the fifth SEC fractions.
Table 1.
HmBRI absorption maxima in different environments (nm).
Figure 2.
HmBRI crystals and diffraction.
(A) HmBRI crystals in the crystallization well. Two types of crystals are observed: regular shaped hexagonal crystals and bunches of needles. (B) Example of the diffraction from the hexagonal crystals. Resolution at the detector edge is 2.4 Å. (C) Example of the diffraction from needle-shaped crystals. Resolution at the detector edge is 2.5 Å. The diffraction patterns are smeared and observed up to the resolution of 3.5 Å.
Table 2.
Crystallographic data collection and refinement statistics.
Figure 3.
HmBRI packing in hexagonal crystals.
(A) Packing in the membrane plane. There are two trimers in the unit cell (shown in yellow and green) that are oriented in opposite directions. (B) Packing of the membrane-like layers. (C) Crystal contacts between the layers. The contacts rely on the ionic interactions between the SO42− ions and the R172 and K176 side-chains, and on the hydrogen bond between the N166 side-chain of one protomer and the G71 backbone oxygen of another. A second putative SO42− ion is observed close to the first one that interacts with K176 and R179 side-chains. Potential interactions are marked by dashed lines. Fo-Fc difference electron densities before the insertion of the ions into the model are shown at the level of 3 σ. The symbols ', ” and ’” denote different crystallographic symmetry-related HmBRI molecules. The black oval denotes the crystallographic symmetry rotation axis (C2).
Figure 4.
Crystallographic structure of the HmBRI D94N mutant.
(A) Comparison of the HmBRI backbone structure (green) with that of HsBR [23] (yellow). (B) 2Fo-Fc electron density maps in the vicinity of the retinal. The maps are contoured at the level of 1.5 σ. (C) 2Fo-Fc electron density maps in the proton release region. The maps are contoured at the level of 1.2 σ. (D) Comparison of the HmBRI proton release group (green) with that of HsBR [23] (yellow). Overall, the conformations of the side-chains and positions of water molecules are very similar. However, the water accessible space is larger in HmBRI, and additional water molecules are observed (WHm). One of the reasons for this difference might be the substitution of HsBR’ proline 200 with the glycine 204 in HmBRI, that allows unlatching of the extracellular part of the helix G. (E) Comparison of the HmBRI proton donor region with that of wild-type HsBR [23] (green) and its D96N [30] (orange) and P50A [32] (magenta) mutants. It appears that in HmBRI the effects of the D94N mutation and the P50Hs → T48Hm substitution combine and result in a larger displacement of the helix B relative to the helices C and G (black arrows), as similar displacements are present in the P50A and D96N mutants of HsBR, albeit with a smaller amplitude.
Figure 5.
Phylogenetic tree and sequence alignment of HsBR, ar-1, ar-2, dr-3 and HmBRI.
The unique HmBRI D-E loop elongation, the residues that result in novel inter-helical hydrogen bonds, and the P50Hs/T48Hm position are highlighted. The propeptide residues that are cleaved in the mature protein in vivo are shown in grey. The asterisk indicates the fully conserved residues, the colon and period indicate conservation between groups of strongly similar properties scoring> 0.5 and < = 0.5 correspondingly in the Gonnet PAM 250 matrix [49].
Figure 6.
The novel inter-helical hydrogen bonds in HmBRI (green) relative to HsBR (yellow).
In each case, the structure alignment was done locally to emphasize the local effects. (A) The substitution A126Hs → T124Hm results in two novel hydrogen bonds connecting the helix D to helices C and E. (B) The coupled substitution M56Hs → N54Hm, A84Hs → T82Hm results in introduction of the hydrogen bond between the helices B and C. Interestingly, the intra-helical hydrogen bond between the I52 backbone oxygen and T55 is replaced with the hydrogen bond between the homologous I50 backbone oxygen and N54 side-chain amine. (C) Coupled substitution V188Hs → W192Hm, L207Hs → G211Hm results in introduction of the hydrogen bond between the helices F and G (E208 backbone oxygen and W192 indole nitrogen). Interestingly, the glycine is the only possible amino acid at the position 211, as the Cβ atom of any other amino acid would create a steric conflict with W192 side-chain. The hypothetical position of the residue 211 Cβ atom is shown by the green sphere, and its van der Waals radius, as well as those of proximal W192 heavy atoms, is shown as a black circle. Introduction of the bulky tryptophan at the position 192 might be another reason for the divergence of the helices F and G in HmBRI, and, as a consequence, the bigger water-accessible volume of the proton release region.
Figure 7.
Structure of the HmBRI trimer and its D-E loop.
(A) Comparison of the HmBRI trimer structure (green) with that of HsBR [23] (yellow). HmBRI trimer aligns well in the extracellular region, but the protomers are slightly rotated at the cytoplasmic side. (B) Magnification of the D-E loop. Unlike in other trimerizing retinylidene proteins, in HmBRI the loop is extended and makes contact to the adjacent protomer. (C) Wall-eyed stereogram of the HmBRI D-E loop. The adjacent protomer is shown in orange and its residues are marked by a prime. Three structural water molecules are observed that stabilize the loop structure.
Figure 8.
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.