PDBsum entry 1fbb

Proton transport

PDB id: 1fbb

Contents

Protein chain

224 a.a. *

Ligands

RET

* Residue conservation analysis

PDB id:

1fbb

Name:

Proton transport

Title:

Crystal structure of native conformation of bacteriorhodopsin

Structure:

Bacteriorhodopsin. Chain: a

Source:

Halobacterium salinarum. Organism_taxid: 2242

Biol. unit:

Trimer (from PDB file)

Authors:

S.Subramaniam,R.Henderson

Key ref:

S.Subramaniam and R.Henderson (2000). Molecular mechanism of vectorial proton translocation by bacteriorhodopsin. Nature, 406, 653-657. PubMed id: 10949309 DOI: 10.1038/35020614

Date:

15-Jul-00 Release date: 09-Aug-00

Protein chain

P02945  (BACR_HALSA) -  Bacteriorhodopsin from Halobacterium salinarum (strain ATCC 700922 / JCM 11081 / NRC-1)

Seq: 262 a.a.

Struc: 224 a.a.

DOI no: 10.1038/35020614

Nature 406:653-657 (2000)

PubMed id: 10949309

Molecular mechanism of vectorial proton translocation by bacteriorhodopsin.

S.Subramaniam, R.Henderson.

ABSTRACT

Bacteriorhodopsin, a membrane protein with a relative molecular mass of 27,000, is a light driven pump which transports protons across the cell membrane of the halophilic organism Halobacterium salinarum. The chromophore retinal is covalently attached to the protein via a protonated Schiff base. Upon illumination, retinal is isomerized. The Schiff base then releases a proton to the extracellular medium, and is subsequently reprotonated from the cytoplasm. An atomic model for bacteriorhodopsin was first determined by Henderson et al, and has been confirmed and extended by work in a number of laboratories in the last few years. Here we present an atomic model for structural changes involved in the vectorial, light-driven transport of protons by bacteriorhodopsin. A 'switch' mechanism ensures the vectorial nature of pumping. First, retinal unbends, triggered by loss of the Schiff base proton, and second, a protein conformational change occurs. This conformational change, which we have determined by electron crystallography at atomic (3.2 A in-plane and 3.6 A vertical) resolution, is largely localized to helices F and G, and provides an 'opening' of the protein to protons on the cytoplasmic side of the membrane.

Selected figure(s)

Figure 1.
Figure 1: Three-dimensional difference density map showing structural changes in the D96G, F171C, F219L triple mutant compared with the unilluminated native wild-type bacteriorhodopsin. The view is from the cytoplasmic side along an axis perpendicular to the plane of the membrane. Diffraction patterns from two-dimensional crystals of the D96G, F171C, F219L triple mutant^3 (provided by J. Tittor and D. Oesterhelt) embedded in glucose or trehalose were recorded on a Philips CM-12 electron microscope at a specimen temperature of -180 °C using a CCD camera system^25. About 1,000 diffraction patterns were included in the initial set, from which 402 patterns were chosen by visual inspection, and merged together (merging R-factor of 17.3%) to generate a set of lattice lines covering 87% of (three-dimensional) reciprocal space, with a resolution of 3.2 Å in-plane and 3.6 Å vertically. Diffraction intensities from the merged data set were scaled to those of native bacteriorhodopsin. Three-dimensional difference maps ( F[triple] - F[native]) [native] were calculated using experimental amplitudes for the triple mutant (this work) and previously measured amplitudes and phases for wild-type bacteriorhodopsin^ 26. The difference densities are contoured at 3 . Yellow, positive densities; purple, negative densities. a, Section close to cytoplasmic boundary where the largest structural differences are observed, with prominent features in the vicinity of helices F and G. The difference densities near helix F indicate an outward displacement of helix F in the triple mutant. b, Section close to extracellular boundary. The main change in this region is a positive feature localized to the vicinity of Arg 82. The differences here are considerably smaller than in the section in a.

Figure 2.
Figure 2: Comparison of atomic models for wild-type bacteriorhodopsin and the D96G, F171C, F219L triple mutant. a,b, Sections of [A] weighted^27 2F[o]-F[c] density maps for wild-type bacteriorhodopsin (purple) and the D96G, F171C, F219L triple mutant (yellow) near the cytoplasmic (a) and extracellular (b) boundaries. The maps are superimposed on the structure of wild-type bacteriorhodopsin. Significant rearrangements of helices F and G occur in the cytoplasmic, but not in the extracellular region. c, Superposition of C atoms of wild-type bacteriorhodopsin (purple) and the D96G, F171C, F219L triple mutant (yellow) illustrating differences in the cytoplasmic portions of helices F and G. Initially, a minimal starting model containing only the transmembrane regions of bacteriorhodopsin was used in a simplified least squares refinement. Coordinates from six different starting models (PDB entries 2brd, 1brr, 1ap9, 1brx, 1at9 and 2at9) were tested using diffraction data sets obtained both from wild-type bacteriorhodopsin and the triple mutant. The 1brr coordinates^28 were used as a starting model for the next stage of refinement using the crystallography and NMR system (CNS)^27 suite of programs, involving simulated annealing followed by temperature factor refinement. The wild-type structure was refined to a final R-factor of 23.9% (R[free] 31%), and the triple mutant structure was refined to a final R-factor of 27.2% (R[ free] 32.1%). The final map was tested by completely omitting from the starting model the side chains from a series of test residues such as Phe 42, Trp 86, Trp 189 and Phe 208, or various combinations of residues at the cytoplasmic ends of helix F or helix G. In each case, difference maps (F[o]–F[c]) obtained at the end of the refinement were unambiguous and clear density peaks were observed for each of the omitted regions in complete agreement with the atomic models reported here. As the diffraction data from the triple mutant and wild-type bacteriorhodopsin are completely independent of the 1brr starting coordinates, these omit maps constitute stringent and objective tests of the entire refinement procedure.

The above figures are reprinted by permission from Macmillan Publishers Ltd: Nature (2000, 406, 653-657) copyright 2000.

Figures were selected by the author.