Crystal Structure of the Bromide-Bound D85S Mutant of Bacteriorhodopsin: Principles of Ion Pumping

Abstract

We report the crystal structure of a bromide-bound form of the D85S mutant of bacteriorhodopsin, bR(D85S), a protein that uses light energy rather than ATP to pump halide ions across the cell membrane. Comparison of the structure of the halide-bound and halide-free states reveals that both displacements of individual side-chain positions and concerted helical movements occur on the extracellular side of the protein. Analysis of these structural changes reveals how this ion pump first facilitates ion uptake deep within the cell membrane and then prevents the backward escape of ions later in the pumping cycle. Together with the information provided by structures of intermediate states in the bacteriorhodopsin photocycle, this study also suggests the overall design principles that are necessary for ion pumping.

Specificity of Anion Binding in the Substrate Pocket of Bacteriorhodopsin

“…This proposal is supported by the experimental observation that the D85S/D212N mutant seems to have two distinguishable binding events that occur at low pH. A second binding site for anions on the cytoplasmic face of the protein has already been observed in the structure of bR(D85S) (8). The reason why the D85S/D212N double mutant seems to be spectroscopically sensitive to the binding of anions at a second site, while the D85S single mutant is not, is not clear from the available structural data.…”

“…Crystals were released from the continuous bilayer gel formed by mono-olein by lipase treatment of aliquots of gel (8,13). All of the liberated crystals, initially grown in the presence 0.2M KCl, 100mM…”

“…Since part of the experimental procedure for soaking bromide (8) or nitrate ions (current work) into crystals that were grown in the presence of chloride ions involves first soaking the protein crystals in a halide-free solution to remove the chloride present during crystallization, we also had the opportunity to solve the structure of this new halide-free form of bR(D85S). This new structure allows us to observe structural changes that occur in the protein during anion binding within the same crystal form.…”

Membrane-protein stability in a phospholipid-based crystallization medium

“…In the course of previous structural studies of intermediates in the bacteriorhodopsin photocycle (Facciotti et al, , 2003Rouhani et al, 2001), we found that some mutants of bacteriorhodopsin are actually less stable when reconstituted into MO. This observation contradicted the generally accepted hypothesis that incorporation of the solubilized protein into the MO bilayer might lead to a more favorable crystallization environment.…”

Genome sequence of Haloarcula marismortui: A halophilic archaeon from the Dead Sea

“…It also contains a tryptophan residue at position 138, which is known from high-resolution crystal structures of bacteriorhodopsin and its mutants to be a key component of the complex hydrogen-bond network on the extracellular side of the protein, which undergoes substantial rearrangements late in the photocycle (Supplemental Fig. E; Luecke et al 2001;Rouhani et al 2001;Facciotti et al 2003). Thus, although it seems that Xop1 may function as a second bacteriorhodopsin, the significance of evolutionarily maintaining two such genes is difficult to explain unless there is some further differentiation of function.…”