Specificity of anion binding in the substrate pocket of bacteriorhodopsin.
The structure of the D85S mutant of bacteriorhodopsin with a nitrate anion bound in the Schiff base binding site and the structure of the anion-free protein have been obtained in the same crystal form. Together with the previously solved structures of this anion pump, in both the anion-free state and bromide-bound state, these new structures provide insight into how this mutant of bacteriorhodopsin is able to bind a variety of different anions in the same binding pocket. The structural analysis reveals that the main structural change that accommodates different anions is the repositioning of the polar side chain of S85. On the basis of these X-ray crystal structures, the prediction is then made that the D85S/D212N double mutant might bind similar anions and do so over a broader pH range than does the single mutant. Experimental comparison of the dissociation constants, K(d), for a variety of anions confirms this prediction and demonstrates, in addition, that the binding affinity is dramatically improved by the D212N substitution.
Facciotti
Marc T
MT
Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA.
Cheung
Vincent S
VS
Lunde
Christopher S
CS
Rouhani
Shahab
S
Baliga
Nitin S
NS
Glaeser
Robert M
RM
D019943:Amino Acid Substitution; D027321:Anion Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000838:Anions / Q000378:metabolism*; D001216:Asparagine / Q000378:metabolism; D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D001965:Bromides / Q000378:metabolism; D018360:Crystallography, X-Ray; D006217:Halobacterium / Q000737:chemistry / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D009566:Nitrates / Q000378:metabolism; D017354:Point Mutation; D011487:Protein Conformation; D012545:Schiff Bases / Q000737:chemistry / Q000378:metabolism; D012680:Sensitivity and Specificity; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014867:Water / Q000737:chemistry
D027321:Anion Transport Proteins; D000838:Anions; D001965:Bromides; D009566:Nitrates; D012545:Schiff Bases; D014867:Water; D001436:Bacteriorhodopsins; D001216:Asparagine