STRUCTURAL STUDIES OF BINDING-SITE TRYPTOPHAN MUTANTS IN THE HIGH-AFFINITY STREPTAVIDIN-BIOTIN COMPLEX

Previous thermodynamic and computational studies have pointed to the i mportant energetic role of aromatic contacts in generating the excepti onal binding free energy of streptavidin-biotin association. We report here the crystallographic characterization of single site tryptophan mutants in investigating structural consequences of alterations in the se aromatic contacts. Four tryptophan residues, Trp79, Trp92, Trp108 a nd Trp120, play an important role in the hydrophobic binding contribut ions, which along with a hydrogen bonding network curd a flexible bind ing loop give rise to tight ligand binding (K-a similar to 10(13) M-1) . The crystal structures of ligand-free and biotin-bound mutants, W79F , W108F, W120F and W120A, in the resolution range from 1.9 to 2.3 Angs trom were determined. Nine data sets for these four different mutants were collected, and structural models were refined to X-values ranging from 0.15 to 0.20. The major question addressed here is how these mut ations influence the streptavidin binding site and in particular how t hey affect the binding mode of biotin in the complex. The overall fold ing of streptavidin was not significantly altered in any of the trypto phan mutants. With one exception, only minor deviations in the unbound structures were observed. In one crystal form of unbound W79F, there is a coupled shift in the side-chains of Phe29 and Tyr43 toward the mu tation site, although in a different crystal form these shifts are not observed. In the bound structures, the orientation of biotin in the b inding pocket was not significantly altered in the mutant complex. Com pared with the wild-type streptavidin-biotin complex, there were no ad ditional crystallographic water molecules observed for any of the muta nts in the binding pocket. These structural studies thus suggest that the thermodynamic alterations can be attributed to the local alteratio ns in binding residue composition, rather than a rearrangement of bind ing site architectures. (C) 1998 Academic Press Limited.