BINDING OF BIOTIN TO STREPTAVIDIN STABILIZES INTERSUBUNIT SALT BRIDGES BETWEEN ASP61 AND HIS87 AT LOW PH

The remarkable stability of the streptavidin tetramer towards subunit dissociation becomes even greater upon binding of biotin. At two equiv alent extensive monomer-monomer interfaces, monomers tightly associate into dimers that in turn associate into the tetramer at a less extens ive dimer-dimer interface. To probe the structural basis for the enhan cement of the stability of streptavidin by biotin, the crystal structu res of apostreptavidin and its complexes with biotin and other small m olecule and cyclic peptide ligands were determined and compared at res olutions as high as 1.36 Angstrom over a range of pH values from as lo w as 1.39. At low pH dramatic changes occur in the conformation and in tersubunit hydrogen bonds involving the loop comprising Asp61 to Ser69 . The hydrogen-bonded salt bridge between Asp61 O-delta 2 and His87 N- delta 1, observed at higher pH, is replaced with a strong hydrogen bon d between Asp61 O-delta 1 and Asn85 O-delta 1. Through crystallography at multiple pH values, the pH where this conformational change occurs , and thus the pK(a) of Asp61, was determined in crystals of space gro up I222 and/or I4(1)22 of apostreptavidin and complexes. A range in pK , values for Asp61 was observed in these structures, the lowest being 1.78 +/- 0.19 for I222 streptavidin-biotin in 2.9 M (NH4)(2)SO4. At lo w pH the decrease in pk(a), of Asp61 and preservation of the intersubu nit Asp61 O-delta 2-N-delta 1 His87 hydrogen-bonded salt bridge in str eptavidin-biotin versus apostreptavidin or streptavidin-peptide comple xes is associated with an ordering of the flexible flap comprising res idues Ala46 to Glu51, that in turn orders the Arg84 side-chain of a ne ighboring loop through resulting hydrogen bonds. Ordering of Arg84 in close proximity to the strong intersubunit interface appears to stabil ize the conformation associated with the Asp61 O-delta 2-N-delta 1 His 87 hydrogen-bonded salt bridge. Thus, in addition to the established r ole of biotin in tetramer stabilization by direct mediation of intersu bunit interactions at the weak interface through contact with Trp120, biotin may enhance tetramer stability at the strong interface more ind irectly by ordering loop residues. (C) 1997 Academic Press Limited.