A structural snapshot of an intermediate on the streptavidin-biotin dissociation pathway

It is currently unclear whether small molecules dissociate from a protein b inding site along a defined pathway or through a collection of dissociation pathways, We report herein a joint crystallographic, computational, and bi ophysical study that suggests the Asp-128 --> Ala (D128A) streptavidin muta nt closely mimics an intermediate on a well-defined dissociation pathway. A sp-128 is hydrogen bonded to a ureido nitrogen of biotin and also networks with the important aromatic binding contacts Trp-92 and Trp-108, The Asn-23 hydrogen bond to the ureido oxygen of biotin is lengthened to 3.8 Angstrom in the D128A structure, and a water molecule has moved into the pocket to replace the missing carboxylate interaction, These alterations are accompan ied by the coupled movement of biotin, the flexible binding loop containing Ser-45, and the loop containing the Ser-27 hydrogen bonding contact. This structure closely parallels a key intermediate observed in a potential of m ean force-simulated dissociation pathway of native streptavidin, where the Asn-23 hydrogen bond breaks first, accompanied by the replacement of the As p-128 hydrogen bond by an entering water molecule, Furthermore, both biotin and the flexible loop move in a concerted conformational change that close ly approximates the D128A structural changes. The activation and thermodyna mic parameters for the D128A mutant were measured and are consistent with a n intermediate that has traversed the early portion of the dissociation rea ction coordinate through endothermic bond breaking and concomitant gain in configurational entropy. These composite results suggest that the D128A mut ant provides a structural "snapshot" of an early intermediate on a relative ly well-defined dissociation pathway for biotin.