A DOMINANT-NEGATIVE MUTANT OF E. COLI MALTOSE-BINDING PROTEIN: X-RAY STRUCTURES AND GENERAL LESSONS. Sherry L. Mowbray and Brian H. Shilton, Department of Molecular Biology, Swedish Agricultural University, S-751 24 Uppsala, Sweden

Studies of a dominant-negative mutant of E. coli maltose-binding protein shed new light on the mechanism of binding protein-dependent transport systems. X-ray structures and small-angle X-ray scattering studies of open, ligand-free forms of the mutant show that the unliganded protein is essentially identical with the wild type protein, while only a small region of the protein located between the two structural domains is altered in the closed, ligand-bound form. The physiological effects of the mutant can be explained by the kinetic scheme:

where P, L and M are binding protein, ligand, and membrane permease complex, with M* representing the activated form necessary to transport. It is proposed that wild type binding protein promotes the M to M* transition through binding more tightly to M*, in the same way that enzymes function by binding more tightly to transition states. In the mutant, the binding to M* is weaker, and though binding to M can occur, the transition to M* is in that case not favored. This proposal explains the basic function of the transport system, as well as a number of previously puzzling results. The structures of both binding protein and membrane permease are likely to be more open in the activated complex, a suggestion supported by the observation that multiple forms are possible within the primarily "closed" and "open populations in solution.