Structure of a variant of lac repressor with increased thermostability anddecreased affinity for operator

A single amino acid substitution, K84L, in the Escherichia coh lac represso r produces a protein that has substantially increased stability compared to wild-type. However, despite the increased stability, this altered tetramer ic repressor has a tenfold reduced affinity for operator and greatly decrea sed rate-constants of inducer binding as well as a reduced phenotypic respo nse to inducer in vivo. To understand the dramatic increase in stability an d altered functional properties, we have determined the X-ray crystal struc tures of a dimeric repressor with and without the K84L substitution at reso lutions of 1.7 and 3.0 Angstrom, respectively. In the wild-type dimer, K84- 11, Lys84 forms electrostatic interactions at the monomer-monomer interface and is partially exposed to solvent. In the K84L-11 substituted protein th ere is reorientation of the N-subdomains, which allows the leucine to becom e deeply buried at the monomer-monomer interface. This reorientation of the N-subdomains, in turn, results in an alteration of hydrogen bonding, ion p airing, and van der Waals interactions at the monomer-monomer interface. Th e lysine residue at position 84 appears to exert its key effects by destabi lizing the "optimal" conformation of the repressor, effectively loosening t he dimer interface and allowing the repressor to adopt the conformations ne cessary to function as a molecular switch. (C) 2001 Academic Press.