Srisunder Subramaniam

My Story:

Thesis title:
Studies of conformational changes and dynamics accompanying substrate recognition, allostery and catalysis in bacteriophage lambda integrase

Abstract:
Bacteriophage lambda integrase catalyzes the site specific recombination of the viral DNA into the host genome, via a Holliday junction intermediate, and has been the subject of extensive study. It is a 40 kDa protein with three major domains: the N-terminal arm binding domain, the central core binding domain (CBD) and the C-terminal catalytic domain (C170). Biochemical and structural data suggest that substantial conformational change in the loop containing the tyrosine nucleophile is required to position it in conjunction with the rest of the conserved catalytic residues in the domain in order to generate the active site and enable DNA strand cleavage. The nature of these conformational changes, however, remain poorly understood. We used NMR spectroscopy together with limited trypsin proteolysis to examine the dynamics and conformations of C170 in the presence and absence of DNA half-site substrates. Although the C-terminus was indeed flexible in the absence of DNA, we found that conformational changes in the tyrosine-containing loop are not coupled to DNA binding. This led us to studies involving CBD. CBD was found to stimulate the activity of C170 even when the two domains were not linked. Moreover, mutation of two base pairs in the recognition sequence abolished this stimulation in activity, but the basal catalytic activity of C170 was unperturbed by these mutations. Since the two domains bind on either side of the DNA and do not significantly interact with each other, this finding implied that allosteric activation (coupled to conformational changes in the C-terminus) results from structural deformation of the DNA substrate. In addition, CBD is poorly structured in the absence of DNA but becomes folded upon binding its cognate sequence (Hari Kamadurai). The present study stands out in that this is the first instance in which a substrate binding-coupled folding event leads to allosteric enhancement of catalytic activity. Given the prevalence of bipartite structures in DNA cleaving enzymes we propose that allosteric activation may be a general mechanistic feature of such enzymes. These results and a new paradigm for site recognition and catalytic activation by large multidomain enzymes form the heart of the dissertation.