Solution NMR studies of acetohydroxy acid synthase I: Identification of the sites of inter-subunit interactions using multidimensional NMR methods

Abstract

The novel multidomain organization in the multimeric Escherichia coli AHAS I (ilvBN) enzyme has been dissected to generate polypeptide fragments. These fragments when cloned, expressed and purified reassemble in the presence of cofactors to yield a catalytically competent enzyme. Structural characterization of AHAS has been impeded due to the fact that the holoenzyme is prone to dissociation leading to heterogeneity in samples. Our approach has enabled the structural characterization using high-resolution nuclear magnetic resonance methods. Near complete sequence specific NMR assignments for backbone H N, 15N, 13C α and 13C β atoms of the FAD binding domain of ilvB have been obtained on samples isotopically enriched in 2H, 13C and 15N. The secondary structure determined on the basis of observed 13C α secondary chemical shifts and sequential NOEs indicates that the secondary structure of the FAD binding domain of E. coli AHAS large subunit (ilvB) is similar to the structure of this domain in the catalytic subunit of yeast AHAS. Protein–protein interactions involving the regulatory subunit (ilvN) and the domains of the catalytic subunit (ilvB) were studied using circular dichroic and isotope edited solution nuclear magnetic resonance spectroscopic methods. Observed changes in circular dichroic spectra indicate that the regulatory subunit (ilvN) interacts with ilvBα and ilvBβ domains of the catalytic subunit and not with the ilvBγ domain. NMR chemical shift mapping methods show that ilvN binds close to the FAD binding site in ilvBβ and proximal to the intrasubunit ilvBα/ilvBβ domain interface. The implication of this interaction on the role of the regulatory subunit on the activity of the holoenzyme is discussed. NMR studies of the regulatory domains show that these domains are structured in solution. Preliminary evidence for the interaction of ilvN with the metabolic end product of the pathway, viz., valine is also presented.