Associate Professor, Department of Clinical Laboratory Sciences
University of Kansas Medical Center
Atomic structure of a multi-domain redox enzyme Ncb5or implicated in diseases (2015-2016)
Elucidation of the structural basis of Ncb5or function is expected to increase understanding of redox pathways involved in diabetes and breast cancer. Thus, the proposed research is relevant to the part of NIH’s mission that pertains to developing fundamental knowledge that will help to reduce the burdens of human disability.
NADH cytochrome b5 oxidoreductase (Ncb5or) is a multi-domain redox enzyme that has been implicated in disease processes. Studies of Ncb5or null mice show that they develop early-onset diabetes and neurodegeneration and exhibit altered lipid and iron metabolism. In addition, genetic studies have identified two missense mutations in Ncb5or associated with human breast cancer. Ncb5or contains two redox domains, b5 and b5R, that are homologous to microsomal cytochrome b5 (Cyb5A) and its reductase Cyb5R3, respectively. These are separated by a domain that belongs to the CS (CHORD-SGT1) family, whose members are known for mediating protein-protein interactions. In addition, Ncb5or features a novel N-terminal region (N) that is intrinsically disordered, but folds when connected to the b5 domain. Our long-term goals in studies of Ncb5or are to (1) elucidate the biological roles of Ncb5or; (2) determine how lost or compromised function lead to diabetes, cancer and other observed health effects; and (3) identify ways in which function can be recovered for therapeutic benefit. In order to make progress on any of these goals, it is essential to obtain atomic level structural information about Ncb5or that reveals how its various components are assembled. On the basis of preliminary data, we hypothesize that the CS domain and the N-terminal polypeptide operate in tandem to facilitate the electron transfer role of Ncb5or, by mediating docking between the redox domains.
Efforts to provide evidence for direct interactions between the CS domain and the N-terminal polypeptide comprise Specific Aim 1 of this proposal. We plan to obtain a solution structure of a fragment of Ncb5or (designated N/CS/b5) that lacks the b5R domain, using 3-dimensional NMR experiments performed on triple labeled protein (13C/15N/2H). Ultimately, we hope to obtain a high-resolution structure of the protein by X-ray crystallography that will reveal full details of interactions among all domains.
Specific Aim 2 describes plans to extend and augment previous attempts to obtain X-ray quality crystals of Ncb5or that will enable us to accomplish this key objective.