Grants and Contributions:

Grant or Award spanning more than one fiscal year. (2017-2018 to 2022-2023)

Nitric oxide (NO) is an important endogenous messenger in a variety of physiological and pathophysiological processes. NO is synthesized by the three isoforms of nitric oxide synthase (NOS) that are encoded by different genes, neuronal (nNOS), endothelial (eNOS) and inducible (iNOS). The dual role of NO in the body as a second messenger or cytotoxic agent shows the importance for tight regulation of the different NOS isoforms. Each subunit of NOS is composed of a reductase domain that contains the binding sites for NADPH, and two flavins as well as an oxygenase domain that contains the binding sites for heme, L-arginine and tetrahydrobiopterin. The two domains are linked by a calmodulin (CaM) binding domain that plays an important role in the activation of the enzyme. CaM is the primary protein mediator of the calcium cation that is also an important secondary messenger. CaM undergoes calcium-dependent conformational changes that allow it to bind and activate scores of target proteins including all three NOS isozymes. Two of the long-term goals of this research program are to: 1) understand the mechanism(s) of CaM binding and control for all three NOS isozymes; 2) study the selective ligand binding and inhibition of human NOS isozymes.

A critical structural determinant of NOS activity is the CaM controlled electron transfer from NADPH to the heme via two flavin cofactors. How CaM controls electron transfer is not fully understood and several differences have been noted between the three isoforms of NOS. Due to the large size and dynamic properties of the NOS enzymes, it is very difficult to get information on their full length protein structure. Structural information on NOS isozymes is limited to truncations encompassing their heme domains, reductase domains and the iNOS FMN subdomain bound to CaM. This is most likely due to the large conformational shift of the FMN subdomain believed to occur due to electron transfer during catalysis. As part of our proposed research program, we will perform NMR investigations on full-length as well as judiciously truncated forms of the enzymes to decipher the role of CaM and the different domains in the regulatory mechanism of the enzyme. These NMR experiments when combined with other biophysical studies and enzymes assays will provide a better understanding of the CaM dependent activation of NOS enzymes.

The second major goal of our research program is to study selective binding and inhibition of human NOS isozymes. While considerable research has been performed on mammalian NOS enzymes, there is limited information on the human isozymes. We propose to use the human isozymes in our investigation and compare the results with previous studies performed on the corresponding mammalian enzymes. Our rational is based on the possibility that small differences between human and mammalian enzymes may be important in our search for isoform selective binding and enzyme inhibition.