Nitric oxide (NO) is an important endogenous messenger in a variety of physiological and pathophysiological processes. NO is synthesized by the three isoforms of a homodimeric enzyme, nitric oxide synthase (NOS). Each NOS subunit is composed of a reductase domain that contains the binding sites for NADPH and two flavins. The reductase domain is linked via a 20 amino acid calmodulin (CaM) binding motif to an oxygenase domain that contains the binding sites for heme, L-arginine and tetrahydrobiopterin. The calcium cation (Ca2+) is also an important secondary messenger and the primary protein mediator of calcium is CaM. CaM undergoes Ca2+ concentration [Ca2+] dependent conformational changes that allow it to bind and activate scores of target proteins including NOS isozymes and voltage-gated calcium channels (VGCCs).
The long-term objectives of our research program is to understand:
- the mechanism and control of NO synthesis by NOS;
- the CaM-dependent activation of its numerous target proteins including NOS isozymes and VGCCs.
A) One of our objectives is to understand how calmodulin binds to NOS and affects structure, function and electron transfer within the different domains and the holoprotein.
The NOS isozymes are activated by the binding of CaM, the ubiquitous Ca2+-binding regulatory protein. We are investigating the role of calmodulin in the differential regulation of the various NOS isoforms. Recombinant DNA techniques are used to obtain the native and selectively modified forms of the human NOS enzymes. The mechanism of calmodulin binding and activation is being investigated using native and mutant forms of recombinant calmodulin. These enzymes are investigated for enzymatic activity and characterized using a variety of biophysical techniques including various forms of spectroscopy and calorimetry. These investigations will provide a better understanding of the control of NOS enzymes.
B) Investigation of electron transfer within the different domains of NOS enzymes.
The two mono-oxygenase reactions catalyzed by NOS enzymes require electron transfer within and between molecules that form the active NOS dimer. A fundamental understanding of the electron transfer processes in these enzymes may provide novel methods to control enzyme activity. Biophysical techniques are being used to investigate the rapid transfer of electrons within the molecule.
C) Investigation of NOS isoform selective binding.
The three mammalian NOS isozyme active sites share nearly full amino acid conservation and structural similarity making the design of isoform specific ligands and inhibitors very difficult. There are numerous examples of the failure of gaining improved potency from modified ligands and inhibitors whose designs were based upon information from the static X-ray structure of the docked complexes. Structure based studies also provide little information on the thermodynamics of binding and desolvation. Furthermore, since most studies have been performed on enzymes that are not of human origin, another reason for the non-availability of NOS isoform-selective compounds for clinical use comes from the inherent differences in the amino acid sequences between human and other mammalian isoforms. We are using a biophysical based approach in the development of molecules capable of isoform-specific binding to the active site of the human NOS isozymes.
D) Investigation of Calmodulin Dependent Signal Transduction.
Calcium is a ubiquitous intracellular messenger responsible for controlling numerous cellular processes including fertilization, mitosis, neuronal transmission, contraction and relaxation of muscles, gene transcription, and cell death. Many of the cellular effects of calcium are mediated by CaM. Upon binding up to four calcium ions, CaM undergoes conformational changes that enable it to bind and regulate many different protein targets. This process is a key component of a calcium-dependent signal transduction pathway. Our lab is investigating the fundamental properties of CaM that enables it to selectively bind and activate over three hundred proteins. Our investigation of the “Calmodulome” involves the characterization of specifically designed CaM mutants. We are using several biophysical and biochemical methods to map the binding and investigate the activation properties of CaM. The results of our research will provide a better understanding of protein-protein interactions, signal transduction in mammalian cells and may lead to the development of novel therapeutic compounds.
E) Regulation of VGCCs by intracellular Ca2+ concentration.
CaM undergoes Ca2+ concentration [Ca2+] dependent conformational changes that allow it to bind and activate scores of target proteins including NOS isozymes and voltage-gated calcium channels (VGCCs). Cav1.2s are high-voltage-activated or L-type calcium channels that control Ca2+ influx in response to membrane depolarization and are found in numerous cell types including muscle and neurons. These channels are modulated by membrane potential (voltage-dependent inactivation, VDI) as well as Ca2+-dependent feedback mechanisms known as Ca2+-dependent inactivation (CDI). While not fully understood, CDI is suggested to be dependent on the channel-associated CaM that detects intracellular Ca2+ in the vicinity of the channel pore. We are investigating how intracellular Ca2+ concentration regulates VGCCs and more specifically how CaM is involved in this regulation mechanism.
F) Investigation of Class II aldolases.
Class II aldolases in fungi have a unique active site that is significantly different from the one found in plants and humans making this enzyme a good target for the development of antifungal agents. The recombinant forms of Class II aldolases from diverse plant pathogenic fungi including Phytophthora infestans (responsible for the Irish potato famine) are being used in our investigation with the goal of developing novel antifungal agents. A similar approach is being used for the investigation of the Class II aldolase from Mycobacterium tuberculosis. Tuberculosis is a leading infectious killer worldwide, and new treatments are needed to combat drug-resistant tuberculosis. The investigation of the enzymatic mechanism is being pursued with the goal of gaining insight into the development of novel inhibitors.
