Cloning, Expression, and Purification of a Nitric Oxide Synthase-Like Protein from Bacillus cereus

The nitric oxide synthase-like protein from Bacillus cereus (bcNOS) has been cloned, expressed, and characterized. This small hemeprotein (356 amino acids in length) has a mass of 43 kDa and forms a dimer. The recombinant protein showed similar spectral shifts to the mammalian NOS proteins and could bind the substrates L-arginine and NG-hydroxy-L-arginine as well as the ligand imidazole. Low levels of activity were recorded for the hydrogen peroxide-dependent oxidation of NG-hydroxy-L-arginine and L-arginine by bcNOS, while a reconstituted system with the rat neuronal NOS reductase domain showed no activity. The recombinant bcNOS protein adds to the complement of bacterial NOS-like proteins that are used for the investigation of the mechanism and function of NO in microorganisms

Solution Structure of Calmodulin Bound to the Target Peptide of Endothelial Nitric Oxide Synthase Phosphorylated at Thr495

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Biochemistry, copyright © American Chemical Society after peer review and technical editing by publisher. To access the final edited and published work see http://dx.doi.org/10.1021/bi401466sNitric oxide synthase (NOS) plays a major role in a number of key physiological and pathological processes, and it is important to understand how this enzyme is regulated. The small acidic calcium binding protein, calmodulin (CaM), is required to fully activate the enzyme. The exact mechanism of how CaM activates NOS is not fully understood at this time. Studies have shown CaM to act like a switch that causes a conformational change in NOS to allow for the transfer of an electron between the reductase and oxygenase domains through a process that is thought to be highly dynamic and at least in part controlled by several possible phosphorylation sites. We have determined the solution structure of CaM bound to a peptide that contains a phosphorylated threonine corresponding to Thr495 in full size endothelial NOS (eNOS) to investigate the structural and functional effects that the phosphorylation of this residue may have on nitric oxide production. Our biophysical studies show that phosphorylation of Thr495 introduces electrostatic repulsions between the target sequence and CaM as well as a diminished propensity for the peptide to form an α-helix. The calcium affinity of the CaM–target peptide complex is reduced because of phosphorylation, and this leads to weaker binding at low physiological calcium concentrations. This study provides an explanation for the reduced level of NO production by eNOS carrying a phosphorylated Thr495 residue.National Science and Engineering Research Council (NSERC) [326911-2009, 183521

Chemical shift perturbations induced by residue specific mutations of CaM interacting with NOS peptides

The final publication is available at Springer via http://dx.doi.org/10.1007/s12104-015-9596-0The regulation of nitric oxide synthase (NOS) by calmodulin (CaM) plays a major role in a number of key physiological and pathological processes. A detailed molecular level picture of how this regulation is achieved is critical for drug development and for our understanding of protein regulation in general. CaM is a small acidic calcium binding protein and is required to fully activate NOS. The exact mechanism of how CaM activates NOS is not fully understood at this time. Studies have shown CaM to act like a switch that causes a conformational change in NOS to allow for the electron transfer between the reductase and oxygenase domains through a process that is thought to be highly dynamic. The interaction of CaM with NOS is modified by a number of post-translation modifications including phosphorylation. Here we present backbone and sidechain 1H, 15N NMR assignments of modified CaM interacting with NOS peptides which provides the basis for a detailed study of CaM–NOS interaction dynamics using 15N relaxation methods

Chemical shift assignments of calmodulin constructs with EF hand mutations

The final publication is available at Springer via http://dx.doi.org/10.1007/s12104-015-9665-4Calmodulin (CaM) is a ubiquitous cytosolic Ca2+-binding protein able to bind and regulate hundreds of different proteins. It consists of two globular domains joined by a flexible central linker region. Each one of these domains contains two EF hand pairs capable of binding to Ca2+. Upon Ca2+ binding CaM undergoes a conformational change exposing hydrophobic patches that interact with its intracellular target proteins. CaM is able to bind to target proteins in the Ca2+-replete and Ca2+-deplete forms. To study the Ca2+-dependent/independent properties of binding and activation of target proteins by CaM, CaM constructs with Ca2+ binding disrupting mutations of Asp to Ala at position one of each EF hand have been used. One target protein of CaM is nitric oxide synthase, which catalyzes the production of nitric oxide. At elevated Ca2+ concentrations, CaM binds to neuronal NOS and endothelial NOS, making them the Ca2+-dependent NOS enzymes. In contrast, inducible NOS is transcriptionally regulated in vivo and binds to CaM at basal levels of Ca2+. Here we report the NMR backbone and sidechain resonance assignments of C-lobe Ca2+-replete and deplete CaM12, N-lobe Ca2+-replete and deplete CaM34, CaM1234 in the absence of Ca2+ and N-lobe Ca2+-replete CaM34 with the iNOS CaM-binding domain peptide

Structural Consequences of Calmodulin EF Hand Mutations

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Biochemistry, copyright © American Chemical Society after peer review and technical editing by publisher. To access the final edited and published work see http://dx.doi.org/10.1021/acs.biochem.6b01296Calmodulin (CaM) is a cytosolic Ca2+-binding protein that serves as a control element for many enzymes. It consists of two globular domains, each containing two EF hand pairs capable of binding Ca2+, joined by a flexible central linker region. CaM is able to bind and activate its target proteins in the Ca2+-replete and Ca2+-deplete forms. To study the Ca2+-dependent/independent properties of binding and activation of target proteins by CaM, CaM constructs with Ca2+-binding disrupting mutations of Asp to Ala at position one of each EF hand have been used. These CaM mutant proteins are deficient in binding Ca2+ in either the N-lobe EF hands (CaM12), C-lobe EF hands (CaM34), or all four EF hands (CaM1234). To investigate potential structural changes these mutations may cause, we performed detailed NMR studies of CaM12, CaM34, and CaM1234 including determining the solution structure of CaM1234. We then investigated if these CaM mutants affected the interaction of CaM with a target protein known to interact with apoCaM by determining the solution structure of CaM34 bound to the iNOS CaM binding domain peptide. The structures provide direct structural evidence of changes that are present in these Ca2+-deficient CaM mutants and show these mutations increase the hydrophobic exposed surface and decrease the electronegative surface potential throughout each lobe of CaM. These Ca2+-deficient CaM mutants may not be a true representation of apoCaM and may not allow for native-like interactions of apoCaM with its target proteins.Natural Sciences and Engineering Research Council of Canada (NSERC) [326911, 183521

Structural Studies of a Complex Between Endothelial Nitric Oxide Synthase and Calmodulin at Physiological Calcium Concentration

The small acidic protein calmodulin (CaM) serves as a Ca²⁺ sensor and control element for many enzymes including nitric oxide synthase (NOS) enzymes that play major roles in key physiological and pathological processes. CaM binding causes a conformational change in NOS to allow for the electron transfer between the reductase and oxygenase domains through a process that is thought to be highly dynamic. In this report, NMR spectroscopy was used to determine the solution structure of the endothelial NOS (eNOS) peptide in complex with CaM at the lowest Ca²⁺ concentration (225 nM) required for CaM to bind to eNOS and corresponds to a physiological elevated Ca²⁺ level found in mammalian cells. Under these conditions, the CaM–eNOS complex has a Ca²⁺-replete C-terminal lobe bound to the eNOS peptide and a Ca²⁺ free N-terminal lobe loosely associated with the eNOS peptide. With increasing Ca²⁺ concentration, the binding of Ca²⁺ by the N-lobe of CaM results in a stronger interaction with the C-terminal region of the eNOS peptide and increased α-helical structure of the peptide that may be part of the mechanism resulting in electron transfer from the FMN to the heme in the oxygenase domain of the enzyme. Surface plasmon resonance studies performed under the same conditions show Ca²⁺ concentration-dependent binding kinetics were consistent with the NMR structural results. This investigation shows that structural studies performed under more physiological relevant conditions provide information on subtle changes in structure that may not be apparent when experiments are performed in excess Ca²⁺ concentrations