Understanding the structural dynamics of metallo-β-lactamase, cystatin related epididymal spermatogenic (CRES) protein, and bacterial inwardly rectifying potassium channel (KirBac1.1) by NMR

Proteins play central role in virtually all biological processes. Enzymes, a class of protein with catalytic properties mediates almost all biochemical reactions. Cellular structure, transport, storage, growth and differentiation are also dictated by different kind of proteins. Many proteins are related with several diseases. Activity of a protein is closely related with its three-dimensional structure. Therefore, studying protein structure provides valuable information about its functional mechanism. Among several high standard structural biology methods, we used Nuclear magnetic resonance (NMR) to study three proteins: metallo-β-lactamase enzyme, cystatin related epididymal spermatogenic (CRES) protein and bacterial homolog of eukaryotic inwardly rectifying potassium (KirBac1.1) channel. Metallo-β-lactamase, a globular protein, is responsible for catalyzing the hydrolysis of β-lactam ring of β-lactam antibiotics, thus conferring the antibiotic resistance, a global health threat. We studied a 10-nucleotide single stranded DNA aptamer (10-mer) mediated inhibition of Bacillus cereus 5/B/6 metallo-β-lactamase (5/B/6-MBL) by kinetic assay and solution-state NMR and site directed mutagenesis. Kinetic studies reveal an uncompetitive inhibition of 5/B/6-MBL by 10-mer during cefuroxime hydrolysis, indicating an allosteric type of inhibition. By solution-state NMR studies we identified three lysine residues as the enzyme-aptamer interaction site far away from the active site. By molecular docking, we showed a model of enzyme-aptamer complex. Mutation of these three positively charged lysine residues to uncharged showed decreased enzyme activity, which confirmed 10-mer mediated allosteric inhibition of 5/B/6-MBL. CRES (define) is an amyloidogenic protein and exhibits highly restricted expression in the epididymis of the male reproductive system. Although it is believed that CRES has putative roles in sperm maturation and protection, its mechanism of action is still unclear. CRES is produced as secreted protein and assembles with other CRES subgroup proteins into a web-like amyloid matrix in the epididymal lumen. To elucidate the mechanism of CRES-amyloid formation, we performed solution and solid-state NMR experiments. We present here a chemical shift based solution NMR structural model upon completion of backbone assignments for ~87% residues. The structure showed typical cystatin fold with an α-helix lying on top of four β-sheets. After completing the SSNMR assignments, we calculated a CRES domain swapped dimer by using CS-rosetta fold and dock and ROSETTA. Then we compared both solution and solid-state NMR chemical shifts. Chemical shift perturbations (CSP) for Cα and Cβ between solution and solid-state NMR were calculated. Significant chemical perturbations were observed for the residues comprising the N-terminal α helix in solution NMR model. Upon comparing the predicted chemical shift based secondary structural components, the N-terminal α-helical region of the solution NMR model exhibited significant differences according to solid-state NMR data. The N-terminal α-helix and the CRES-loop according to solution NMR model predicted to be more rigid according to solid-state NMR when random coil index (RCI) was calculated. Amyloid propensity prediction showed that 88 residues are amyloid prone. Based on calculated CSPs, predicted differences in secondary structural components, RCIs, and amyloid prone sites we propose two mechanism of CRES-amyloidogeneis: conformational switch of flexible loop into a more rigid β-sheet and traditional domain swapping. We also propose the domain swapped dimer of CRES as the basic unit of amyloid matrix. Potassium channels are membrane proteins that are responsible for transporting potassium ions (K+) in and out of cell. Inwardly rectifying potassium (Kir) channels unidirectionally transport K+ from outside to inside of the cell. KirBac1.1 is a prokaryotic homolog of eukaryotic Kir channel. Structure of KirBac1.1 in its closed state was studied by x-ray crystallography in details. We initiated a study to explore the KirBac1.1 structure in its open-activated state by solid-state NMR. Here we report ssNMR chemical shift assignment of KirBac1.1. With the help of two-dimensional and three-dimensional experiments we have assigned 309 out 333 residues of this 37.1 KD protein.