Tai-he Xia

Classical protein kinase C (PKC) family members are activated by the binding of various ligands to one of several cysteine-rich domains of the enzyme. The natural agonist, diacylglycerol (DAG), and the natural product superagonist, phorbol dibutyrate (PDB), activate the enzyme to produce wide-ranging physiological effects. The second cysteine-rich (Cys2) domain of rat brain PKC-gamma was expressed and labeled with 15N and 13C, and the solution structure was determined to high resolution using multidimensional heteronuclear NMR methods. The phorbol binding site was identified by titrating this domain with phorbol-12,13-dibutyrate (PDB) in the presence of organic cosolvents. Titrations of this domain with lipid micelles, in the absence and presence of phorbols, indicate selective broadening of some resonances. The observed behavior indicates conformational exchange between bound and free states upon protein-micelle interaction. The data also suggest that half of the domain, including the phorbol site and one of the zinc sites, is capable of inserting into membranes.

The determination of the NMR structure of oxidized Escherichia coli glutaredoxin in aqueous solution is described, and comparisons of this structure with that of reduced E. coli glutaredoxin and the related proteins E. coli thioredoxin and T4 glutaredoxin are presented. Based on nearly complete sequence-specific 1H-NMR assignments, 804 nuclear Overhauser enhancement distance constraints and 74 dihedral angle constraints were obtained as the input for the structure calculations, for which the distance geometry program DIANA was used followed by simulated annealing with the program X-PLOR. The molecular architecture of oxidized glutaredoxin is made up of three helices and a four-stranded beta-sheet. The three-dimensional structures of oxidized and the recently described reduced glutaredoxin are very similar. Quantitative analysis of the exchange rates of 34 slowly exchanging amide protons from corresponding series of two-dimensional [15N,1H]-correlated spectra of oxidized and reduced glutaredoxin showed close agreement, indicating almost identical hydrogen-bonding patterns. Nonetheless, differences in local dynamics involving residues near the active site and the C-terminal alpha-helix were clearly manifested. Comparison of the structure of E. coli glutaredoxin with those of T4 glutaredoxin and E. coli thioredoxin showed that all three proteins have a similar overall polypeptide fold. An area of the protein surface at the active site containing Arg 8, Cys 11, Pro 12, Tyr 13, Ile 38, Thr 58, Val 59, Pro 60, Gly 71, Tyr 72, and Thr 73 is proposed as a possible site for interaction with other proteins, in particular ribonucleotide reductase. It was found that this area corresponds to previously proposed interaction sites in T4 glutaredoxin and E. coli thioredoxin. The solvent-accessible surface area at the active site of E. coli glutaredoxin showed a general trend to increase upon reduction. Only the sulfhydryl group of Cys 11 is exposed to the solvent, whereas that of Cys 14 is buried and solvent inaccessible.