Superoxide dismutases rely on protein structural elements to adjust the redox potential of the metallocenter to an optimum value near 300 mV (vs. NHE), to provide a source of protons for catalysis, and to control the access of anions to the active site. These aspects of the catalytic mechanism are examined herein for recombinant preparations of the nickel-dependent SOD (NiSOD) from Streptomyces coelicolor, and for a series of mutants that affect a key tyrosine residue, Tyr9 (Y9F-, Y62F-, Y9FY62F- and D3A-NiSOD). Structural aspects of the nickel sites are examined by a combination of EPR and x-ray absorption spectroscopies, and by single crystal x-ray diffraction at ~ 1.9 Å resolution in the case of Y9F- and D3A-NiSODs. The functional effects of the mutations are examined by kinetic studies employing pulse radiolytic generation of O2− and by redox titrations. These studies reveal that although the structure of the nickel center in NiSOD is unique, the ligand environment is designed to optimize the redox potential at 290 mV and results in the oxidation of 50% of the nickel centers in the oxidized hexamer. Kinetic investigations show that all of the mutant proteins have considerable activity. In the case of Y9F-NiSOD, the enzyme shows saturation behavior that is not observed in WT-NiSOD and suggests that release of peroxide is inhibited. The crystal structure of Y9F-NiSOD reveals an anion binding site that is occupied by either Cl− or Br− and is located close to, but not within bonding distance of the nickel center. The structure of D3A-NiSOD reveals that in addition to affecting the interaction between subunits, this mutation repositions Y9 and leads to altered chemistry with peroxide. Comparisons with Mn(SOD) and Fe(SOD) reveal that although different strategies are employed to adjust the redox potential and supply of protons, NiSOD has evolved a similar strategy to control the access of anions to the active site.

MHCII proteins bind peptide antigens in endosomal compartments of antigen-presenting cells. The non-classical MHCII protein HLA-DM chaperones peptide-free MHCII against inactivation and catalyzes peptide exchange on loaded MHCII. Another non-classical MHCII protein, HLA-DO, binds HLA-DM and influences the repertoire of peptides presented by MHCII proteins. However, the mechanism by which HLA-DO functions is unclear. Here we use x-ray crystallography, enzyme kinetics and mutagenesis approaches to investigate human HLA-DO structure and function. In complex with HLA-DM, HLA-DO adopts a classical MHCII structure, with alterations near the alpha subunit 310 helix. HLA-DO binds to HLA-DM at the same sites implicated in MHCII interaction, and kinetic analysis demonstrates that HLA-DO acts as a competitive inhibitor. These results show that HLA-DO inhibits HLA-DM function by acting as a substrate mimic and place constraints on possible functional roles for HLA-DO in antigen presentation.

The enzyme ␣-galactosidase (␣-GAL, also known as ␣-GAL A; E.C. 3.2.

Background: HLA-DM-mediated peptide exchange is a key factor in epitope selection, but how HLA-DM selects peptides for editing is not known. Results: Peptide complexes sensitive to HLA-DM editing exhibited conformational alterations. Conclusion: HLA-DM efficiently identifies unstable complexes by sensing MHCII-peptide conformations. Significance: These data emphasize HLA-DM as a conformational editor and provide novel mechanistic insight into its function.