Catalytic cycle of human glutathione reductase near 1 A resolution.

Efficient enzyme catalysis depends on exquisite details of structure beyond those resolvable in typical medium- and high-resolution crystallographic analyses. Here we report synchrotron-based cryocrystallographic studies of natural substrate complexes of the flavoenzyme human glutathione reductase (GR) at nominal resolutions between 1.1 and 0.95 A that reveal new aspects of its mechanism. Compression in the active site causes overlapping van der Waals radii and distortion in the nicotinamide ring of the NADPH substrate, which enhances catalysis via stereoelectronic effects. The bound NADPH and redox-active disulfide are positioned optimally on opposite sides of the flavin for a 1,2-addition across a flavin double bond. The new structures extend earlier observations to reveal that the redox-active disulfide loop in GR is an extreme case of sequential peptide bonds systematically deviating from planarity--a net deviation of 53 degrees across five residues. But this apparent strain is not a factor in catalysis, as it is present in both oxidized and reduced structures. Intriguingly, the flavin bond lengths in oxidized GR are intermediate between those expected for oxidized and reduced flavin, but we present evidence that this may not be due to the protein environment but instead due to partial synchrotron reduction of the flavin by the synchrotron beam. Finally, of more general relevance, we present evidence that the structures of synchrotron-reduced disulfide bonds cannot generally be used as reliable models for naturally reduced disulfide bonds.

The EBP50-moesin interaction involves a binding site regulated by direct masking on the FERM domain.

Members of the ezrin-radixin-moesin (ERM) protein family serve as regulated microfilament-membrane crosslinking proteins that, upon activation, bind the scaffolding protein ERM-phosphoprotein of 50 kDa (EBP50). Here we report a 3.5 A resolution diffraction analysis of a complex between the active moesin N-terminal FERM domain and a 38 residue peptide from the C terminus of EBP50. This crystallographic result, combined with sequence and structural comparisons, suggests that the C-terminal 11 residues of EBP50 binds as an alpha-helix at the same site occupied in the dormant monomer by the last 11 residues of the inhibitory moesin C-terminal tail. Biochemical support for this interpretation derives from in vitro studies showing that appropriate mutations in both the EBP50 tail peptide and the FERM domain reduce binding, and that a peptide representing just the C-terminal 14 residues of EBP50 also binds to moesin. Combined with the recent identification of the I-CAM-2 binding site on the ERM FERM domain (Hamada, K., Shimizu, T., Yonemura, S., Tsukita, S., and Hakoshima, T. (2003) EMBO J. 22, 502-514), this study reveals that the FERM domain contains two distinct binding sites for membrane-associated proteins. The contribution of each ligand to ERM function can now be dissected by making structure-based mutations that specifically affect the binding of each ligand.

Structural Aspects of Plant Ferredoxin : NADP(+) Oxidoreductases.

Ferredoxin reductase (FNR) is ubiquitous among photosynthetic organisms as the enzyme directly responsible for the generation of NADPH. Structural studies over the last 15 years have generated over 30 crystal structures of wild-type and mutant FNRs that have yielded a great deal of insight into its structure-function relations. These insights are summarized and combined to propose a structurally informed cycle for FNR catalysis in vivo.