The mammalian complement system is a phylogenetically ancient cascade system that has a major role in innate and adaptive immunity. Activation of component C3 (1,641 residues) is central to the three complement pathways and results in inflammation and elimination of self and non-self targets. Here we present crystal structures of native C3 and its final major proteolytic fragment C3c. The structures reveal thirteen domains, nine of which were unpredicted, and suggest that the proteins of the alpha2-macroglobulin family evolved from a core of eight homologous domains. A double mechanism prevents hydrolysis of the thioester group, essential for covalent attachment of activated C3 to target surfaces. Marked conformational changes in the alpha-chain, including movement of a critical interaction site through a ring formed by the domains of the beta-chain, indicate an unprecedented, conformation-dependent mechanism of activation, regulation and biological function of C3.

Re-evaluation of the crystallographic data of the molybdenum-containing E. coli formate dehydrogenase H (Boyington et al. Science 275:1305-1308, 1997), reported in two redox states, reveals important structural differences for the formate-reduced form, with large implications for the reaction mechanism proposed in that work. We have re-refined the reduced structure with revised protocols and found substantial rearrangement in some parts of it. The original model is essentially correct but an important loop close to the molybdenum active site was mistraced, and, therefore, catalytic relevant residues were located in wrong positions. In particular selenocysteine-140, a ligand of molybdenum in the original work, and essential for catalysis, is no longer bound to the metal after reduction of the enzyme with formate. These results are incompatible with the originally proposed reaction mechanism. On the basis of our new interpretation, we have revised and proposed a new reaction mechanism, which reconciles the new X-ray model with previous biochemical and extended X-ray absorption fine structure data.

Desulfovibrio gigas formate dehydrogenase is the first representative of a tungsten-containing enzyme from a mesophile that has been structurally characterized. It is a heterodimer of 110 and 24 kDa subunits. The large subunit, homologous to E. coli FDH-H and to D. desulfuricans nitrate reductase, harbors the W site and one [4Fe-4S] center. No small subunit ortholog containing three [4Fe-4S] clusters has been reported. The structural homology with E. coli FDH-H shows that the essential residues (SeCys158, His159, and Arg407) at the active site are conserved. The active site is accessible via a positively charged tunnel, while product release may be facilitated, for H(+) by buried waters and protonable amino acids and for CO(2) through a hydrophobic channel.

Phage T4 endonuclease VII (Endo VII), the first enzyme shown to resolve Holliday junctions, recognizes a broad spectrum of DNA substrates ranging from branched DNAs to single base mismatches. We have determined the crystal structures of the Ca 2⍣ -bound wild-type and the inactive N62D mutant enzymes at 2.4 and 2.1 Å, respectively. The Endo VII monomers form an elongated, highly intertwined molecular dimer exhibiting extreme domain swapping. The major dimerization elements are two pairs of antiparallel helices forming a novel 'four-helix cross' motif. The unique monomer fold, almost completely lacking β-sheet structure and containing a zinc ion tetrahedrally coordinated to four cysteines, does not resemble any of the known junctionresolving enzymes, including the Escherichia coli RuvC and λ integrase-type recombinases. The S-shaped dimer has two 'binding bays' separated by~25 Å which are lined by positively charged residues and contain near their base residues known to be essential for activity. These include Asp40 and Asn62, which function as ligands for the bound calcium ions. A pronounced bipolar charge distribution suggests that branched DNA substrates bind to the positively charged face with the scissile phosphates located near the divalent cations. A model for the complex with a four-way DNA junction is presented.

Here we describe the 1.95 Å structure of the clinically used antiprogestin RU486 (mifepristone) in complex with the progesterone receptor (PR). The structure was obtained by taking a crystal of the PR ligand binding domain containing the agonist norethindrone and soaking it in a solution containing the antagonist RU486 for extended times. Clear ligand exchange could be observed in one copy of the PR ligand binding domain dimer in the crystal. RU486 binds while PR is in an agonistic conformation without displacing helix 12. Although this is probably because of the constraints of the crystal lattice, it demonstrates that helix 12 displacement is not a prerequisite for RU486 binding. Interestingly, B-factor analysis clearly shows that helix 12 becomes more flexible after RU486 binding, suggesting that RU486, being a model antagonist, does not induce one fixed conformation of helix 12 but changes its positional equilibrium. This conclusion is confirmed by comparing the structures of RU486 bound to PR and RU486 bound to the glucocorticoid receptor.

The progesterone receptor is able to bind to a large number and variety of ligands that elicit a broad range of transcriptional responses ranging from full agonism to full antagonism and numerous mixed profiles inbetween. We describe here two new progesterone receptor ligand binding domain x-ray structures bound to compounds from a structurally related but functionally divergent series, which show different binding modes corresponding to their agonistic or antagonistic nature. In addition, we present a third progesterone receptor ligand binding domain dimer bound to an agonist in monomer A and an antagonist in monomer B, which display binding modes in agreement with the earlier observation that agonists and antagonists from this series adopt different binding modes.

Background: Understanding the molecular basis for the mixed profiles of progesterone receptor (PR) ligands will benefit future drug design. Results: Two differing mechanisms for the induction of mixed profiles by 11␤-steroids are described. Conclusion: Subtle electrostatic and steric factors explain the differing PR activities of 11␤-steroids. Significance: These observations will impact future drug-design strategies for PR and potentially other nuclear receptors.

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