D-amino acid oxidase is the prototype of the FAD-dependent oxidases. It catalyses the oxidation of Damino acids to the corresponding a-ketoacids. The reducing equivalents are transferred to molecular oxygen with production of hydrogen peroxide. We have solved the crystal structure of the complex of D-amino acid oxidase with benzoate, a competitive inhibitor of the substrate, by single isomorphous replacement and eightfold averaging. Each monomer is formed by two domains with an overall topology similar to that ofp-hydroxybenzoate hydroxylase. The benzoate molecule lays parallel to the flavin ring and is held in position by a salt bridge with Arg-283. Analysis of the active site shows that no side chains are properly positioned to act as the postulated base required for the catalytic carboanion mechanism. On the contrary, the benzoate binding mode suggests a direct transfer of the substrate a-hydrogen to the flavin during the enzyme reductive half-reaction. The active site of D-amino acid oxidase exhibits a striking similarity with that of flavocytochrome b2, a structurally unrelated FMN-dependent flavoenzyme. The active site groups of these two enzymes are in fact superimposable once the mirror-image of the flavocytochrome b2 active site is generated with respect to the flavin plane. Therefore, the catalytic sites of D-amino acid oxidase and flavocytochrome b2 appear to have converged to a highly similar but enantiomeric architecture in order to catalyze similar reactions (oxidation of a-amino acids or a-hydroxy acids), although with opposite stereochemistry.Since the description of D-amino acid oxidase (EC 1.4.3.3; DAAO) activity in mammalian tissues by Krebs in 1935 (1), DAAO has been the subject of a number of biochemical, spectroscopic, and kinetic investigations, becoming the prototype for the oxidase class of the flavin-containing enzymes [for a recent review, see ref. 2]. Its primary structure has been determined and its gene has been cloned (3, 4). Its kinetic and mechanistic properties have been studied in detail by a variety of techniques, while information on the topology of the active site and on its three-dimensional structure have only been derived from chemical modification studies and site-directed mutagenesis of selected residues. Based on these approaches, a catalytic mechanism for DAAO has been proposed, although definitive evidence against alternative mechanisms has not been found (refs. 2 and 5 and references therein).The enzyme catalyzes the oxidation of D-a-amino acids into the corresponding a-ketoacids. The reaction formally proceeds according to the following scheme:E-FADH2 + 02-*E-FAD + H202[2]The reductive half reaction (Eq. 1), in which the noncovalently bound FAD becomes reduced, is followed by the oxidative step in which FAD is reoxidized by molecular oxygen, with the release of hydrogen peroxide (Eq. 2). The imino acid product spontaneously hydrolyzes to the ketoacid in a nonenzymatic process (Eq. 3). DAAO displays a broad substrate specificity, with a preference for D-amin...

The three-dimensional structure of the complex formed by two plasma proteins, transthyretin and retinol-binding protein, was determined from x-ray diffraction data to a nominal resolution of 3.1 angstroms. One tetramer of transthyretin was bound to two molecules of retinol-binding protein. The two retinol-binding protein molecules established molecular interactions with the same transthyretin dimer, and each also made contacts with one of the other two monomers. Thus, the other two potential binding sites in a transthyretin tetramer were blocked. The amino acid residues of the retinol-binding protein that were involved in the contacts were close to the retinol-binding site.

5CINECA Supercomputing Centre. 6University of Modena and Reggio Emilia.7Christian-Albrechts-University Kiel.8Michigan State University. International course and report were conceived by Pietro Cozzini and Glen E. Kellogg. * To whom correspondence should be addressed. For G.E.K.: Department of Medicinal Chemistry, Virginia Commonwealth University, Box 980540, Richmond, VA 23298-0540; (phone) 804-828-6452; (fax) 804-827-3664; (e-mail) glen.kellogg@vcu.edu. For P.C.: Department of General and Inorganic Chemistry, University of Parma, Via G.P. Usberti 17/A 43100, Parma, Italy; (phone) +39-0521-905669; (fax) +39-0521-905556; (e-mail) pietro.cozzini@unipr.it. NIH Public Access IntroductionStructure-based drug discovery has played an important role in medicinal chemistry 1 beginning nearly when the first X-ray crystal structure of the myoglobin and hemoglobin proteins at nearatomic resolution were described by Perutz, Kendrew and colleagues. 2-5 Even though only static structures were (and still generally are) used for most Structure-Based Drug Design (SBDD), and indeed most molecular modeling, the importance of flexibility was recognized immediately: hemoglobin has two rather different structures, "tense" and "relaxed", depending on its oxygenation, although in recent years a family of relaxed hemoglobin structures with different tertiary structure conformations have been reported. 6 In fact, all proteins are inherently flexible systems. This flexibility is frequently essential for function (e.g., as in hemoglobin). Proteins have an intrinsic ability to undergo functionally relevant conformational transitions under native state conditions, 7,8 on a wide range of scales, both in time and space. 9 In adenylate kinase large conformational changes due to movements of the nucleotide 'lids'-rate-limiting for overall catalytic turnover 10,11 -are 'linked' with relatively small-amplitude atomic fluctuations on the ps timescale such that changes in the local backbone conformation are required for lid closure. 12 Nuclear receptors are modular proteins where a significant degree of conformational flexibility is essential to biological function. Most of the pharmacology of nuclear receptor ligands has been discussed on the basis of their ability to stabilize (or displace) a short α-helix segment (known as H12 or AF-2) localized at the carboxy terminus of the receptor in (or from) its conformation in the protein "active" form. 13-15 Available X-ray crystal structures show a surprisingly wide range of structural diversity in ligands binding to, and inhibiting, nuclear receptor proteins such as the farnesoid X-receptor (FXR). 16,17 Protein dynamics is also a key component of intramolecular and intermolecular communication/signaling mechanisms and an essential requirement for the function of Gprotein coupled receptors (GPCRs), which are the largest known superfamily of membrane proteins. GPCRs regulate cell activity by transmitting extracellular signals to the inside of cells and respond to these signals by catalyzing nucleotide e...

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