Imidazole glycerol phosphate synthase catalyzes formation of the imidazole ring in histidine biosynthesis. The enzyme is also a glutamine amidotransferase, which produces ammonia in a glutaminase active site and channels it through a 30-A internal tunnel to a cyclase active site. Glutaminase activity is impaired in the resting enzyme, and stimulated by substrate binding in the cyclase active site. The signaling mechanism was investigated in the crystal structure of a ternary complex in which the glutaminase active site was inactivated by a glutamine analogue and the unstable cyclase substrate was cryo-trapped in the active site. The orientation of N(1)-(5'-phosphoribulosyl)-formimino-5-aminoimidazole-4-carboxamide ribonucleotide in the cyclase active site implicates one side of the cyclase domain in signaling to the glutaminase domain. This side of the cyclase domain contains the interdomain hinge. Two interdomain hydrogen bonds, which do not exist in more open forms of the enzyme, are proposed as molecular signals. One hydrogen bond connects the cyclase domain to the substrate analogue in the glutaminase active site. The second hydrogen bond connects to a peptide that forms an oxyanion hole for stabilization of transient negative charge during glutamine hydrolysis. Peptide rearrangement induced by a fully closed domain interface is proposed to activate the glutaminase by unblocking the oxyanion hole. This interpretation is consistent with biochemical results [Myers, R. S., et al., (2003) Biochemistry 42, 7013-7022, the accompanying paper in this issue] and with structures of the free enzyme and a binary complex with a second glutamine analogue.

This is the first structure in which all the components of the ubiquitous (beta/alpha)(8) barrel fold, top, bottom, and interior, take part in enzymatic function. Intimate contacts between the barrel domain and the glutaminase active site appear to be poised for crosstalk between catalytic centers in response to substrate binding at the cyclase active site. The structure provides a number of potential sites for inhibitor development in the active sites and in a conserved interdomain cavity.

Conformational changes play a vital role in the biological function of many proteins. The wide spectrum of conformational changes observed in crystal structures can be broadly classified as small amplitude shear motion, large amplitude hinge bending motion, or some combination of the two (1, 2). Shear motion generally represents the sliding movement of secondary structural elements on other parts of the tertiary structure, whereas hinge-bending motion is characterized by a few localized torsional rotations that combine to produce dramatic changes in the protein as a whole.A classic example of the involvement of hinge-bending motion in protein function is found in the periplasmic receptors of the bacterial ABC transporter systems. Such systems use the energy of ATP to carry small ligands and ions across the cytoplasmic membranes of both prokaryotes and eukaryotes (3-5).A typical ABC system consists of an membrane-bound permease, an ATP-binding component, and, in most bacterial systems, a periplasmic receptor. Binding of a small molecule ligand to the periplasmic proteins favors their closure via large scale hinge-bending motions (6). These movements are required for productive interactions with the cognate membrane permeases and, in some cases, with membrane-bound chemotaxis receptors as well. The periplasmic sugar-binding proteins belong to a subfamily (pentose/hexose sugar receptors) of the larger family of periplasmic receptors (7). Crystal structures of several members of this subfamily have been reported in the closed, ligand-bound form, including allose-binding protein (ALBP 1 (8)), ribose-binding protein (RBP (9)), arabinose-binding protein (ABP (10)), and glucose-galactose-binding protein (GBP (11, 12)). Each consists of two similar Rossmann fold domains linked by a three-stranded hinge region. The binding site is located at the domain interface; extensive hydrogen bonding and hydrophobic interactions of the ligand with both domains of the protein stabilize the closed form. Although the periplasmic receptors can assume similar closed forms in the ligand-free state (e.g. Ref. 13), experimental data suggest that more open forms will predominate in the absence of ligand (6, 14 -16).The structures of three ligand-free forms of RBP provided the first picture of the conformational changes in this subfamily of receptors (17). The two domains of each open RBP were shown to move as nearly rigid bodies at the hinge that joins them; the observed structures were opened by 43, 53, and 64°w ith respect to the closed receptor. Most structural changes involved only a few torsional changes in the hinge segments, although some minor repacking was observed where domaindomain interactions were lost in the opened receptors. Further, the three structures represented discrete points along a conformational trajectory, thus describing the motion that should apply to ligand capture as well as ligand release into the permease. In each open form, the two domains had a similar set of packing interactions that were not present in the...

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Imidazole glycerol-phosphate dehydratase (IGPD) catalyzes the sixth step of histidine biosynthesis. The enzyme is of fundamental biochemical interest, because it catalyzes removal of a non-acidic hydrogen atom in the dehydration reaction. It is also a potential target for development of herbicides. IGPD is a metalloenzyme in which transition metals induce aggregation and are required for catalysis. Addition of 1 equivalent of Mn 2؉ / subunit is shown by analytical ultracentrifugation to induce the formation of 24-mers from trimeric IGPD. Two histidine-rich motifs may participate in metal binding and aggregation. The 2.3-Å crystal structure of metal-free trimeric IGPD from the fungus Filobasidiella neoformans reveals a novel fold containing an internal repeat, apparently the result of gene duplication. The 95-residue ␣/␤ half-domain occurs in a few other proteins, including the GHMP kinase superfamily (galactohomoserine-mevalonate-phosphomevalonate), but duplication to form a compact domain has not been seen elsewhere. Conserved residues cluster at two types of sites in the trimer, each site containing a conserved histidine-rich motif. A model is proposed for the intact, active 24-mer in which all highly conserved residues, including the histidine-rich motifs in both the N-and C-terminal halves of the polypeptide, cluster at a common site between trimers. This site is a candidate for the active site and also for metal binding leading to aggregation of trimers. The structure provides a basis for further studies of enzyme function and mechanism and for development of more potent and specific herbicides.