Multifunctional proteins play a variety of roles in metabolism.Here, we examine the catalytic function of the combined 3-deoxy-D-arabino heptulosonate-7-phosphate synthase (DAH7PS) and chorismate mutase (CM) from Geobacillus sp. DAH7PS operates at the start of the biosynthetic pathway for aromatic metabolites, whereas CM operates in a dedicated branch of the pathway for the biosynthesis of amino acids tyrosine and phenylalanine. In line with sequence predictions, the two catalytic functions are located in distinct domains, and these two activities can be separated and retain functionality. For the full-length protein, prephenate, the product of the CM reaction, acts as an allosteric inhibitor for the DAH7PS. The crystal structure of the full-length protein with prephenate bound and the accompanying small angle x-ray scattering data reveal the molecular mechanism of the allostery. Prephenate binding results in the tighter association between the dimeric CM domains and the tetrameric DAH7PS, occluding the active site and therefore disrupting DAH7PS function. Acquisition of a physical gating mechanism to control catalytic function through gene fusion appears to be a general mechanism for providing allostery for this enzyme.

Ezrin is a multidomain protein providing regulated membrane-cytoskeleton contacts that play a role in cell differentiation, adhesion, and migration. Within the cytosol of resting cells ezrin resides in an autoinhibited conformation in which the Nand C-terminal ezrin/radixin/moesin (ERM) association domains (ERMADs) interact with one another. Activation of the ezrin membrane-cytoskeleton linker function requires an opening of this interdomain association that can result from phosphatidylinositol 4,5-bisphosphate binding to the N-ERMAD and threonine 567 phosphorylation in the C-ERMAD. We have shown that ezrin can also be activated by Ca 2؉ -dependent binding of the EF-hand protein S100P. We now provide a quantitative analysis of this interaction and map the respective binding sites to the F2 lobe in the ezrin N-ERMAD and a stretch of hydrophobic residues in the C-terminal extension of S100P. Phospholipid binding assays reveal that S100P and phosphatidylinositol 4,5-bisphosphate compete to some extent for at least partially overlapping binding sites in N-ERMAD. Using interaction-competent as well as interaction-incompetent S100P derivatives and permanently active ezrin mutants, we also show that the protein interaction and a resulting activation of ezrin promote the transendothelial migration of tumor cells. Thus, a prometastatic role of ezrin and S100P that had been proposed based on their overexpression in highly metastatic cancers is probably due to a direct interaction between the two proteins and the S100P-mediated activation of ezrin. ERM2 (ezrin/radixin/moesin) family proteins function as membrane-cytoskeleton linkers in a number of dynamic processes ranging from the formation and maintenance of actinrich surface structures such as microvilli to the control of cellcell and cell-cell matrix contacts and the regulation of cell migration (for review, see Refs. 1-3). Often these processes are accompanied by the establishment of cellular polarity, one example being the front-rear asymmetry in motile cells. The latter is particularly evident in leukocyte migration that requires the regulation of ERM protein activity at the rear end of the cells for efficient migration (4, 5). In addition to providing direct physical scaffolds in the membrane skeleton, ERM proteins also function in diverse aspects of intracellular signaling, for example by directly interacting with effectors of Rho GTPase signaling (for review, see Ref. 6).The three-modular structure of ERM proteins is ideally suited for carrying out cross-linking and signaling functions in a regulated manner. The N-terminal domain, also known as N-terminal ERM association domain (N-ERMAD) or FERM domain (for Four point one ERM), is a conserved element shared with other members of the band 4.1 superfamily of proteins. It is followed by a central ␣-helical domain with the propensity to form coiled-coils and a C-terminal ERMAD. Binding sites for PtdIns(4,5)P 2 and several plasma membrane-resident proteins like the hyaluronate receptor CD44, and different intercellula...

Adenosine triphosphate phosphoribosyltransferase (ATP-PRT) catalyzes the first committed step of the histidine biosynthesis in plants and microorganisms. Here, we present the functional and structural characterization of the ATP-PRT from the pathogenic e-proteobacteria Campylobacter jejuni (CjeATP-PRT). This enzyme is a member of the long form (HisG L ) ATP-PRT and is allosterically inhibited by histidine, which binds to a remote regulatory domain, and competitively inhibited by AMP. In the crystalline form, CjeATP-PRT was found to adopt two distinctly different hexameric conformations, with an open homohexameric structure observed in the presence of substrate ATP, and a more compact closed form present when inhibitor histidine is bound. CjeATP-PRT was observed to adopt only a hexameric quaternary structure in solution, contradicting previous hypotheses favoring an allosteric mechanism driven by an oligomer equilibrium.Abbreviations: ATP-PRT, adenosine triphosphate phosphoribosyltransferase; BTP, 1,3-bis[tris(hydroxymethyl)methylamino]propane; CjeATP-PRT, ATP-PRT from Campylobacter jejuni; EcoATP-PRT, Escherichia coli ATP-PRT; His, l-histidine; ITC, isothermal titration calorimetry; MtuATP-PRT, Mycobacterium tuberculosis ATP-PRT; PDB, Protein Data Bank; PR-ATP, phosphoribosyl ATP; PRPP, phosphoribosyl pyrophosphate; PRT, phosphoribosyltransferase; RMSD, root-mean-square difference; SenATP-PRT, Salmonella enterica subsp. enterica Typhimurium ATP-PRT Additional Supporting Information may be found in the online version of this article.Significance Statement: ATP-phosphoribosyltransferase catalyzes the first dedicated step in the biosynthesis of the essential amino acid histidine in microorganisms. We report the functional characterization of this enzyme from human pathogen Campylobacter jejuni. The enzyme is inhibited by histidine, allowing for tuned production of histidine in response to cellular demands. Our results reveal how the enzyme structure becomes compressed when histidine binds and exposes the molecular details of how this enzyme performs its function. Instead, this study supports the conclusion that the ATP-PRT long form hexamer is the active species; the tightening of this structure in response to remote histidine binding results in an inhibited enzyme.

Background: Two enzymes from Mycobacterium tuberculosis involved in aromatic amino acid biosynthesis form a heterooctameric complex. Results: Complex formation boosts the catalytic activity of both enzymes and greatly extends the allosteric effector sensitivity. Conclusion: Enzyme interactions allow complex allosteric machinery of one of the complex partners to be shared. Significance: Sophisticated allosteric responses are delivered through protein-protein interactions, allowing enhanced metabolic control.