Structural Analysis of Streptococcus pyogenes NADH Oxidase: Conformational Dynamics Involved in Formation of the C(4a)-Peroxyflavin Intermediate.

In probing the oxygen reactivity of an Enterococcus faecalis NADH oxidase (Nox; O2 → 2H2O) C42S mutant lacking the Cys42-sulfenic acid (Cys42-SOH) redox center, we provided direct evidence of a C(4a)-peroxyflavin intermediate in the oxidative half-reaction and also described a conformational or chemical change that is rate-limiting for full reoxidation of the homodimer. In this work, the Nox from Streptococcus pyogenes (SpyNox) has been expressed and crystallized, and the overoxidized wild-type [Cys44-SOH → Cys44-sulfinic acid (Cys44-SO2H)] and C44S mutant enzyme structures have been refined at 2.0 and 2.15 Å, respectively. We show that azide binds to the two-electron reduced wild-type (EH2) enzyme and to the mutant enzyme in solution, but with a significantly higher affinity for the mutant protein. The spectral course of the titration with the SpyNox EH2 form clearly indicates progressive displacement of the Cys44-S(-) → FAD charge-transfer interaction. An azide soak with C44S Nox crystals led to the structure of the complex, as refined at 2.10 Å. The active-site N3(-) ligand is proximal to the Ser44 and His11 side chains, and a significant shift in the Ser44 side chain also appears. This provides an attractive explanation for the azide-induced loss of charge-transfer absorbance seen with the wild-type EH2 form and also permits accommodation of a C(4a)-peroxyflavin structural model. The conformation of Ser44 and the associated helical element, and the resulting steric accommodation, appear to be linked to the conformational change described in the E. faecalis C42S Nox oxidative half-reaction.

Crystal structure and catalytic properties of Bacillus anthracis CoADR-RHD: implications for flavin-linked sulfur trafficking.

Rhodanese homology domains (RHDs) play important roles in sulfur trafficking mechanisms essential to the biosynthesis of sulfur-containing cofactors and nucleosides. We have now determined the crystal structure at 2.10 A resolution for the Bacillus anthracis coenzyme A-disulfide reductase isoform (BaCoADR-RHD) containing a C-terminal RHD domain; this is the first structural representative of the multidomain proteins class of the rhodanese superfamily. The catalytic Cys44 of the CoADR module is separated by 25 A from the active-site Cys514' of the RHD domain from the complementary subunit. In stark contrast to the B. anthracis CoADR [Wallen, J. R., Paige, C., Mallett, T. C., Karplus, P. A., and Claiborne, A. (2008) Biochemistry 47, 5182-5193], the BaCoADR-RHD isoform does not catalyze the reduction of coenzyme A-disulfide, although both enzymes conserve the Cys-SSCoA redox center. NADH titrations have been combined with a synchrotron reduction protocol for examination of the structural and redox behavior of the Cys44-SSCoA center. The synchrotron-reduced (Cys44 + CoASH) structure reveals ordered binding for the adenosine 3'-phosphate 5'-pyrophosphate moiety of CoASH, but the absence of density for the pantetheine arm indicates that it is flexible within the reduced active site. Steady-state kinetic analyses with the alternate disulfide substrates methyl methanethiolsulfonate (MMTS) and 5,5'-dithiobis(2-nitrobenzoate) (DTNB), including the appropriate Cys --> Ser mutants, demonstrate that MMTS reduction occurs within the CoADR active site. NADH-dependent DTNB reduction, on the other hand, requires communication between Cys44 and Cys514', and we propose that reduction of the Cys44-SSCoA disulfide promotes the transfer of reducing equivalents to the RHD, with the swinging pantetheine arm serving as a ca. 20 A bridge.

Pyridine nucleotide complexes with Bacillus anthracis coenzyme A-disulfide reductase: a structural analysis of dual NAD(P)H specificity.

We have recently reported that CoASH is the major low-molecular weight thiol in Bacillus anthracis [Nicely, N. I. , Parsonage, D., Paige, C., Newton, G. L., Fahey, R. C., Leonardi, R., Jackowski, S., Mallett, T. C., and Claiborne, A. (2007) Biochemistry 46, 3234-3245], and we have now characterized the kinetic and redox properties of the B. anthracis coenzyme A-disulfide reductase (CoADR, BACoADR) and determined the crystal structure at 2.30 A resolution. While the Staphylococcus aureus and Borrelia burgdorferi CoADRs exhibit strong preferences for NADPH and NADH, respectively, B. anthracis CoADR can use either pyridine nucleotide equally well. Sequence elements within the respective NAD(P)H-binding motifs correctly reflect the preferences for S. aureus and Bo. burgdorferi CoADRs, but leave questions as to how BACoADR can interact with both pyridine nucleotides. The structures of the NADH and NADPH complexes at ca. 2.3 A resolution reveal that a loop consisting of residues Glu180-Thr187 becomes ordered and changes conformation on NAD(P)H binding. NADH and NADPH interact with nearly identical conformations of this loop; the latter interaction, however, involves a novel binding mode in which the 2'-phosphate of NADPH points out toward solvent. In addition, the NAD(P)H-reduced BACoADR structures provide the first view of the reduced form (Cys42-SH/CoASH) of the Cys42-SSCoA redox center. The Cys42-SH side chain adopts a new conformation in which the conserved Tyr367'-OH and Tyr425'-OH interact with the nascent thiol(ate) on the flavin si-face. Kinetic data with Y367F, Y425F, and Y367,425F BACoADR mutants indicate that Tyr425' is the primary proton donor in catalysis, with Tyr367' functioning as a cryptic alternate donor in the absence of Tyr425'.

Structure of alpha-glycerophosphate oxidase from Streptococcus sp.: a template for the mitochondrial alpha-glycerophosphate dehydrogenase.

The FAD-dependent alpha-glycerophosphate oxidase (GlpO) from Enterococcus casseliflavus and Streptococcus sp. was originally studied as a soluble flavoprotein oxidase; surprisingly, the GlpO sequence is 30-43% identical to those of the alpha-glycerophosphate dehydrogenases (GlpDs) from mitochondrial and bacterial sources. The structure of a deletion mutant of Streptococcus sp. GlpO (GlpODelta, lacking a 50-residue insert that includes a flexible surface region) has been determined using multiwavelength anomalous dispersion data and refined at 2.3 A resolution. Using the GlpODelta structure as a search model, we have also determined the intact GlpO structure, as refined at 2.4 A resolution. The first two domains of the GlpO fold are most closely related to those of the flavoprotein glycine oxidase, where they function in FAD binding and substrate binding, respectively; the GlpO C-terminal domain consists of two helix bundles and is not closely related to any known structure. The flexible surface region in intact GlpO corresponds to a segment of missing electron density that links the substrate-binding domain to a betabetaalpha element of the FAD-binding domain. In accordance with earlier biochemical studies (stabilizations of the covalent FAD-N5-sulfite adduct and p-quinonoid form of 8-mercapto-FAD), Ile430-N, Thr431-N, and Thr431-OG are hydrogen bonded to FAD-O2alpha in GlpODelta, stabilizing the negative charge in these two modified flavins and facilitating transfer of a hydride to FAD-N5 (from Glp) as well. Active-site overlays with the glycine oxidase-N-acetylglycine and d-amino acid oxidase-d-alanine complexes demonstrate that Arg346 of GlpODelta is structurally equivalent to Arg302 and Arg285, respectively; in both cases, these residues interact directly with the amino acid substrate or inhibitor carboxylate. The structural and functional divergence between GlpO and the bacterial and mitochondrial GlpDs is also discussed.

Structure of the type III pantothenate kinase from Bacillus anthracis at 2.0 A resolution: implications for coenzyme A-dependent redox biology.

Coenzyme A (CoASH) is the major low-molecular weight thiol in Staphylococcus aureus and a number of other bacteria; the crystal structure of the S. aureus coenzyme A-disulfide reductase (CoADR), which maintains the reduced intracellular state of CoASH, has recently been reported [Mallett, T.C., Wallen, J.R., Karplus, P.A., Sakai, H., Tsukihara, T., and Claiborne, A. (2006) Biochemistry 45, 11278-89]. In this report we demonstrate that CoASH is the major thiol in Bacillus anthracis; a bioinformatics analysis indicates that three of the four proteins responsible for the conversion of pantothenate (Pan) to CoASH in Escherichia coli are conserved in B. anthracis. In contrast, a novel type III pantothenate kinase (PanK) catalyzes the first committed step in the biosynthetic pathway in B. anthracis; unlike the E. coli type I PanK, this enzyme is not subject to feedback inhibition by CoASH. The crystal structure of B. anthracis PanK (BaPanK), solved using multiwavelength anomalous dispersion data and refined at a resolution of 2.0 A, demonstrates that BaPanK is a new member of the Acetate and Sugar Kinase/Hsc70/Actin (ASKHA) superfamily. The Pan and ATP substrates have been modeled into the active-site cleft; in addition to providing a clear rationale for the absence of CoASH inhibition, analysis of the Pan-binding pocket has led to the development of two new structure-based motifs (the PAN and INTERFACE motifs). Our analyses also suggest that the type III PanK in the spore-forming B. anthracis plays an essential role in the novel thiol/disulfide redox biology of this category A biodefense pathogen.

Structure of coenzyme A-disulfide reductase from Staphylococcus aureus at 1.54 A resolution.

Coenzyme A (CoASH) replaces glutathione as the major low molecular weight thiol in Staphylococcus aureus; it is maintained in the reduced state by coenzyme A-disulfide reductase (CoADR), a homodimeric enzyme similar to NADH peroxidase but containing a novel Cys43-SSCoA redox center. The crystal structure of S. aureus CoADR has been solved using multiwavelength anomalous dispersion data and refined at a resolution of 1.54 A. The resulting electron density maps define the Cys43-SSCoA disulfide conformation, with Cys43-S(gamma) located at the flavin si face, 3.2 A from FAD-C4aF, and the CoAS- moiety lying in an extended conformation within a cleft at the dimer interface. A well-ordered chloride ion is positioned adjacent to the Cys43-SSCoA disulfide and receives a hydrogen bond from Tyr361'-OH of the complementary subunit, suggesting a role for Tyr361' as an acid-base catalyst during the reduction of CoAS-disulfide. Tyr419'-OH is located 3.2 A from Tyr361'-OH as well and, based on its conservation in known functional CoADRs, also appears to be important for activity. Identification of residues involved in recognition of the CoAS-disulfide substrate and in formation and stabilization of the Cys43-SSCoA redox center has allowed development of a CoAS-binding motif. Bioinformatics analyses indicate that CoADR enzymes are broadly distributed in both bacterial and archaeal kingdoms, suggesting an even broader significance for the CoASH/CoAS-disulfide redox system in prokaryotic thiol/disulfide homeostasis.

Crystallization and preliminary X-ray crystallographic studies of the alkanesulfonate FMN reductase from Escherichia coli.

The alkanesulfonate FMN reductase (SsuE) from Escherichia coli catalyzes the reduction of FMN by NADPH to provide reduced flavin for the monooxygenase (SsuD) enzyme. The vapor-diffusion technique yielded single crystals that grow as hexagonal rods and diffract to 2.9 A resolution using synchrotron X-ray radiation. The protein crystallizes in the primitive hexagonal space group P622. The SsuE protein lacks any cysteine or methionine residues owing to the role of the SsuE enzyme in the acquisition of sulfur during sulfate starvation. Therefore, substitution of two leucine residues (Leu114 and Leu165) to methionine was performed to obtain selenomethionine-containing SsuE for MAD phasing. The selenomethionine derivative of SsuE has been expressed and purified and crystals of the protein have been obtained with and without bound FMN. These preliminary studies should lead to the structure solution of SsuE. It is anticipated that this new protein structure will provide detailed structural information on specific active-site regions of the protein and insight into the mechanism of flavin reduction and transfer of reduced flavin.

Ligand binding promotes the entropy-driven oligomerization of integrin alpha IIb beta 3.

Integrin alpha(IIb)beta(3) clusters on the platelet surface after binding adhesive proteins in a process that regulates signal transduction. However, the intermolecular forces driving integrin self-association are poorly understood. This work provides new insights into integrin clustering mechanisms by demonstrating how temperature and ligand binding interact to affect the oligomeric state of alpha(IIb)beta(3). The ligand-free receptor, solubilized in thermostable octyl glucoside micelles, exhibited a cooperative transition at approximately 43 degrees C, monitored by changes in intrinsic fluorescence and circular dichroism. Both signals changed in a direction opposite to that for global unfolding, and both were diminished upon binding the fibrinogen gamma-chain ligand-mimetic peptide cHArGD. Free and bound receptors also exhibited differential sensitivity to temperature-enhanced oligomerization, as measured by dynamic light scattering, sedimentation velocity, and sedimentation equilibrium. Van't Hoff analyses of dimerization constants for alpha(IIb)beta(3) complexed with cHArGD, cRGD, or eptifibatide yielded large, favorable entropy changes partly offset by unfavorable enthalpy changes. Transmission electron microscopy showed that ligand binding and 37 degrees C incubation enhanced assembly of integrin dimers and larger oligomers linked by tail-to-tail contacts. Interpretation of these images was aided by threading models for alpha(IIb)beta(3) protomers and dimers based on the ectodomain structure of alpha(v)beta(3). We propose that entropy-favorable nonpolar interactions drive ligand-induced integrin clustering and outside-in signaling.