YvoA is a GntR/HutC transcription regulator from Bacillus subtilis implicated in the regulation of genes from the N-acetylglucosamine-degrading pathway. Its 2.4-Å crystal structure reveals a homodimeric assembly with each monomer displaying a two-domain fold. The C-terminal domain, which binds the effector N-acetylglucosamine-6-phosphate, adopts a chorismate lyase fold, whereas the N-terminal domain contains a winged helix–turn–helix DNA-binding domain. Isothermal titration calorimetry and site-directed mutagenesis revealed that the effector-binding site in YvoA coincides with the active site of related chorismate lyase from Escherichia coli. The characterization of the DNA- and effector-binding properties of two disulfide-bridged mutants that lock YvoA in two distinct conformational states provides for the first time detailed insight into the allosteric mechanism through which effector binding modulates DNA binding and, thereby regulates transcription in a representative GntR/HutC family member. Central to this allosteric coupling mechanism is a loop-to-helix transition with the dipole of the newly formed helix pointing toward the phosphate of the effector. This transition goes in hand with the emergence of internal symmetry in the effector-binding domain and, in addition, leads to a 122° rotation of the DNA-binding domains that is best described as a jumping-jack-like motion.

In phylogenetically diverse bacteria, the conserved protein RapZ plays a central role in RNA-mediated regulation of amino-sugar metabolism. RapZ contributes to the control of glucosamine phosphate biogenesis by selectively presenting the regulatory small RNA GlmZ to the essential ribonuclease RNase E for inactivation. Here, we report the crystal structures of full length Escherichia coli RapZ at 3.40 Å and 3.25 Å, and its isolated C-terminal domain at 1.17 Å resolution. The structural data confirm that the N-terminal domain of RapZ possesses a kinase fold, whereas the C-terminal domain bears closest homology to a subdomain of 6-phosphofructokinase, an important enzyme in the glycolytic pathway. RapZ self-associates into a domain swapped dimer of dimers, and in vivo data support the importance of quaternary structure in RNA-mediated regulation of target gene expression. Based on biochemical, structural and genetic data, we suggest a mechanism for binding and presentation by RapZ of GlmZ and the closely related decoy sRNA, GlmY. We discuss a scenario for the molecular evolution of RapZ through re-purpose of enzyme components from central metabolism.

Carbon monoxide dehydrogenase from the bacterium Oligotropha carboxidovorans catalyzes the oxidation of CO to CO(2) at a unique [CuSMoO(2)] cluster. In the bacteria the cluster is assembled post-translational. The integration of S, and particularly of Cu, is rate limiting in vivo, which leads to CO dehydrogenase preparations containing the mature and fully functional enzyme along with forms of the enzyme deficient in one or both of these elements. The active sites of mature and immature forms of CO dehydrogenase were converted into a [MoO(3)] centre by treatment with potassium cyanide. We have established a method, which rescues 50% of the CO dehydrogenase activity by in vitro reconstitution of the active site through the supply of sulphide first and subsequently of Cu(I) under reducing conditions. Immature forms of CO dehydrogenase isolated from the bacterium, which were deficient in S and/or Cu at the active site, were similarly activated. X-ray crystallography and electron paramagnetic resonance spectroscopy indicated that the [CuSMoO(2)] cluster was properly reconstructed. However, reconstituted CO dehydrogenase contains mature along with immature forms. The chemical reactions of the reconstitution of CO dehydrogenase are summarized in a model, which assumes resulphuration of the Mo-ion at both equatorial positions at a 1:1 molar ratio. One equatorial Mo-S group reacts with Cu(I) in a productive fashion yielding a mature, functional [CuSMoO(2)] cluster. The other Mo-S group reacts with Cu(I), then Cu(2)S is released and an oxo group is introduced from water, yielding an inactive [MoO(3)] centre.

The zona pellucida (ZP) domain is present in extracellular proteins such as the zona pellucida proteins and tectorins and participates in the formation of polymeric protein networks. However, the ZP domain also occurs in the cytokine signaling co-receptor transforming growth factor β (TGF-β) receptor type 3 (TGFR-3, also known as betaglycan) where it contributes to cytokine ligand recognition. Currently it is unclear how the ZP domain architecture enables this dual functionality. Here, we identify a novel major TGF-β-binding site in the FG loop of the C-terminal subdomain of the murine TGFR-3 ZP domain (ZP-C) using protein crystallography, limited proteolysis experiments, surface plasmon resonance measurements and synthetic peptides. In the murine 2.7 Å crystal structure that we are presenting here, the FG-loop is disordered, however, well-ordered in a recently reported homologous rat ZP-C structure. Surprisingly, the adjacent external hydrophobic patch (EHP) segment is registered differently in the rat and murine structures suggesting that this segment only loosely associates with the remaining ZP-C fold. Such a flexible and temporarily-modulated association of the EHP segment with the ZP domain has been proposed to control the polymerization of ZP domain-containing proteins. Our findings suggest that this flexibility also extends to the ZP domain of TGFR-3 and might facilitate co-receptor ligand interaction and presentation via the adjacent FG-loop. This hints that a similar C-terminal region of the ZP domain architecture possibly regulates both the polymerization of extracellular matrix proteins and cytokine ligand recognition of TGFR-3.

Today's proteome is the result of innumerous gene duplication, mutagenesis, drift and selection processes. Whereas random mutagenesis introduces predominantly only gradual changes in protein function, a case can be made that an abrupt switch in function caused by single amino acid substitutions will not only considerably further evolution but might constitute a prerequisite for the appearance of novel functionalities for which no promiscuous protein intermediates can be envisaged. Recently, tetracycline repressor (TetR) variants were identified in which binding of tetracycline triggers the repressor to associate with and not to dissociate from the operator DNA as in wild-type TetR. We investigated the origin of this activity reversal by limited proteolysis, CD spectroscopy and X-ray crystallography. We show that the TetR mutant Leu17Gly switches its function via a disorder–order mechanism that differs completely from the allosteric mechanism of wild-type TetR. Our study emphasizes how single point mutations can engender unexpected leaps in protein function thus enabling the appearance of new functionalities in proteins without the need for promiscuous intermediates.

Two analogues of the discontinued tumor vascular-disrupting agent verubulin (Azixa®, MPC-6827, 1) featuring benzo-1,4-dioxan-6-yl (compound 5 a) and N-methylindol-5-yl (compound 10) residues instead of the para-anisyl group on the 4-(methylamino)-2-methylquinazoline pharmacophore, were prepared and found to exceed the antitumor efficacy of the lead compound. They were antiproliferative with single-digit nanomolar IC50 values against a panel of nine tumor cell lines, while not affecting nonmalignant fibroblasts. Indole 10 surpassed verubulin in seven tumor cell lines including colon, breast, ovarian, and germ cell cancer cell lines. In line with docking studies indicating that compound 10 may bind the colchicine binding site of tubulin more tightly (Ebind =-9.8 kcal mol(-1) ) than verubulin (Ebind =-8.3 kcal mol(-1) ), 10 suppressed the formation of vessel-like tubes in endothelial cells and destroyed the blood vessels in the chorioallantoic membrane of fertilized chicken eggs at nanomolar concentrations. When applied to nude mice bearing a highly vascularized 1411HP germ cell xenograft tumor, compound 10 displayed pronounced vascular-disrupting effects that led to hemorrhages and extensive central necrosis in the tumor.

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Edited by Patrick SungRNA degradation is crucial for regulating gene expression in all organisms. Like the decapping of eukaryotic mRNAs, the conversion of the 5-terminal triphosphate of bacterial transcripts to a monophosphate can trigger RNA decay by exposing the transcript to attack by 5-monophosphate-dependent ribonucleases. In both biological realms, this deprotection step is catalyzed by members of the Nudix hydrolase family. The genome of the gastric pathogen Helicobacter pylori, a Gramnegative epsilonproteobacterium, encodes two proteins resembling Nudix enzymes. Here we present evidence that one of them, HP1228 (renamed HpRppH), is an RNA pyrophosphohydrolase that triggers RNA degradation in H. pylori, whereas the other, HP0507, lacks such activity. In vitro, HpRppH converts RNA 5-triphosphates and diphosphates to monophosphates. It requires at least two unpaired nucleotides at the 5 end of its substrates and prefers three or more but has only modest sequence preferences. The influence of HpRppH on RNA degradation in vivo was examined by using RNA-seq to search the H. pylori transcriptome for RNAs whose 5-phosphorylation state and cellular concentration are governed by this enzyme. Analysis of cDNA libraries specific for transcripts bearing a 5-triphosphate and/or monophosphate revealed at least 63 potential HpRppH targets. These included mRNAs and sRNAs, several of which were validated individually by half-life measurements and quantification of their 5-terminal phosphorylation state in wild-type and mutant cells. These findings demonstrate an important role for RppH in post-transcriptional gene regulation in pathogenic Epsilonproteobacteria and suggest a possible basis for the phenotypes of H. pylori mutants lacking this enzyme.