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Abstract: The enzyme ForT catalyzes C–C bond formation between 5′-phosphoribosyl-1′-pyrophosphate (PRPP) and 4-amino-1H-pyrazole-3,5-dicarboxylate to make a key intermediate in the biosynthesis of formycin A 5′-phosphate by Streptomyces kaniharaensis. We report the 2.5 Å resolution structure of the ForT/PRPP complex and locate active site residues critical for PRPP recognition and catalysis.

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Abstract: Nowadays it is possible to combine X-ray crystallography and fragment screening in a medium throughput fashion to chemically probe the surfaces used by proteins to interact and use the outcome of the screens to systematically design protein-protein inhibitors. To prove it we first performed a bioinformatics analysis of the Protein Data Bank protein complexes, which revealed over 400 cases where the crystal lattice of the target in the free form is such that large portions of the interacting surfaces are free from lattice contacts and therefore accessible to fragments during soaks. Among the tractable complexes identified, we then performed single fragment crystal screens on two particular interesting cases: the Il1β-ILR and p38α-TAB1 complexes. The result of the screens showed that fragments tend to bind in clusters, highlighting the small-molecule hotspots on the surface of the target protein. In most of the cases the hotspots overlapped with the binding sites of the interacting proteins.

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Abstract: While most SAM riboswitches strongly discriminate between SAM and SAH, the SAM/SAH riboswitch responds to both ligands with similar apparent affinities. We have determined crystal structures of the SAM/SAH riboswitch bound to SAH, SAM and other variant ligands at high resolution. The riboswitch forms an H-type pseudoknot structure with coaxial alignment of the stem–loop helix (P1) and the pseudoknot helix (PK). An additional three base pairs form at the non-open end of P1, and the ligand is bound at the interface between the P1 extension and the PK helix. The adenine nucleobase is stacked into the helix and forms a trans Hoogsteen–Watson–Crick base pair with a uridine, thus becoming an integral part of the helical structure. The majority of the specific interactions are formed with the adenosine. The methionine or homocysteine chain lies in the groove making a single hydrogen bond, and there is no discrimination between the sulfonium of SAM or the thioether of SAH. Single-molecule FRET analysis reveals that the riboswitch exists in two distinct conformations, and that addition of SAM or SAH shifts the population into a stable state that likely corresponds to the form observed in the crystal. A model for translational regulation is presented whereby in the absence of ligand the riboswitch is largely unfolded, lacking the PK helix so that translation can be initiated at the ribosome binding site. But the presence of ligand stabilizes the folded conformation that includes the PK helix, so occluding the ribosome binding site and thus preventing the initiation of translation.

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Abstract: The β-barrel assembly machinery (BAM) inserts outer membrane β-barrel proteins (OMPs) in the outer membrane of Gram-negative bacteria. In Enterobacteriacea, BAM also mediates export of the stress sensor lipoprotein RcsF to the cell surface by assembling RcsF–OMP complexes. Here, we report the crystal structure of the key BAM component BamA in complex with RcsF. BamA adopts an inward-open conformation, with the lateral gate to the membrane closed. RcsF is lodged deep within the lumen of the BamA barrel, binding regions proposed to undergo outward and lateral opening during OMP insertion. On the basis of our structural and biochemical data, we propose a push-and-pull model for RcsF export following conformational cycling of BamA, and provide a mechanistic explanation for how RcsF uses its interaction with BamA to detect envelope stress. Our data also suggest that the flux of incoming OMP substrates is involved in the control of BAM activity.

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Abstract: SAMHD1 regulates cellular 2′-deoxynucleoside-5′-triphosphate (dNTP) homeostasis by catalysing the hydrolysis of dNTPs into 2′-deoxynucleosides and triphosphate. In CD4+ myeloid lineage and resting T-cells, SAMHD1 blocks HIV-1 and other viral infections by depletion of the dNTP pool to a level that cannot support replication. SAMHD1 mutations are associated with the autoimmune disease Aicardi–Goutières syndrome and hypermutated cancers. Furthermore, SAMHD1 sensitises cancer cells to nucleoside-analogue anti-cancer therapies and is linked with DNA repair and suppression of the interferon response to cytosolic nucleic acids. Nevertheless, despite its requirement in these processes, the fundamental mechanism of SAMHD1-catalysed dNTP hydrolysis remained unknown. Here, we present structural and enzymological data showing that SAMHD1 utilises an active site, bi-metallic iron-magnesium centre that positions a hydroxide nucleophile in-line with the Pα-O5′ bond to catalyse phosphoester bond hydrolysis. This precise molecular mechanism for SAMHD1 catalysis, reveals how SAMHD1 down-regulates cellular dNTP and modulates the efficacy of nucleoside-based anti-cancer and anti-viral therapies.