Graham Cheetham

Three dimensional structure of human C-reactive protein.

The structure of the classical acute phase reactant human C-reactive protein provides evidence that phosphocholine binding is mediated through calcium and a hydrophobic pocket centred on Phe 66. The residue Glu 81 is suitably positioned to interact with the choline group. A cleft on the pentameric face opposite to that containing the calcium site may have an important functional role. The structure provides insights into the molecular mechanisms by which this highly conserved plasma protein, for which no polymorphism or deficiency state is known, may exert its biological role.

Structural basis for initiation of transcription from an RNA polymerase-promoter complex.

Although the single-polypeptide-chain RNA polymerase from bacteriophage T7 (T7RNAP), like other RNA polymerases, uses the same mechanism of polymerization as the DNA polymerases, it can also recognize a specific promoter sequence, initiate new RNA chains from a single nucleotide, abortively cycle the synthesis of short transcripts, be regulated by a transcription inhibitor, and terminate transcription. As T7RNAP is homologous to the Pol I family of DNA polymerases, the differences between the structure of T7RNAP complexed to substrates and that of the corresponding DNA polymerase complex provides a structural basis for understanding many of these functional differences. T7RNAP initiates RNA synthesis at promoter sequences that are conserved from positions −17 to +6 relative to the start site of transcription. The crystal structure at 2.4 Å resolution of T7RNAP complexed with a 17-base-pair promoter shows that the four base pairs closest to the catalytic active site have melted to form a transcription bubble. The T7 promoter sequence is recognized by interactions in the major groove between an antiparallel β-loop and bases. The amino-terminal domain is involved in promoter recognition and DNA melting. We have also used homology modelling of the priming and incoming nucleoside triphosphates from the T7 DNA-polymerase ternary complex structure to explain the specificity of T7RNAP for ribonucleotides, its ability to initiate from a single nucleotide, and the abortive cycling at the initiation of transcription.

Insights into transcription: structure and function of single-subunit DNA-dependent RNA polymerases

Single-subunit RNA polymerases are widespread throughout prokaryotic and eukaryotic organisms, and also viruses. T7 RNA polymerase is one of the simplest DNA-dependent enzymes, capable of transcribing a complete gene without the need for additional proteins. During the past two years, three illuminating crystal structures of T7 RNA polymerase complexed to either T7 lysozyme, which is a transcription inhibitor, an open promoter DNA fragment or a promoter DNA fragment being transcribed into RNA at initiation have been determined. For the first time, these structures describe in detail the intricate mechanism of transcription initiation by T7 RNA polymerase, which is likely to be a general model for other related RNA polymerases.

VPI-5: Structure refinement with single crystal synchrotron radiation diffraction data

Single crystal diffraction data have been recorded with synchrotron radiation for a very small crystal (70 × 10 × 10 urn) of the aluminophosphate VPI-5 ([Al 18 P 18 O 72 ] · 42H 2 O) and used for structure refinement. All of the framework atom positions in the model of McCusker, Baerlocher, Jahn and Bulow ( Zeolites 1991, 11, 308–313) in space group P 6 3 are fully confirmed and determined with slightly better e.s.d.s. Some additional detail on the network of water molecule positions is provided. The single crystal and powder methods are compared briefly.

Structure determination from small crystals of two aluminophosphates, CrAPO-14 and SAPO-43

Small single crystals, ca 10 5 μm 3 , of two alumino-phosphates have been used to determine their structures. Both compounds were synthesized with the aim of incorporating chromium into the framework, and for CrAPO-14 the X-ray diffraction results show that this has been achieved. For CrAPO-14, Al 3.96 Cr 0.04 (PO 4 ) 4 (OH)-C 3 H 10 N-H 2 O, data recorded on a Rigaku AFC-SR diffractometer (Cu Kα) and, independently, on an Enraf-Nonius FAST diffractometer (Mo Kα), showed the structure to be like that of GaPO-14 [Parise (1986). Acta Cryst. C42, 670-673]; aluminium atoms occupy four-, five- and six-coordinated sites, and 45% of the aluminium in the six-coordinate site is replaced by chromium

Crystal structure of the Pseudomonas aeruginosa MurG : UDP-GlcNAc substrate complex

MurG is an essential bacterial glycosyltransferase enzyme in Pseudomonas aeruginosa performing one of the key membrane steps of peptidoglycan synthesis catalyzing the transfer of N-acetyl glucosamine (GlcNAc) from its donor substrate, UDP-GlcNAc, to the acceptor substrate Lipid I. We have solved the crystal structure of the complex between Pseudomonas aeruginosa MurG and UDP-GlcNAc and compared it with the previously solved complex from E. coli. The structure reveals a large-scale conformational change in the relative orientations of the N- and C-terminal domains, which has the effect of widening the cofactor binding site and displacing the UDP-GlcNAc donor. These results suggest new opportunities to design potent inhibitors of peptidoglycan biosynthesis.

Synthesis and microcrystal structure determination of [Au10(PPh3)7{S2C2(CN)2}2] with monochromatic synchrotron radiation

The structure of this high nuclearity gold cluster has been determined from a single microcrystal of dimensions 10 × 10 × 30 µm (the X-ray diffraction data were recorded with synchrotron radiation using an Enraf-Nonius area detector diffractometer at Daresbury Laboratory); in the cluster three trigonal bipyramidal Au5 groups share a common central gold atom and share edges; the group often gold atoms has symmetry near to D3h, but when the seven triphenyl phosphine and two dithiolene ligands are taken into account the molecular symmetry is very approximately C2.

Synchrotron Radiation Laue Diffraction for the Time-Resolved Study of a Transformation in Crystals of P4N4Cl8

Experiments are described to show some of the potential of the synchrotron radiation Laue method for the study of structural change within single crystals. In the metastable tetragonal crystals of P(4)N(4)Cl(8) the eight-membered P(4)N(4) ring is in a boat conformation, with symmetry {\bar 4}. On heating to ca 340 K the crystals transform, slowly to a second tetragonal form in which the ring conformation is a chair, its symmetry {\bar 1}. Both structures are known [Hazekamp, Migchelsen & Vos (1962). Acta Cryst. 15, 539-543; Wagner & Vos (1968). Acta Cryst. B24, 707-713]. In the transformation the molecular packing, unit-cell dimensions and crystal quality remain almost unchanged. To study this transformation, series of Laue diffraction patterns were recorded at 2-3 min intervals over a period of 30-40 min, while the temperature was raised to 373 K. For two series, reflection intensities were measured and they allowed determination and refinement of the fraction of boat and chair molecules present in a mixed boat/chair model of the structure. No significant change in the crystal occurs below ca 340 K; at or above 340 K, 40-50% of the molecules are converted from boat to chair conformations within 5 min, but the remainder of the conversion is much slower, even when the temperature is raised towards 370 K.

Crystal structures and inhibitors of proteins involved in IL-2 release and T cell signaling

Publisher Summary This chapter presents a structural overview of several important enzymes involved in T cell signaling and Interleukin‐2 (IL‐2) release, and a summary of potential opportunities for the design of inhibitors that might improve upon Cyclosporin and FK‐506. The chapter describes the process through which potent inhibition of calcineurin readily translates into potent in vivo inhibition of IL‐2 secretion and immunosupression. The chapter identifies several other protein kinases that are implicated in signaling downstream of the T cell receptor (TCR) receptor and secretion of IL‐2 by T cells. While Zap‐70 is an established target for treating immune disease, the TEC‐family kinases are new and promising targets for treating immune disease. Inducible T cell kinase (ITK) is the most recent of these to be highlighted and is located immediately downstream of Lymphocyte specific kinase (Lck). It appears to be an attractive alternative to calcineurin as a therapeutic target, although inhibitors suitable for in vivo and clinical use are yet to be presented.

Correction: Structural basis for initiation of transcription from an RNA polymerase|[ndash]|promoter complex

This corrects the article DOI: 10.1038/19999