... understanding life in molecular detail

Prof Simon Phillips

Protein-DNA Interactions, Enzyme Structure, X-ray Crystallography

Research interests: The laboratory group is mainly working on structural and functional studies of biological macromolecules, with particular interests centring on the critical biological problem of how these molecules recognize each other. This highly specific molecular recognition underpins the function of all complex biological systems. The approach is three-dimensional structure determination with X-ray crystallography as the main technique, although other structural techniques are employed, as well as routine protein chemistry and molecular biology.

Research projects:

Protein-DNA interactions -

Some proteins recognize specific DNA sequences that stimulate them to carry out particular functions, such as turning transcription of genes on and off, or transferring genes from one DNA molecule to another in recombination events. We can to obtain a detailed atomic structure of complexes, such as the met repressor/DNA complex shown below, by using X-ray crystallography. The determination of the metJ/DNA complex led to a model which involves a novel "electrically operated molecular switch", turning genes on and off in response to the cellular levels of the electrically charged co-repressor SAM (S-adenosylmethionine). In collaboration with Peter Stockley’s group we are dissecting the molecular details of metJ/DNA recognition using structural and functional studies of mutant repressors and their complexes.

The structure of the E.coli met-repressor/DNA-operator complex determined by X-ray crystallography. (The DNA is shown in atom colours, the co-repressor SAM in cyan)

We are also interested in the molecular structure of junctions formed by two DNA molecules as they come together in genetic recombination events. This "Holliday Junction" structure, and the way specific enzymes cleave (resolve) it to produce separate DNA duplexes, is poorly understood at the molecular level. The structural analysis is underway of a resolving enzyme and of its complex with a synthetic DNA junction. Further projects on structures of protein-nucleic acid complexes are aimed at restriction endonucleases, RNA-methyltransferases and transcriptional activators.

Plasmid replication and antibiotic resistance-

Another project, in collaboration with Chris Thomas’ group is concerned with proteins that control replication of plasmids, small circular DNA molecules that carry foreign genes into bacteria. Such plasmids can harbour genes for antibiotic resistance apart from a few other genes dedicated to their own replication. Antibiotic resistance is becoming a major public health problem. The Rep proteins, which govern replication by binding to the replication origin on a plasmid, are potential targets for new antibiotic compounds, but structural information on members of the Rep protein family is scarce. The long-term goals are to understand the replication-initiation process and provide structural targets for rational design of new generations of antibiotics. We are also interested in development of new potential antibiotics by structure determination of essential enzymes in bacterial pathogens followed by structure based drug design.

RNA strructure and RNA-protein interactions -

The structures of RNA motifs are currently of great interest, not least because some viruses depend on them for viability. Such RNA molecules are, therefore, targets for development of antiviral drugs. RNA structures are more difficult to crystallise than proteins, but, in collaboration with Peter Stockley and Nicola Stonehouse’s groups we have developed a method for determining such structures using X-ray crystallography by immobilising them in pre-existing virus crystals. We have recently published as series of structures of small RNA "aptamers" generated by in vitro selection techniques, thus validating the method. This should allow us in future to carry crystal structure determinations of small RNA fragments, and their complexes with potential drug molecules, routinely.

Structural enzymology -

The ultimate goal of structural molecular biology must be to understand the function as well as the structure of biological molecules. In the case of enzymes this entails the elucidation of all the steps along the pathway of the catalytic reaction. In collaboration with Peter Knowles’ and Mike McPherson’s groups we have been studying a class of copper-containing enzymes that oxidise various substrates using molecular oxygen as the oxidant. These copper oxidases are able to harness the oxidising power of atmospheric oxygen to carry out chemically difficult reactions such as cleavage of carbon-hydrogen bonds. Copper-containing amine oxidase is a ubiquitous enzyme that occurs in all classes of organisms from bacteria to humans. It catalyses the oxidation of primary amines to the corresponding aldehydes while reducing oxygen to hydrogen peroxide, making use of a novel quinone cofactor that is derived from a tyrosine side-chain. We have succeeded in trapping several reaction intermediates by passing substrate into crystals of the enzyme and trapping the reaction at various stages by rapid freezing to cryogenic temperatures (-170C). We are making good progress towards our goal of making a "molecular movie" of catalysis showing each stage of the reaction in atomic detail. We have already determined for the first time the structure of an intermediate where molecular oxygen is bound to a single copper centre. The techniques should also be applicable to other enzyme reactions.

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