Carr Group

Structure and function of enzymes, protein complexes and viruses

 

We are working on structural and functional studies of biological macromolecules and assemblies. One of our particular interests is how enzymes work, for instance the nickel and iron-containing hydrogenase in bacteria, which can split hydrogen to generate electricity without using rare metals such as platinum.  We are also analyzing the detailed molecular structure of viruses, which will help to understand how they are assembled and function in cells, and large protein complexes involved in the processing and repair of DNA.  The approaches used are three-dimensional structure determination with X-ray crystallography or cryo-electron microscopy, biophysical methods, chemistry and molecular biology.

 

Hydrogenase: [NiFe]-Hydrogenases are metalloenzymes that contain nickel and iron ions at their active site, which allow bacteria to use hydrogen as an energy source.  This is achieved   by enzymatic splitting of hydrogen gas (H2) into protons and electrons.  We are using structural analysis and electrochemistry (in collaboration with Prof. Fraser Armstrong at the University of Oxford) to understand how the enzyme tunes the properties of nickel and iron ions bound within the protein in order to safely split hydrogen.  Analysis of a number of variant proteins containing mutated amino acids at the active site of the protein has allowed us to propose a novel mechanism for how [NiFe]-hydrogenases can achieve this.  We aim to use ultra-high resolution X-ray crystallography and neutron diffraction to analyse intermediates of the catalytic cycle to fully understand how these enzymes work with a view to using the active site as inspiration for the design of new, inexpensive chemical catalysts based on Ni/Fe

 

 

Plasmid replication and antibiotic resistance: In collaboration with Dr Chris Thomas at the University of Leeds, we are studying how such plasmids replicate in medically important bacteria.  Plasmids are small circular pieces of DNA that can be transferred between bacteria and are a major source of antibiotic resistance in bacterial populations, representing a serious public health concern, particularly when acquired by bacteria that can cause disease.   Plasmid replication is governed by the Rep family of proteins and we have solved the structure of three family members allowing us to better understand how these proteins enable DNA replication.  We now aim to solve structures of the Rep proteins bound to DNA and a partner protein (PcrA) to enhance our understanding of the replication process.  The Rep proteins are also attractive targets for novel antibiotics and a longer term goal is to use the structures to aid in their rational design.

 

 

Molecular organisation of archaeal viruses : Archaeal viruses form a distinct group of microorganisms with unusual morphologies, genome sequences and protein folds. These unique features are closely linked to the metabolism of their hosts and the particular environment where they live. We are investigating the structures of hyperthermophilic archaeal viruses APBV1 and SSV1 to allow a better understanding of the adaptation of these microorganisms to extreme conditions and their interaction with their host. In collaboration with the group of Dr. David Prangishvilli (Institut Pasteur, Paris) we have recently solved the structure of APBV1 virus with a helical symmetry by cryo-electron microscopy to near atomic resolution which allowed to identify the key determinants of viral thermostability and the organisation of the viral DNA genome.

 

 

Molecular mechanism of DNA repair by the SLX4 complex:  Maintaining genome integrity is essential for normal cellular function and for the accurate propagation of the genome in all organisms. SLX4 is a highly-conserved multi-domain platform protein, binding to and activating a number of structure-selective DNA endonucleases. These endonucleases, MUS81-EME1, SLX1 and XPF-ERCC1 play pivotal roles in homologous recombination, DNA interstrand crosslink repair and telomere maintenance. Recent work has elucidated the action of SLX4 and its associated nucleases at the biochemical, genetic and cellular level, but our understanding of their detailed molecular mechanism remains incomplete owing to limited structural information. The aim of our project is to produce a comprehensive structural understanding of SLX4 and its nuclease partners using cryo-electron microscopy (in collaboration with Prof Peter McHugh, Weatherall Institute of Molecular Medicine, University of Oxford).

 

 

Anti-cancer drug design: In a collaboration led by Prof Terry Rabbitts group at Oxford, we are using protein crystallography, along with medicine chemistry and cell biology, aimed at structure-based drug design to inhibit RAS.  Human RAS is a protein in cells that has a key role in signalling networks, such as instructing a cell to grow in response to exposure of the outside of the cell to a growth hormone.  The signalling is mediated by the RAS protein interacting specifically with other proteins in the cell.  Mutations in the RAS pro

tein are present in many human cancers, and can affect the interactions with other proteins, resulting in inappropriate signalling, such as uncontrolled growth.  We are developing small molecules that bind to mutant forms of RAS to inhibit such interactions and switch off the growth signal. The long-term goal is to provide lead compounds that could result in the development of new anti-cancer drugs.

 


Recent publications
Evans, R.M., Brooke, E.J., Wehlin, S.A.M., Nomerotskaia, E., Sargent, F., Carr, S.B., Phillips, S.E.V. and Armstrong, F.A. (2016)
“Mechanism of Hydrogen Activation by [NiFe]-hydrogenases”
Nature Chemical Biology 12, 46-50. doi: 10.1038/nchembio.1976

Carr, S.B., Phillips, S.E.V.  and Thomas, C.D. (2016)
“Crystal Structures of Rolling-Circle Replication Initiator Proteins from Geobacillus
stearothermophilus and Staphylococcus aureus”
NAR 44(5), 2417-2428. doi: 10.1093/nar/gkv1539

 

Carr, S.B., Evans, R.M., Brooke, E.J., Wehlin, S.A.M., Nomerotskaia, E., Sargent, F., Armstrong, F.A.  and Phillips, S.E.V. (2016)
“Hydrogen Activation by [NiFe]-hydrogenases”
Biochem. Soc. Trans. 44(3), 863-868. doi:10.1042/BST20160031

Punekar, A.S., Porter, J., Urbanowski, M.L., Stauffer, G.V., Carr, S.B. and Phillips, S.E.V. (2016)
“Structural basis for DNA binding by the transcription regulator MetR”
Acta Cryst. F72, 417–426.  doi:10.1107/S2053230X16006828

Patel, N., Dykeman, E., Coutts, R. H.A., Lomonossoff, G.P., Rowlands, D.J., Phillips, S.E.V., Ranson, N.A., Twarock, R., Tuma, R. and Stockley, P.G. (2015)
“Revealing the density of encoded functions in a viral RNA”
PNAS 112, 2227-2232. doi/10.1073/pnas.1420812112

Ma, C., Hao, Z., Huysmans, G., Lesiuk, A., Bullough, P., Wang, Y., Bartlam, M., Phillips, S.E., Young, J.D., Goldman, A., Baldwin, S.A. and Vincent L.G. Postis (2015)
“A versatile strategy for production of membrane proteins with diverse topologies: application to investigation of bacterial homologues of human divalent metal ion and nucleoside transporters.”
PLOS One, 10, e0143010. Doi: 10.1371/journal.pone.0143010

Luger, K. and Phillips, S.E.V. (2014)
“Nucleic acid movers and shakers”
Current Opinion in Structural Biology, 24, v-vii

Daniels, A.D., Campeotto, I., van der Kamp, M.W., Bolt, A.H., Trinh, C.H., Phillips, S.E.V., Pearson, A.R., Nelson, A, Mullholland, A.J. and Berry, A. (2014)
“The reaction mechanism of N-acetylneuraminic acid lyase revealed by a combination of crystallography, QM/MM simulation and mutagenesis”
ACS Chem. Biol, 9, 1025-1032.

Yuzugullu, Y., Trinh, C.H., Smith, M.A., Pearson, A.R., Phillips, S.E.V., Kobacas, D., Bolukbasi, U., Ogel, Z. and McPherson, M.J. (2013)
“Crystal structure, recombinant expression and mutagenesis studies of the bifunctional catalase-phenol oxidase from Scytalidium thermophilum”
Acta Cryst D, 69, 398-408.

Ford, R.J., Barker, A.M., Bakker, S.E., Coutts, R.H., Ranson, N.A., Phillips, S.E.V., Pearson, A.R. and Stockley, P.G. (2013)
“Sequence-specific, RNA-protein interactions overcome electrostatic barriers preventing assembly of Satellite Tobacco Necrosis Virus coat protein”
J.Mol.Biol., 425, 1050-1064. Doi 10.1016/j.jmb.2013.01.004

Stockley, P.G., Twarock, R., Bakker, S., Barker, A.M., Borodavka, O., Dykeman, E.C., Ford, R.J., Pearson, A.R., Phillips, S.E.V. and Tuma, R. (2013)
“Packaging signals in single-stranded RNA viruses: Nature’s alternative to a purely electrostatic assembly mechanism”
J Biol Physics, 39, 277-287.  Doi 10.1007/s10867-013-9313-0

Phillips, S.E.V., Carr, S.B., Mecia, L.B., Stelfox, A.J. and Thomas, C.D. (2013)
“Structural Studies of Rolling Circle Replication Initiator Proteins”
Biophys. J. 104:2, 73A-74A.

Carr, S.B., Mecia, L.B., Stelfox, A.J., Phillips, S.E.V. and Thomas, C.D. (2013)
“Structural Studies of Rolling Circle Replication Initiator Proteins”
J. Biomol. Struct. Dynamics, 31:1, 50-50.

Carr, S.B., Mecia, L.B., Phillips, S.E.V. and Thomas.  C. D. (2013)
“Identification, characterisation and preliminary X-ray diffraction analysis of rolling-circle replication initiator protein from plasmid pSTK1”
Acta Cryst F, 69, 1123-1126.

van Geersdaele, LK, Stead, MA, Harrison, CM, Carr, SB, Close, HJ, Rosbrook, GO, Connell, SD and Wright, SC (2013)
“Structural basis of high-order oligomerization of the cullin-3 adaptor SPOP”
Acta Cryst. D, 69, 1677-1684. Doi 10.1107/S0907444913012687

Cruz-Migoni, A, Fuentes-Fernandez, N, and Rabbitts, TH (2013)
“Peptides: minimal drug surrogates to interrogate and interfere with protein function”
Med. Chem. Comm. 4, 1218-1221. Doi 10.1039/c3md00142c