Center for Protein Engineering

Cambridge, United Kingdom

Center for Protein Engineering

Cambridge, United Kingdom
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Radford I.H.,Center for Protein Engineering | Fersht A.R.,Center for Protein Engineering | Settanni G.,Center for Protein Engineering
Journal of Physical Chemistry B | Year: 2011

Atomistic molecular dynamics simulations of the TZ1 beta-hairpin peptide have been carried out using an implicit model for the solvent. The trajectories have been analyzed using a Markov state model defined on the projections along two significant observables and a kinetic network approach. The Markov state model allowed for an unbiased identification of the metastable states of the system, and provided the basis for commitment probability calculations performed on the kinetic network. The kinetic network analysis served to extract the main transition state for folding of the peptide and to validate the results from the Markov state analysis. The combination of the two techniques allowed for a consistent and concise characterization of the dynamics of the peptide. The slowest relaxation process identified is the exchange between variably folded and denatured species, and the second slowest process is the exchange between two different subsets of the denatured state which could not be otherwise identified by simple inspection of the projected trajectory. The third slowest process is the exchange between a fully native and a partially folded intermediate state characterized by a native turn with a proximal backbone H-bond, and frayed side-chain packing and termini. The transition state for the main folding reaction is similar to the intermediate state, although a more native like side-chain packing is observed. © 2011 American Chemical Society.


Joerger A.C.,Center for Protein Engineering
Cold Spring Harbor perspectives in biology | Year: 2010

Even 30 years after its discovery, the tumor suppressor protein p53 is still somewhat of an enigma. p53's intimate and multifaceted role in the cell cycle is mirrored in its equally complex structural biology that is being unraveled only slowly. Here, we discuss key structural aspects of p53 function and its inactivation by oncogenic mutations. Concerted action of folded and intrinsically disordered domains of the highly dynamic p53 protein provides binding promiscuity and specificity, allowing p53 to process a myriad of cellular signals to maintain the integrity of the human genome. Importantly, progress in elucidating the structural biology of p53 and its partner proteins has opened various avenues for structure-guided rescue of p53 function in tumors. These emerging anticancer strategies include targeting mutant-specific lesions on the surface of destabilized cancer mutants with small molecules and selective inhibition of p53's degradative pathways.


Rajagopalan S.,Center for Protein Engineering | Andreeva A.,Medical Research Council Center | Rutherford T.J.,Medical Research Council Center | Fersht A.R.,Center for Protein Engineering
Proceedings of the National Academy of Sciences of the United States of America | Year: 2010

p53 maintains genome integrity either by regulating the transcription of genes involved in cell cycle, apoptosis, and DNA repair or by interacting with partner proteins. Here we provide evidence for a direct physical interaction between the tumor suppressors p53 and BRCA2. We found that the transactivation domain of p53 made specific interactions with the C-terminal oligonucleotide/oligosaccharide-binding-fold domains of BRCA2 (BRCA2 CTD). A second distinct site situated on the p53 DNA-binding domain, bound to a region containing BRC repeats of BRCA2 (BRCA2[BRC1-8]) and may contribute synergistically for high affinity association of intact full-length proteins. Overexpression of BRCA2 and BRCA2CTD suppressed the transcriptional activity of p53 with a concomitant reduction in the expression of p53-target genes such as Bax and p21. Consequently, p53-mediated apoptosis was significantly attenuated by BRCA2. The observed physical association of p53 and BRCA2 may have important functional implications in the p53 transactivation-independent suppression of homologous recombination and suggests a possible interregulatory role for both proteins in apoptosis and DNA repair.


van Dieck J.,Center for Protein Engineering | Lum J.K.,Center for Protein Engineering | Teufel D.P.,Center for Protein Engineering | Fersht A.R.,Center for Protein Engineering
FEBS Letters | Year: 2010

S100 proteins interact with the transactivation domain and the C-terminus of p53. Further, S100B has been shown to interact with MDM2, a central negative regulator of p53. Here, we show that S100B bound directly to the folded N-terminal domain of MDM2 (residues 2-125) by size exclusion chromatography and surface plasmon resonance experiments. This interaction with MDM2 (2-125) is a general feature of S100 proteins; S100A1, S100A2, S100A4 and S100A6 also interact with MDM2 (2-125). These interactions with S100 proteins do not result in a ternary complex with MDM2 (2-125) and p53. Instead, we observe the ability of a subset of S100 proteins to disrupt the extent of MDM2-mediated p53 ubiquitylation in vitro. © 2010 Federation of European Biochemical Societies.


Rossmann M.,University of Cambridge | Sukumaran M.,University of Cambridge | Penn A.C.,University of Cambridge | Penn A.C.,French National Center for Scientific Research | And 3 more authors.
EMBO Journal | Year: 2011

The assembly of AMPA-type glutamate receptors (AMPARs) into distinct ion channel tetramers ultimately governs the nature of information transfer at excitatory synapses. How cells regulate the formation of diverse homo- and heteromeric AMPARs is unknown. Using a sensitive biophysical approach, we show that the extracellular, membrane-distal AMPAR N-terminal domains (NTDs) orchestrate selective routes of heteromeric assembly via a surprisingly wide spectrum of subunit-specific association affinities. Heteromerization is dominant, occurs at the level of the dimer, and results in a preferential incorporation of the functionally critical GluA2 subunit. Using a combination of structure-guided mutagenesis and electrophysiology, we further map evolutionarily variable hotspots in the NTD dimer interface, which modulate heteromerization capacity. This flexibility of the NTD not only explains why heteromers predominate but also how GluA2-lacking, Ca2+-permeable homomers could form, which are induced under specific physiological and pathological conditions. Our findings reveal that distinct NTD properties set the stage for the biogenesis of functionally diverse pools of homo- and heteromeric AMPAR tetramers. © 2011 European Molecular Biology Organization.


Mittal R.,University of Cambridge | Peak-Chew S.Y.,University of Cambridge | Sade R.S.,Center for Protein Engineering | Vallis Y.,University of Cambridge | McMahon H.T.,University of Cambridge
Journal of Biological Chemistry | Year: 2010

Plague, one of the most devastating diseases in human history, is caused by the bacterium Yersinia pestis. The bacteria use a syringe-like macromolecular assembly to secrete various toxins directly into the host cells they infect. One such Yersinia outer protein, YopJ, performs the task of dampening innate immune responses in the host by simultaneously inhibiting the MAPK and NFκB signaling pathways. YopJ catalyzes the transfer of acetyl groups to serine, threonine, and lysine residues on target proteins. Acetylation of serine and threonine residues prevents them from being phosphorylated thereby preventing the activation of signaling molecules on which they are located. In this study, we describe the requirement of a host-cell factor for full activation of the acetyltransferase activity of YopJ and identify this activating factor to be inositol hexakisphosphate (IP6). We extend the applicability of our results to show that IP6 also stimulates the acetyltransferase activity of AvrA, the YopJ homologue from Salmonella typhimurium. Furthermore, an IP6-induced conformational change in AvrA suggests that IP 6 acts as an allosteric activator of enzyme activity. Our results suggest that YopJ-family enzymes are quiescent in the bacterium where they are synthesized, because bacteria lack IP6; once injected into mammalian cells by the pathogen these toxins bind host cell IP6, are activated, and deregulate the MAPK and NFκB signaling pathways thereby subverting innate immunity. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.


Vezzoli A.,Center for Protein Engineering | Bonadies N.,University of Cambridge | Allen M.D.,Center for Protein Engineering | Freund S.M.V.,Medical Research Council Laboratory of Molecular Biology | And 5 more authors.
Nature Structural and Molecular Biology | Year: 2010

Trimethylation of Lys36 in histone H3 (H3K36me3) coordinates events associated with the elongation phase of transcription and is also emerging as an important epigenetic regulator of cell growth and differentiation. We have identified the PWWP domain of bromo and plant homeodomain (PHD) finger-containing protein 1 (BRPF1) as a H3K36me3 binding module and have determined the structure of this domain in complex with an H3K36me3-derived peptide. © 2010 Nature America, Inc. All rights reserved.


Gossage L.,CRUK Cambridge Research Institute | Gossage L.,Center for Protein Engineering | Eisen T.,CRUK Cambridge Research Institute | Eisen T.,Addenbrookes Hospital
Clinical Cancer Research | Year: 2010

Anticancer drugs that target protein kinases include small molecule inhibitors and monoclonal antibodies. Feedback loops and cross talk between signaling pathways impact significantly on the efficacy of cancer therapeutics, and resistance to targeted agents is a major barrier to effective treatments. Increasingly, therapies are being designed to target multiple kinase pathways. This can be achieved using a single agent that inhibits multiple signaling pathways or a combination of highly selective agents. In this review we discuss the principles of specifically targeting multiple kinase pathways with particular reference to angiogenic signaling pathways. ©2010 AACR.


Van Den Ent F.,University of Cambridge | Johnson C.M.,Center for Protein Engineering | Persons L.,Case Western Reserve University | De Boer P.,Case Western Reserve University | Lowe J.,University of Cambridge
EMBO Journal | Year: 2010

Bacterial actin homologue MreB is required for cell shape maintenance in most non-spherical bacteria, where it assembles into helical structures just underneath the cytoplasmic membrane. Proper assembly of the actin cytoskeleton requires RodZ, a conserved, bitopic membrane protein that colocalises to MreB and is essential for cell shape determination. Here, we present the first crystal structure of bacterial actin engaged with a natural partner and provide a clear functional significance of the interaction. We show that the cytoplasmic helix-turn-helix motif of Thermotoga maritima RodZ directly interacts with monomeric as well as filamentous MreB and present the crystal structure of the complex. In vitro and in vivo analyses of mutant T. maritima and Escherichia coli RodZ validate the structure and reveal the importance of the MreB-RodZ interaction in the ability of cells to propagate as rods. Furthermore, the results elucidate how the bacterial actin cytoskeleton might be anchored to the membrane to help constrain peptidoglycan synthesis in the periplasm. © 2010 European Molecular Biology Organization | All Rights Reserved.


Rajagopalan S.,Center for Protein Engineering | Rajagopalan S.,University of Washington | Huang F.,Center for Protein Engineering | Huang F.,China University of Petroleum - East China | Fersht A.R.,Center for Protein Engineering
Nucleic Acids Research | Year: 2011

The state of oligomerization of the tumor suppressor p53 is an important factor in its various biological functions. It has a well-defined tetramerization domain, and the protein exists as monomers, dimers and tetramers in equilibrium. The dissociation constants between oligomeric forms are so low that they are at the limits of measurement by conventional methods in vitro. Here, we have used the high sensitivity of single-molecule methods to measure the equilibria and kinetics of oligomerization of full-length p53 and its isolated tetramerization domain, p53tet, at physiological temperature, pH and ionic strength using fluorescence correlation spectroscopy (FCS) in vitro. The dissociation constant at 37°C for tetramers dissociating into dimers for full-length p53 was 50 ± 7 nM, and the corresponding value for dimers into monomers was 0.55 ± 0.08 nM. The half-lives for the two processes were 20 and 50 min, respectively. The equivalent quantities for p53tet were 150 ± 10 nM, 1.0 ± 0.14 nM, 2.5 ± 0.4 min and 13 ± 2 min. The data suggest that unligated p53 in unstressed cells should be predominantly dimeric. Single-molecule FCS is a useful procedure for measuring dissociation equilibria, kinetics and aggregation at extreme sensitivity. © The Author(s) 2010. Published by Oxford University Press.

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