Structural Biology, Structural Bioinformatics and Structure-Guided Drug Discovery
Living cells - their growth and multiplication - are regulated by messengers, such as growth factors, which are recognised by cell surface receptors. This leads to signals that are transduced inside the cell by further messengers or by chemical modification. DNA damage activates similar signalling pathways. These must all have high signal-to-noise ratios, just like electrical circuits; indeed these systems have switches, transducers, adaptors and so on.
But how do molecular systems in cells achieve the required sensitivity and specificity? The answer cannot be in terms of very tight, enduring molecular complexes, as the signals could not be turned off. On the other hand, weak binary complexes would lack specificity and give rise to a noisy system. The answer is to be found in multicomponent protein complexes, where weak binary interactions are transformed through cooperativity into very specific control systems. Some components lack structure on their own, but assemble into an ordered whole, sometimes through zipper mechanisms.
The objective of our research programme is to define the architecture and describe the interactions that characterise these often transient molecular complexes. The research includes many systems that are responsible for mediating disease processes and are therefore important for a rational approach to therapeutic intervention.
- Blundell, T.L., Bolanos-Garcia, V., Chirgadze, D.Y., Harmer, N.J., Lo, T., Pellegrini, L., Sibanda, L.B. (2002) Asymmetry in the multiprotein systems of molecular biology (2002) Structural Chemistry 13 (3-4), 405-412.
- Blundell TL, Burke DF, Chirgadze D, Dhanaraj D, Hyvonen M, Innis A, Parisini E, Pellegrini L, Sayed M and Sibanda BL (2000). Protein-protein interactions in receptor activation and intracellular signalling. Biol. Chem. 381, 955-959.
Cell Surface Receptors
Investigators: Dr Anja Winter, Dr Michal Blaszczyk, Anna Sigurdardottir.
Collaborators: Ermanno Gherardi, Cambridge
Research has concentrated on receptor tyrosyl kinases with a single transmembrane helix and a stable and globular ectodomain that is amenable to crystallisation. In each case we have been interested in the way the ligands organise the receptors in multiprotein complexes.
Fibroblast Growth Factors and their Receptors:
Members of the FGF family, implicated in cell proliferation, differentiation, survival, and migration, demonstrate an exquisite pattern of affinities for both protein and proteoglycan receptors. We have solved structures of both FGF18 (2.55 resolution) and FGF19 (1.3 resolution; Harmer et al.,
2004a): FGF19 is one of the most divergent human FGFs that binds only to FGFR4.
Our structure of fibroblast growth factor receptor 2 (FGFR2) in complex with its ligand (FGF1) and heparin at 2.8 resolution (Pellegrini et al
., 2000) (Figure 1a) reveals a series of interactions between FGF2, FGFR1 and heparin. These include heterotetramers in which two FGF:FGFR heterodimers pack back to back, and interact with one heparin, thus including a unit similar to that identified by Schlessinger and coworkers. The structure also revealed a 2:2:1 FGF1-FGFR2-heparin decasaccharide complex, in which a single heparin chain bridges two FGF1-FGFR2 complexes (Pellegrini et al.,
2000). Analyses of complexes prepared using different protocols with nanospray mass spectrometry (with Carol Robinson) and analytical ultracentrifugation (Harmer et al.,
2004b) showed that the stoichiometries of both were 2 FGF1: 2 FGFR2: 1 heparin. We have suggested that both complexes contribute to the formation of a larger focal complex (Figure 1B). With John Gallagher we found that 2:2:1 complex formed spontaneously in solution between FGF1, FGFR2 and heparin decasaccharide and less efficiently with octasaccharide (Robinson et al.,
- Goodger SJ, Robinson CJ, Murphy KJ, Gasiunas N, Harmer NJ, Blundell TL, Pye DA, Gallagher JT (2008) Evidence that heparin saccharides promote FGF2 mitogenesis through two distinct mechanisms. J Biol Chem 283(19) 09 May 2008
- Ryu EK, Cho KJ, Kim JK, Harmer NJ, Blundell TL, Kim KH (2006) Expression and purification of recombinant human Fibroblast growth factor receptor in Escherichia coli. Science Direct Protein Expression and Purification 49: 15-22
- Robinson CJ, Harmer NJ, Goodger SJ, Blundell TL, Gallagher JT. (2005) Cooperative dimerization of fibroblast growth factor 1 (FGF1) upon a single heparin saccharide may drive the formation of 2 : 2 : 1 FGF1:FGFR2c:heparin ternary complexes. J Biol Chem 2005, 280(51):42274-42282
- Harmer NJ, Pellegrini L, Chirgadze D, Fernandez-Recio J, Blundell TL (2004) The crystal structure of fibroblast growth factor (FGF) 19 reveals novel features of the FGF family and offers a structural basis for its unusual receptor affinity. Biochemistry 43, 629-40.
- Harmer NJ, Ilag LL, Mulloy B, Pellegrini L, Robinson CV, Blundell TL (2004). Towards a resolution of the stoichiometry of the fibroblast growth factor (FGF)-FGF receptor-heparin complex. Journal of Molecular Biology 339, 821-34
- Pellegrini L., Burke D.F., von Delft F., Mulloy B., Blundell TL (2000) Crystal Structure of fibroblast growth factor receptor ectodomain bound to ligand and heparin. Nature 407, 1029-1034
- Pellegrini L, Burke DF and Blundell TL (2002) Activation mechanism of fibroblast growth factor receptor tyrosine kinase revealed by crystal structure of fibroblast growth factor receptor ectodomain bound to fibroblast growth factor and heparin. In Insulin and related proteins structure to function and pharmacology. 189-200.
- Harmer NJ, Chirgadze D, Kim K-H, Pellegrini L, Blundell TL (2002) The structural biology of growth factor receptor activation. Biophysical Chemistry 100, 545 - 553
- Nagendra, H.G., Harrington, A.E., Harmer, N.J., Pellegrini, L., Blundell, T.L. and Burke, D.F. (2001). Sequence analyses and comparative modeling of fly and worm fibroblast growth factor receptors indicate that the determinants for FGF and heparin binding are retained in evolution. FEBS Letters 501, 51-58.
Hepatocyte Growth Factor / Scatter Factor (HGF/SF) and Met receptor
HGF/SF causes cell movement and growth, playing essential roles in development of placenta, liver, and distinct groups of skeletal muscle, motor and sensory neurons. HGF/SF signals through the Met receptor tyrosyl kinase, first discovered as a re-arranged oncogene. Single-chain HGF/SF binds but does not activate MET and behaves as a receptor antagonist in vivo
. The process of activation of single-chain HGF/SF is functionally equivalent to that of the complex serine proteinases of the blood coagulation and fibrinolytic pathways.
With Ermanno Gherardi we defined structures of the partial agonist NK1 (N + K1) (Chirgadze et al.
, 1999; Watanabe et al
., 2002) and its complexes with heparin (Leitha et al
., 2001), which define a major heparin N-domain interaction that leaves some heparin surface free for interactions with K2 and Met (Figure 1c). We have determined the crystal structure at 1.85 resolution of the serine proteinase homology domain (SPH) of HGFl/MSP, which contains the high-affinity binding site for the Met homologue, the Ron receptor (Carafoli et al.,
The MET ectodomain
behaves as an elongated monomer with a large Stoke's radius in solution. We showed that the N-terminal ligand-binding domain was likely to adopt a beta-propeller fold similar to the N-terminal domain of aV integrin, and that the C-terminal half contains four immunoglobin domains of the unusual structural E set. We provided three-dimensional models and a functional map of the MET ectodomain (Gherardi et al
., 2003). Small-angle X-ray scattering of the complex of HGF/SF and a truncated form of MET (MET567) indicates. that the basic signalling unit may be a 2:2 complex formed around a dimerisation interface similar to that of the NK1 crystal structure (Gherardi et al.,
- Gherardi E, Sandin S, Petoukhov MV, Finch J, Youles ME, Ofverstedt L-G, Miguel RN, Blundell TL, Vande Woude GF, Skoglund U, Svergun DI (2006) Structural basis of hepatocyte growth factor/scatter factor and MET signalling. Proc Natl Acad Sci USA 103:4046-405
- Carafoli F, Chirgadze DY, Blundell TL, Gherardi E (2005) Crystal Structure of the b-Chain of Hgfl/Msp. FEBS Journal 272 (2005) 5799-5807
- Gherardi E, Youles MY, Miguel RN, Blundell TL, Iamele L, Gough J, Bandyopadhyay A, Hartmann G, Butler JG (2003) Functional map and domain structure of the MET, the product of the c- met protooncogene and receptor for hepatocyte growth factor scatter factor, receptor. Proc. Natl. Acad. Sci (USA) 100, 12039-12044
- Watanabe K, Chirgadze DY, Lietha D, .de Jonge H, Blundell TL and Gherardi E (2002). A new crystal form of the NK1 splice variant of HGF/SF demonstrates extensive hinge movement and suggests that the NK1 dimer originates by domain swapping. J.Mol.Biol 319, 283-288.
- Leitha D, Chirgadze DY, Mulloy B, Blundell TL, Gherardi E (2001) Crystal structures of NK1 heparin complexes reveal the basis for NK1 activity and enable engineering of potent agonists and the MET receptor. EMBO J. 20, 5543-5555
- Chirgadze, D.Y., Hepple, J.P., Zhou, H., Byrd, R.A., Blundell, T.L. & Gherardi, E. (1999). Crystal structure of the NK1 fragment of HGF/SF suggests a novel mode for growth factor dimerization and receptor binding . Nature Structural Biology 6, 72-79.
- Nagendra, H.G., Harrington, A.E., Harmer, N.J., Pellegrini, L., Blundell, T.L. and Burke, D.F. (2001). Sequence analyses and comparative modeling of fly and worm fibroblast growth factor receptors indicate that the determinants for FGF and heparin binding are retained in evolution. FEBS Letters 501, 51-58.
Nerve Growth Factor (NGF) and its low affinity p75 receptor
Neurotrophins are important in development and survival of certain neuronal populations in both central and peripheral nervous systems. Our early work on NGF was the first to describe a cystine-knot and defined an extensive hydrophobic interface in a symmetrical dimer (Figure 1d: McDonald et al.,
1991). We showed that this was protected in the symmetrical hetero-hexameric 7S NGF complex comprising b-NGF dimer surrounded by two a-NGF and two gNGF subunits (Figure 1e: Bax et al.,
The structures of the b-NGF dimer, the 7S-NGF complex and the TrkA receptor domain complex show that a stable symmetrical dimer is very efficient organiser of the symmetry of multiprotein complexes in which it is involved, and this brings together receptor dimers in a way that would facilitate transphosphorylation of receptor tyrosyl kinases and activation of the downstream signalling pathway. The structures also provide us with insights into how specific signalling can arise in multiprotein complexes, allowing reliable interactions in crucial developmental pathways and immune cell development.
We have now expressed human low affinity receptor p75NTR
ligand-binding domain as a glycosylated secreted protein; mass spectrometry, analytical ultracentrifugation and solution X-ray scattering measurements indicate a 2:2 stoichiometry (Figure 1f: Aurikko et al.,
- Loew, A., Ho Y-K., Blundell, TL. and Bax (1998) Phosducin induces a structural change in transducin bg. Structure 6, 1007-1019 184.
- Bax, B., Blundell, TL., Murray-Rust, J. and McDonald N.Q. (1997) Structure of mouse 7S NGF: a complex of nerve growth factor with four binding proteins. Structure. 5: 1275-1285.
- McDonald N, Lapatto R, Murray-Rust J, Gunning J, Wlodawer A and Blundell TL (1991) New Protein fold revealed by a 2.3 resolution crystal structure of nerve growth factor. Nature 345: 411-414
Investigators: Dr Victor Bolanos Garcia, Ann Lee Ling.
Collaborators: Prof Ernest Laue, Prof Alfonso Martinez Arias, Cambridge
Cdk6 protein kinase in complex with cell cycle inhibitor p19INK4
The structure of the cyclin D-dependent kinase Cdk6 complexed with cell cycle inhibitor p19INK4d at 1.9 resolution, defined in collaboration with Ernest Laue (Brotherton et al.,
1998), (Figure 1g) provided the first structural information for a cyclin D-dependent protein kinase and showed that the INK4 family of Cdk inhibitors do not interfere directly with ATP or substrate binding but rather stabilise a conformation that does not bind ATP.
Brotherton, DH., Dhanaraj, V., Wick, S., Brizuela, L., Domaille, PJ., Voyanik, E., Xu, X., Parisini, E., Smith, OB., Archer, SJ., Serrano, M., Brenner, SL., Blundell, TL. and Laue, ED. (1998) Crystal structure of the complex of the cyclin D-dependent kinase Cdk6 bound to the cell cycle inhibitor p19INK4d
. Nature 395, 244-250
CK2 protein kinase
Protein kinases CK2 (casein kinase 2) are a ubiquitous family of eukaryotic enzymes, usually composed of protein kinase catalytic subunits (α) and (α'), and smaller regulatory subunits (β) arranged as heterotetramers. They may be involved in the regulation of cell division and indeed the α-subunits are most closely related in sequence to the cyclin-dependent kinases. However, the regulatory subunits, CK2b, show no significant sequence similarity to those of various cyclins, except for the "cyclin destruction box". Together with our collaborators Catherine Allende and Jorge Allende (Santiago) we have designed and made site-directed mutants of CK2α, and constructed a three-dimensional model, to investigate the basis for the dual specificity for ATP or GTP. The structural and functional effects of the regulatory subunit are not well understood, although the structures of the two separate subunits and a complex of Zea mays CK2 and a peptide of CK2b subunit have been defined elsewhere. CK2β binds tightly to several substrates of CK2α such as p53, thus contributing to an increase in the effective substrate concentration in the vicinity of the catalytic active site. We have also defined the structure of the regulatory subunit of CK2 in the presence of a p21WAF1 peptide, demonstrating the flexibility of the acidic loop.
- Bolanos-Garcia VM, Fernandez-Recio J, Allende JE and Blundell TL (2006) Identifying interaction motifs in CK2b - a ubiquitous kinase regulatory subunit. Trends in Biochemical Sciences 31, 654-661
- Bertrand L, Sayed MFR, Pei X-Y, Parisini E, Dhanaraj V, Bolanos-Garcia VM, Allende JE, Blundell TL (2004) Structure of the regulatory subunit of CK2 in the presence of a p21WAF1 peptide shows the flexibility of the acidic loop. Acta Cryst. D 60, 1698-1704.
- Srinivasan N, Antonelli M, Jacob G, Korn I, Romero F, Jedlicki A, Dhanaraj V, Sayed M F-R, Blundell TL, Allende CC, Allende JE (1999) Structural interpretation of site-directed mutagenesis and specificity of the catalytic subunit of protein kinase CK2 using comparative modelling. Protein Engineering. 12, 119-127.
- Korn I, Gutkind S, Srinivasan N, Blundell TL, Allende CC, Allende JE (1999) Interactions of protein kinase CK2 subunits. Molecular and Cellular Biochemistry. 191, 75-83.
Phosphatases: Structure, Specificity Mechanism
-dependent phosphatases have been identified in most species on the basis of their two highly conserved sequence motifs. We have defined the crystal structures of inositol phosphatases from yeast Hal2p from Saccharomyces cerevisiae
. and of six Hal2 metal ion complexes were solved at 1.3-1.75 resolution in order to understand ion selectivity and catalytic mechanism (Albert et al.,
The crystal structure of RnPIP from Rattus norvegicus
, a dual specificity enzyme, displays a core fold similar to other Li+
-dependent phosphatases with greatest similarity to that of IPPase whose activity it also shares. Modelling shows that the active site can accommodate both PAP and I1,4
. A mechanism similar to that of Hal2 is proposed (Patel et al. 2002; Patel & Blundell, 2002).
- Patel S, Martinez-Ripoll M, Blundell TL and Albert A (2002) Structural Enzymology of Li + sensitive/MG2 dependent Phosphatases. J. Mol. Biol. 320, 1087-1094.
- Patel S, Blundell TL (2002) Crystal Structure of an Enzyme Displaying both Inositol-Polyphosphate 1-Phosphatase and 3Õ-Phosphoadenosine-5Õ-Phosphate Phosphatase Activities: A Novel Target for Lithium Therapy. J. Mol. Biol. 315, 677-686.
- Albert A, Yenush L, Gil-Mascarell MR, Rodriguez PL, Patel S, Martinez-Ripoll M, Blundell TL, Serrano R (2000) X-ray structure of yeast Hal2p, a major target of lithium and sodium toxicity and identification of framework interactions determining cation sensitivity. Journal of Molecular Biology 295, 927-938.
G-bg and phosducin
The structure of farnesylated transducin-bg from bovine retina complexed with phosducin at 2.8 resolution (Loew et al.,
1998; Figure 1h) led to the unexpected discovery of a conformational change in transducin-bg leading to a binding pocket for farnesyl and stabilisation of the Gbg-phosducin
as a soluble complex. This showed how the complex is sequestered away from the membrane. The surface areas involved are large, with Gb to Gg of 5,100 2
and Gb with phosducin of 4,400 2
, characteristic of stable, adaptor, and inhibitory functions.
- Loew, A., Ho Y-K., Blundell, TL. and Bax (1998) Phosducin induces a structural change in transducin bg. Structure 6, 1007-1019
Notch Receptor Ankyrin Domain:
The Notch receptor is the key element of a highly conserved signalling system of central importance to animal development. As a first step to the structural analysis of Notch and functionally related proteins, we defined the crystal structure of the human Notch 1 ankyrin domain, a key mediator of the activity of Notch. Our 1.9 resolution structure (Figure 1i) shows that the domain has six of the seven ankyrin repeats predicted from sequence (Ehebauer et al.,
2005). The putative first repeat is disordered in both molecules in the asymmetric unit, possibly due to absence of the N-terminal RAM region, which plays a role in the interaction with other effectors.
- Ehebauer MT, Chirgadze DY, Hayward P, Martinez Arias A, Blundell TL. (2005) High-resolution crystal structure of the human Notch 1 ankyrin domain. Biochemical Journal 392, 13
DNA Damage Signalling
The overall aim is to understand the architectures of the transient multicomponent complexes that mediate double strand-break repair through Non-Homologous End Joining and Homologous Recombination in order to understand the molecular basis of the repair and to provide opportunities for therapeutic intervention.
Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR) comprise the two major modes of DNA double-strand break (DSB) repair in human cells. It is predicted that in humans about 50 endogenous DSBs per cell during each cell cycle may occur. DSBs are mainly generated by ionizing radiation, reactive oxygen species and DNA replication across a nick. Unrepaired DSBs can cause catastrophic gene loss during cell division, leading to chromosomal translocations, increased mutation rates and carcinogenesis). HR is dominant in late S/G2 phases because it requires a sister chromatid. On the other hand, NHEJ plays a major role in G1/early S phase, because it does not require a template. The NHEJ system is also responsible for programmed DSBs in V(D)J recombination and class switch recombination (during development of immune diversity.
Non-Homologous End Joining
Lynn Sibanda, Dima Chirgadze, Victor Bolanos Garcia, Takashi Ochi and Qian Wu.
Stephen Jackson, Cambridge
The interaction diagram (Figure 1j ) summarises the current understanding of NHEJ protein interactions and phosphorylation by DNA-PKcs. Colour-filled shapes indicate the proteins and complexes with known 3-D structures. Our objectives are to define: Crystal structure of DNA-PKcs at higher resolution,
which will lead to a better understanding of the target for use in structure-guided drug discovery. Organisation of DNA-PKcs/Ku/DNA complex (known as DNA-PK),
which will shed light on the initial events that take place in the NHEJ pathway.
- 3D structures of DNA ligase IV/XRCC4/XLF/DNA complexes, which should give clues about the binding and functional mechanism of XLF and DNA ligase IV/XRCC4 in NHEJ.
- Spatial arrangement of higher order complexes in order to give a picture of the NHEJ repair system as a whole.
A spatial and temporal understanding of these processes should provide insights into the mechanism of this critical cellular process and give clues for designing chemical tools that could be used to further investigate NHEJ. These tools would also likely contribute to the discovery of lead compounds and design of preclinical candidates that could allow therapeutic intervention at allosteric and other regulatory interaction sites - allosites - in oncology and for patients with defects in the pathway.
The NHEJ pathway comprises three major steps: synapsis, end processing and ligation. Synapsis is carried out by DNA-dependent protein kinase (DNA-PK) consisting of Ku70, Ku80, DNA-PK catalytic subunit (DNA-PKcs) and DNA. Ku70 and Ku80 form a ring-shaped heterodimer around the broken DNA ends and maintain them in proximity. DNA-PKcs, a very large protein belonging to the phosphatidylinositol-3-OH kinase (PI3K)-related kinase (PIKKs) family, is recruited through interaction with the C-terminus of Ku80, and causes the Ku70/80 heterodimer to move about one helical turn inward from the end to make space for DNA-PKcs to bind DNA. Activated DNA-PKcs phosphorylates itself and various proteins, including the other NHEJ components.
The end processing involves nucleases including Artemis, which is capable of cutting an array of DNA overhangs and is thought to be sufficient as a nuclease, although other nucleases in particular PNK, aprataxin and PNK-APTX-like factor may play a role. The final ligation step of rejoining is mediated by DNA ligase IV (LigIV), which is associated with dimeric XRCC4. XRCC4 stimulates adenylation and ligase activity.
Considerable advances have been made in the structural biology of individual components and complexes of the NHEJ repair machinery, but further work is required to understand the spatial organisation of this complex and the dynamic process. The crystal structure of the Ku70/80 (Figure 1k), which does not include the C-terminal DNA-PKcs interaction domain of Ku80 (Ku80CTD), and its complex with a DNA fragment revealed a ring structure that encircles the duplex DNA (see figure). No large conformational changes occur on binding of heterodimeric Ku except for the DNA binding C-terminal domain of Ku70. Indeed, no contacts with DNA bases and only a few interactions with the sugar-phosphate backbone are made.
The structure of DNA-PKcs has proved quite elusive. Some beautiful work performed using cryo-electron microscopy single particle reconstruction of DNA-PKcs Chiu et al.
(1998); River-Calzada et al.
(2005) and Williams et al.
(2008) has given a good impression of the overall structure. This has now been complemented by work in our laboratory. We have shown that DNA-PKcs crystals can be grown and diffract to about 8.5 resolution but the diffraction is better for the for complexes with C-terminal fragments of Ku80. We have solved at 6.6 resolution using multi-wavelength anomalous dispersion method with Ta6
heavy metal cluster (Sibanda et al., 2010) the structure of DNA-PKcs in complex with C-terminal domain of Ku80 (Figure 3). From the N-terminus of DNA-PKcs about 66 helices are arranged as HEAT repeats and folded into a hollow circular structure, which has a concave shape rather like a cradle when viewed from the side. The large C-terminal region forms a crown, comprising the FAT, kinase and FATC structures with the kinase exposed at the very top and accessible to substrates. The ring structure reflects its role as a platform for proteins that engage in the repair of broken DNA and which, together with Ku, holds in place the DNA while it is being repaired.
Human LigIV has also proved difficult to study in isolation due to instability and flexibility but it is stabilised by interaction with XRCC4. LigIV can be divided into catalytic and the interaction regions. The catalytic region is conserved among other human DNA ligases and contains the DNA binding domain, (DBD), the nucleotidyltransferase domain (NTase) and the OB-fold domain (OBD). The region that interacts with XRCC4 and Ku70/80 consists of two BRCT domains connected by a flexible linker. XRCC4 has three distinct domains: the globular head domain, the long coiled-coil tail domain and the C-terminal domain.
In solution, XRCC4 is an equilibrium mixture of dimer and tetramers, but only the dimer can complex with LigIV (Modesti et al., 2003). The structure of the tetramer shows a tail-to-tail interaction of two dimers (Junop et al., 2000). We have solved the structure of the dimer in complex with the linker of LigIV (Figure 1m) (Sibanda et al. 2001) and the complex of a yeast orthologue Lif1p/Lig4p (DorŽ et al., 2006). The structures of the human and yeast complexes show that the two BRCT domains and linker encircle the coiled-coil domain, an arrangement, which is incompatible with interactions in the tetramer. A recently published EM structure of XRCC4/LigIV complex shows the N-terminal of LigIV contacts with the head domain (Recuero-Checa et al., 2009). These observations support the idea that LigIV needs the C-terminal domains and will be highly activated only when it forms the complex with XRCC4.
XLF (XRCC4-Like Factor) was identified through cDNA functional complementation cloning study of a group of NHEJ deficiency patients and independently through the yeast two-hybrid screening for XRCC4 interactors. Full-length human XLF contains 299 residues. At the extreme of the C-terminus, a small conserved basic cluster constitutes the nuclear localization sequence (NLS). The crystal structure of XLF with a C-terminal truncation, solved independently at 2.3 resolution by Andres, Modesti et al. (2007) and in our laboratory (Li, Chirgadze et al.
2008), exists as a homodimer protein containing a globular N-terminal head domain and extended coiled-coil helical tail, which is folded back around the coiled-coil (Figure 1n).
Interestingly XLF and XRCC4 interact through their head regions as shown by yeast two-hybrid study of various mutants (Deshpande and Wilson 2007). A side-by-side interaction model, in which XLF head domains slide into the space created by XRCC4 head domains and N-terminal part of the tail structure, has been proposed according to mutagenesis study of XLF and XRCC4 (Andres, Modesti et al. 2007). The functions and mechanisms of action of XLF in NHEJ are still not fully understood. XLF not only stabilizes LigIV/XRCC4 in broken DNA ends, but also enhance the LigIV/XRCC4 end-joining process. Understanding how XLF functions in NHEJ through studying its interaction with other NHEJ proteins structurally will help unravel the exact role of XLF. It will contribute towards our current understanding of DNA repair in NHEJ and may lead to future therapeutic application for NHEJ defects patients.
- Sibanda BL, Chirgadze DY, Blundell TL. (2010) Crystal structure of DNA-PKcs reveals a large open-ring cradle comprised of HEAT repeats. (2010) Nature. 2010 Jan 7;463(7277):118-21. Epub 2009 Dec 20. PMID: 20023628 [PubMed - indexed for MEDLINE]
- Li Y, Chirgadze DY, Bolanos-Garcia VM, Sibanda BL, Davies OW, Ahnesorg P, Jackson SP, Blundell TL (2007) Crystal structure of human XLF/Cernunnos reveals unexpected differences from XRCC4 with implications for NHEJ. EMBO Journal 27(1):290-300
- Brewerton SC, Dore AS, Drake AC, Leuther KK, Blundell TL. (2004) Structural analysis of DNA-PKcs: modelling of the repeat units and insights into the detailed molecular architecture. J Struct Biol. 145, 295-306
- Sibanda BL, Critchlow S, Begun J, Pei XY, Jackson SP, Blundell TL, Pellegrini L (2001) Insight into the mechanism of DNA end joining from the structure of an Xrcc4 dimer in complex with DNA ligase IV. Nature Structural Biology 8, 1015-1019
Dr May Marsh
Dr Marko Hyvonen, Dr Luca Pellegrini, Prof Ashok Venkitaraman, Cambridge
Interactions of tumor suppressor protein BRCA2 with RAD51:
The breast cancer susceptibility protein BRCA2 controls the activity of the RAD51 recombinase in pathways for the repair of DNA double-strand breaks by genetic recombination. Inheritance of one defective copy of the BRCA2 gene causes increased susceptibility to breast, ovarian and other cancers. BRCA2 encodes a protein of 3,418 amino acids, the sequence of which offers few clues to its biological role.
In a collaboration with Ashok Venkitaraman, the crystal structure of a complex between BRC repeat 4 (BRC4) and the RecA-homology domain of RAD51 at 1.7 resolution was defined in our laboratory (Pellegrini et al.,
2002) The structure shows that BRC4 mimics a motif in RAD51 that serves as an interface for multimerization in the RAD51 nucleoprotein filament, an essential intermediate for strand pairing reactions that underlie DNA recombination. Cancer-associated mutations affecting the BRC repeat are predicted to disrupt its interaction with RAD51, yielding structural insight into mechanisms for cancer susceptibility. Sequence fingerprints in both BRCA2 and RAD51 are consistent with the co-evolution of these proteins (Lo et al.,
- Lo T, Pellegrini L, Venkitaraman AR and Blundell TL. (2003) Sequence fingerprints in BRCA2 and RAD51: implications for DNA repair and cancer. DNA Repair 2. 1015-1028
- Yu DS, Sonoda E, Takeda S, Huang CLH, Pellegrini L, Blundell TL and Venkitaraman AR (2003) Dynamic control of RAD51 in the nucleus of living cells by self-association and interaction with BRCA2. Mol Cell. 12, 1029-1041.
- Shin DS, Pellegrini L, Daniels D, Yelent B, Craig L, Tsurata H, Yu D, Hitomi C, Arvai AS, Blundell TL, Venkitaraman AR and Tainer JA (2003) Full-length archaeal Rad51 structure and mutants: mechanisms for RAD51 assembly and control by BRCA2. EMBO J. 2003 22 4566-76
- Pellegrini L, Yu DS, Lo T, Anand S, Lee M, Blundell TL, Venkitaraman AR (2002) Insights into DNA recombination from the structure of a RAD51-BRCA2 complex. Nature 420, 287-293
Structural Biology of Checkpoint Signalling
Victor Bolanos Garcia
David Spring, Cambridge; Ernesto Cota, IC London; Carol Robinson, Oxford; Hassan Bousbaa, CESPU, Portugal
Every time a cell divides it must duplicate its genetic material and ensure that an intact copy is directed to each daughter cell. If this process fails to occur accurately, the resulting daughter cells can inherit too few or too many chromosomes, leading to developmental defects and cancer. BUB1 and BUBR1 are part of an intricate process known as the mitotic spindle assembly checkpoint, the evolutionary conserved regulatory mechanism that ensures the timely and accurate segregation of chromosomes and mitotic progression in higher organisms.
The arrest of cells in mitosis until defects in chromosome attachment are repaired relies on the communication of BUB1 and BUBR1 with the kinetochore-microtubule network and the correction machinery. Understanding how chromosome bi-orientation and accurate chromosome segregation occur requires definition of the structural details of this process.
The role of BUB1 and BUBR1 in mitotic checkpoint signalling
The mitotic spindle assembly checkpoint is a central control mechanism of the cell cycle, which is active in prometaphase and which prevents the precocious separation of sister chromatids. In higher organisms, failure of this molecular circuitry leads to genome instability, defects in chromosome segregation and cancer. BUB1, BUBR1, BUB3, MAD1, MAD2 and CDC20 are components of the mitotic spindle assembly checkpoint (Fig. 1p).
BUB1 and BUBR1 are central to this process and have distinct roles in mitotic progression. BUB1 is essential for assembly of the functional inner centromere and mediates the recruitment of BUBR1 in cells that have activated the checkpoint. BUBR1 promotes proper kinetochore-microtubule linkages. BUBR1 is involved in the mitotic checkpoint in at least two ways: it associates with unattached/incorrectly attached kinetochores and is a component of the Mitotic Checkpoint Complex (MCC) that inhibits the Anaphase-Promoting Complex or Cyclosome (APC/C) E3 ubiquitin ligase activity towards Cyclin B and Securin. The function of the mitotic checkpoint implies communication of this signalling pathway with the kinetochore-microtubule network, which occurs via the physical interaction of BUB1 and BUBR1 with the protein Blinkin (also referred to as KNL1, Spc105, CASC5, AF15Q14) . Depletion of BubR1 affects the timing of mitosis as it accelerates the onset of anaphase. Furthermore, mutations and changes in expression profiles of BUB1 and BUBR1 have been observed in several cancers and associated with aneuploidy and cancer progression. Thus, regulation of the expression levels of BUB1 and BUBR1 with drugs may be beneficial to arrest proliferation of tumour cells.
A proper understanding of the molecular regulation and communication between the mitotic checkpoint and the kinetochore-microtubule network (Fig. 1p) will help unravel the mechanism of chromosome bi-orientation and accurate chromosome segregation.
Structural basis of checkpoint signalling pathways.
Our work on mitotic checkpoint proteins has included the definition of the crystal structure of the TPR-containing domain of yeast BUB1; the description of a pocket of BUB1 that binds a small molecule ligand with high affinity; the elucidation of the structure of the N-terminal domain of human BUBR1; the demonstration that BUBR1 kinetochore recruitment does not depend on Cdc20 binding to the BUBR1 KEN boxes and the definition of the role in vivo
of specific conserved residues of the TPR-containing domain of human BUB1 and BUBR1 in binding Blinkin.
The structure of the functionally conserved N-terminal domain of BUB1 from budding yeast, which revealed a triple tandem arrangement of the TPR motif repeat (Figure 1q), allowed the mapping of the mutations of human BUB1 associated with chromosome instability and cancer progression; the identification of one region of potential therapeutic interest; the definition of the BUB1 residues engaged in the interaction with Blinkin(Figure 1q C-E), the central component of the kinetochore-microtubule network that recruits BUB1 and BUBR1 to the kinetochore.
We have also solved the crystal structure of the N-terminal domain of human BUBR1 at high resolution (1.8 ) and showed the domain constitutes the structural platform for the interaction of BUBR1 with Blinkin. The entire structure has a right-handed super-helical twist (Figure 1r), similar to BUB1.
Yeast two-hybrid data demonstrate that side chains of L126, E161 and R165 contribute to the Blinkin-binding interface of the BUBR1 TPR domain. These residues occupy a shallow groove on the inner concave surface of the BUBR1 domain defined
by TPR2 and TPR3 (Fig. 4A). The Blinkin-binding groove of the BUBR1 TPR domain thus differs from previous characterised TPR domains .
Importantly, for all BUBR1 mutants tested, the growth of mated yeast on SD/-Leu/-Trp plates (selecting for the presence of both plasmids) was unaffected, and a wild-type level of interaction with BUB3 was detected at all time points (Fig. 1s B-C), thus indicating the mutants had little effect on yeast growth, the expression level or stability of the fusion protein.
Key features define the BUB-protein; TPR domain, which differs from the classical TPR fold, possibly indicating conservation for a specific function. The BUBR1 and BUB1 TPR domains, however, do adopt distinct conformations peripheral to the three TPR motifs (Fig. 1r). A disordered region, a short α-helix and a kinked loop at the N-terminus of the BUBR1 TPR domain are replaced by an α-helix, a 310
-helix and a short loop at the N-terminus of the BUB1 TPR domain. The comparatively flexible conformation of BUBR1 may be required for presenting the upstream KEN box, the motif that is essential for BUBR1 interaction with Cdc20, to APC/C-Cdc20
The structures defined in our group provide new insights into how weak binary interactions lead to specificity and sensitive regulation of cellular processes through cooperative interactions among the components of multiprotein complexes. For example FGF1 and FGFR2 form relatively weak binary complexes but lead to stable 2:2:1 complexes, explaining how signalling complexes form at the cell surface in an environment rich in HS. In a similar way NK1 is monomeric in solution, but in the presence of heparin/HS forms stable dimers, probably through domain swapping.
We have investigated the nature of the interactions in multicomponent signalling systems. Most involve interfaces of around 2000 2
comprised of mixed hydrophobic and polar patches, so that both the individual components and the supramolecular assemblies are stable. These features are shared by the interfaces between FGF and FGFR, between FGFR:FGF and heparin, Cdk2 and p19 inhibitor, and Rad51 and BRC4. The Xrcc4 interactions with the ligase are much more extensive and involve many charged interactions, reflecting the more permanent nature of this pair of proteins that together go on to recruit other components.
We have shown that many individual polypeptide or polysaccharide components become conformationally ordered only in the complex, e.g. ligase IV peptide linker in complex with Xrcc4 and BRC4 repeat in complex with Rad51. In the latter, it is likely that the conserved phenylalanine and alanine of repeat FRTASNKEI dock into pockets before the remainder wraps itself round 0Rad51. Such flexible peptides have been widely discussed (e.g. Nature Rev. Mol. Cell Biol., 6
, 197-208, 2005). They are reminiscent of the flexibility of many polypeptide hormones prior to receptor binding and recognition, for example in glucagon (Sasaki et al., 1975), where we proposed that a helical structure was selected from a population of conformers at the receptor. This would allow the unbound flexible peptide to be cleaved by proteases, so removing it quickly from the circulation, but specificity would be enhanced by the need to nucleate folding during binding. Thus, not only specificity but also transience might be achieved by this mechanism.
A variation on this theme occurs with polysaccharides such as heparan sulphate (HS) chains, which probably bind in a similar way, with nucleation of the correct helical structure followed by recognition of sequence specific sulphation patterns as the complex forms.
- Sasaki K, Dockerill S, Adamiak DA, Tickle IJ and Blundell TL (1975) X-ray analysis of glucagon and its relationship to receptor binding Nature 257, 751-757.
Leo Hernani Silvestre
Dr Chris Abell, Dr Alessio Ciulli, Cambridge
Scheme 1: The Pantothenate Pathway in E. coli
Aspartate decarboxylase (ADC)
We have defined the structure of ADC at 1.9 resolution (Albert et al. 1998) and studied the in vitro
processing of ADC involving an active site pyruvoyl, and isolated ADC tetramers in different states of processing. We have prepared a series of mutants to determine the contributions made by various amino acids to protein processing and catalysis of the decarboxylation reaction.
We have demonstrated that the structure of E. coli
KPHMT is decameric by
ultracentrifugation. We solved the crystal structure of the enzyme to 1.8 resolution using a combination of native and selenomethionine proteins. Solving this structure was a significant technical achievement, as there were 160 selenium atoms in the unit cell! The protomer is a closed b-barrel. The decamer is toroidal comprising two closed superposed circles, each of five protomers. Rather serendipitously, the product, ketopantoate is bound in the active site. We have been able to model in the substrate a-ketoisovalerate, and to propose the position of binding of the cofactor methylenetetrahydrofolate (manuscript in preparation)..
Work in S. typhimurium
established that the apbA
gene, initially identified as a gene involved in the alternative pyrimidine biosynthesis pathway, encoded an enzyme with ketopantoate reductase activity, and was allelic to panE
. Using this information, we were able to amplify the E. coli panE
gene by PCR, and clone it into an expression vector. The 31 kDa enzyme protein is expressed to very high levels (13 mg/L of culture), and was easily purified in three chromatographic steps.
Using the selenomethionine MAD method, we solved the structure of the apo
enzyme to 2.4. The enzyme is monomeric and has two domains, the N-terminal domain has an alpha-beta fold of Rossman type, while the C-terminal domain is all alpha helical. The structure is topographically similar to N-(1-D-carboxylethyl)-L-norvaline dehydrogenase, which catalyses the NADH-dependent reaction between hydrophobic L-amino acids and a-ketoacids. This work has been published in Biochemistry
(Matak-Vinkovic et al.,
Pantothenate synthetase (PtS)
We have solved the unliganded structure of E. coli
PtS to 1.7 . This revealed that pantothenate synthetase is a member of the aminoacyl-tRNA synthetase superfamily, and enabled us to identify the ATP binding site and a putative pantoate binding site. We have proposed a catalytic mechanism that involves initial binding of pantoate, then binding of ATP, with an associated hinge bending conformational change (von Delft et al.
- Schmitzberger F, Kilkenny ML, Lobley CM, Webb ME, Vinkovic M, Matak-Vinkovic D, Witty M, Chirgadze DY, Smith AG, Abell C and Blundell TL (2003) The EMBO Journal 22, 6193-6204.
- von Delft F, Inoue T, Saldanha SA, Ottenhof HH, Schmitzberger F, Birch LM, Dhanaraj V, Witty M, Smith AG, Blundell TL, Abell C. (2003) "Structure of E. coli Ketopantoate Hydroxymethyl Transferase Complexed with Ketopantoate and Mg(2+), Solved by Locating 160 Selenomethionine Sites." Structure 11, 985-96.
- Schmitzberger F, Smith AG, Abell CA, and Blundell TL (2003) Comparative Analysis of the E. coli Ketopantoate Hydroxymethyltransferase Crystal Structure Confirms that It Is a Member of the (ab)8 Phosphoenolpyruvate/Pyruvate Superfamily. J. Bacteriol. 185, 4163-4171
- von Delft F, Lewendon A, Dhanaraj V, Blundell TL, Abell C & Smith A (2001) The crystal structure of E. coli Pantothenate Synthase confirms it as a member of the cytidyltransferase superfamily. Structure 9, 439-450.
- Lobley, C. M., Ciulli, A., Whitney, H. M., Williams, G., Smith, A. G., Abell, C., and Blundell, T. L. (2005). The Crystal Structure of Escherichia coli Ketopantoate Reductase with NADP+ Bound. Biochemistry 44, 8930-8939.
- Matak-Vinkovic, D., Vinkovic, M., Saldanha, S.A., Ashurst, J.L., von Delft, F., Inoue, T., Miguel, R.N., Smith, A.G., Blundell, T.L. and Abell, C. (2001) Crystal structure of Escherichia coli ketopantoate reductase at 1.7 angstrom resolution and insight into the enzyme mechanism. Biochemistry 40: 14493-14500.
- Albert, A., Dhanaraj, V.,Genschel, U., Khan, G., Ramjee, MK., Pulido, R., Sibanda, BL., von Delft, F., Witty, M., Blundell, TL., Smith, AG and Abel, C. (1998) Crystal structure of aspartate decarboxylase at 2.2 resolution provides evidence for an ester in protein self-processing. Nature Structural Biology 5(4): 289-293.
- von Delft, F., Dhanaraj, V., Witty, M., Pulido, R., Blundell, T.L., Smith, A.G. and Abell, C. New insights into the catalytic mechanism of L-aspartate-a-decarboxylase from high resolution structures of enzyme ligand complexes. Manuscript in preparation
Other microbial systems
- Dias MV, Huang F, Chirgadze DY, Tosin M, Spiteller D, Dry EF, Leadlay PF, Spencer JB, Blundell TL.(2010) Structural basis for the activity and substrate specificity of fluoroacetyl-CoA thioesterase FlK. J Biol Chem. 2010 Apr 29. [Epub ahead of print] PMID: 20430898 [PubMed - as supplied by publisher]Free Article
- Sridharan S, Howard N, Kerbarh O, Błaszczyk M, Abell C, Blundell TL. (2010) Crystal structure of Escherichia coli enterobactin-specific isochorismate synthase (EntC) bound to its reaction product isochorismate: implications for the enzyme mechanism and differential activity of chorismate-utilizing enzymes. (2010) J Mol Biol. 2010 Mar 19;397(1):290-300. Epub 2010 Jan 15. PMID: 20079748 [PubMed - indexed for MEDLINE]
We seek to exploit comparative analyses of genomes and protein superfamilies to develop new approaches to fold recognition, 3-D structure modelling and prediction of function. The objective is to use this approach to understand the consequences of genetic variation. In man we are interested in nsSNPs and somatic mutations and their impacts on function. In pathogens we are interested in genetic variation as it manifests itself in resistance to drugs. We are also working on the use of databases of structural information concerning protein interactions to identify targets and to improve screening libraries.
The research involves:
- the organisation of sequence, structure and functional data in relational databases of three-dimensional structures of proteins and their interactions (CREDO, PICCOLO, BIPA),
- software http://camelot.bioc.cam.ac.uk/software for sequence-structure (fold) recognition (FUGUE) http://camelot.bioc.cam.ac.uk/servers and software for comparative modeling (COMPOSER, MODELLER, ORCHESTRAR, RAPPER).
The current focus is on the identification and analysis of drug targets, especially those in Mycobacterium tuberculosis and Man, and on the analysis of the roles of nsSNPs and somatic mutations in human disease.
Our work is described in a series of manuscripts over the past 25 years:
Databases of Protein Interactions
- Higueruelo AP, Schreyer A, Bickerton GRJ, Pitt WR, Groom CR, Blundell TL (2009)
Atomic Interactions and Profile of Small Molecules Disrupting Protein-Protein Interfaces: the TIMBAL Database. Chemical Biology & Drug Design. 74, 457 - 467
Protein Family Databases
Amino Acid Substitution Tables
- Lee S, Blundell TL (2009) BIPA: a database for protein-nucleic acid interaction in 3D structures. Bioinformatics. PMID: 19357098
- Schreyer, A. & Blundell (2009) T. Credo: A protein-ligand interaction database for drug discovery. Chem Biol Drug Des 73: 157-167
- De Bakker P, Bateman A, Burke DF, Miguel RN, Mizuguchi K, Shi J, Shirai H, Blundell TL, (2001) HOMSTRAD: adding sequence information to structure-based alignments of homologous protein families. BIOINFOMATICS 17, 748-749.
Sequence-structure Homology Recognition
- Lee S, Blundell TL (2009) Ulla: a program for calculating environment-specific amino acid substitution tables. Bioinformatics. PMID: 19417059
- Gong S, Blundell TL (2008) Discarding Functional Residues from the Substitution Table Improves Predictions of Active Sites within Three-Dimensional Structures. PLoS Computational Biology 4(10): e1000179 doi:10.1371/journal.pcbi.1000179
- Overington J, Sali Andrej and T L Blundell TL (1990) Tertiary structural constraints on protein evolutionary diversity: templates, key residues and structure prediction. Proc. Roy. Soc. B 241, 132-145
- Sali A, Overington JP, Johnson MS and T L Blundell (1990) From comparisons of protein sequences and structures to protein modelling and design Trends Biochem. Sci. 15, 235 240.
- Overington J, Donnelly, D, Johnson M S, Sali A, Blundell TL (1992) Environment-specific amino acid substitution tables: Tertiary templates and prediction of protein folds Protein Science. 2, 216-226.
- Donnelly D, Overington J, Ruffle S, Nugent J & Blundell TL (1993) Modelling a-helical transmembrane domains: the calculation and use of substitution tables for lipid-facing residues. Protein Science 2:55-70
- Sowdhamini, R., Burke, D., Huang, J-F., Mizuguchi, K., Nagarajaram, H.A., Srinivasan, N., Steward, R.E and Blundell, T.L. (1998). CAMPASS: A Database Of Structurally Aligned Protein Superfamilies. Structure 6, 1087-1094.
- Mizuguchi, K., Deane, C.M., Blundell, T.L., Johnson, M.S. & Overington, J.P. (1998). JOY: protein sequence-structure representation and analysis. BIOINFORMATICS 14, 617-623.
- Sowdhamini, R., Burke, D.F., Deane, C., Huang, J-F., Mizuguchi, K., Nagarajaram, H.A., Overington, J.P., Srinivasan, N., Steward, R.E. & Blundell, T.L. (1998). Protein three-dimensional structural databases: domains, structurally aligned homologues and superfamilies. Acta Cryst D54, 1168-1177.
- Shi J, Blundell TL, and Mizuguchi K (2001) FUGUE: Sequence-structure Homology Recognition Using Environment-specific Substitution Tables and Structure-dependent Gap Penalties. J. Mol. Biol. 310, 243-257.
- Johnson M, Overington J & Blundell TL (1993) Alignment and searching for common protein folds using a Data Bank of structural templates. JMB 231:735-752
- Blundell TL and Johnson MS (1993) Catching a Common Fold. Protein Science 2:877-883
- Rufino SD and Blundell TL (1994) Structure-based identification and clustering of protein families and superfamilies. J. of Computer-Aided Molecular Design 8:5-27
- Sowdhamini R and Blundell TL (1995) An Automatic method involving cluster analysis of secondary structures for the identification of domains in proteins. Protein Science 4:506-521
- Karmali AM, Blundell TL and Furnham N (2009) Model-building strategies for low-resolution X-ray crystallographic data. Acta Crystallographica Section D: Biological Crystallography 65, 121-127
- Gore,S.P., and T.L. Blundell (2008) OPSAX Optimal side-chain packing in proteins and crystallographic refinement, J App Cryst. 41, 319-328
- Montalvao RW, Cavalli A, Salvatella X, Blundell TL & Vendruscolo M (2008): Structure Determination of Protein-Protein Complexes Using NMR Chemical Shifts: Case of an Endonuclease Colicin;Immunity Protein Complex. Journal of the American Chemical Society 130, 15990-15996
- Furnham N., de Bakker P.I.W, Gore S., Burke D.F. and Blundell T.L. (2008) Comparative modelling by restraint-based conformal sampling. BMC Structural Biology 8:7 1186/1472-6807-8-7
- Gore SP, Karmali AM and Blundell TL (2007) RapperTK : a versatile engine for discrete restraint-based conformational sampling of macromolecules BMC Structural Biology 7:13
- Smith RE, Lovell SC, Burke DF, Montalvao RW, Blundell TL (2007) Andante: Reducing side-chain rotamer search space during comparative modeling using environment-specific substitution probabilities. Bioinformatics 2007; doi: 10.1093/bioinformatics/btm073
- N. Furnham, A.S. Dore, D.Y. Chirgadze, P.I.W. de Bakker, M.A. DePristo, T.L. Blundell (2006) Knowledge-based Real-space Explorations for Low Resolution Structure Determination. Structure. 14: 1313-1320
- Furnham N, Blundell TL, DePristo MA, Terwilliger T (2006) Is one solution sufficient? Nature Structural Molecular Biology. 13, 184-185
- Montalvao RW, Smith RE, Lovell SC, Blundell TL. (2005) CHORAL: a differential geometry approach to the prediction of the cores of protein structures Bioinformatics 2005, 21(19):3719-3725
- M.A. DePristo, P.I.W. de Bakker, R.P. Shetty, T.L. Blundell (2003) Discrete restraint-based protein modeling and the Cα-trace problem. Protein Science. 12: 2032-2046
- De Bakker PIW, DePristo MA, Burke DF, Blundell TL (2003) Ab Initio Construction of Polypeptide Fragments: Accuracy of Loop Decoy Discrimination by an All-Atom Statistical Potential and the AMBER Force Field With the Generalized Born Solvation Model. PROTEINS: Structure, Function, and Genetics 51, 21-40.
- R.P. Shetty, P.I.W. de Bakker, M.A. DePristo, T.L. Blundell (2003) Advantages of finegrained side chain conformer libraries. Protein Engineering. 16: 963-969
- Deane CM, Kaas Q and Blundell TL (2001) SCORE: predicting the core of protein models. BIOINFOMATICS 17, 541-550.
- Deane CM, and Blundell TL (2001) CODA: A combined algorithm for predicting the structurally variable regions of protein models. Protein Science 10, 599-612.
- Deane, CM and Blundell TL (2000) Examination of the Less favoured Regions of the Ramachandran plot. Indian Academy of Sciences. 16 196-208
- Deane CM, and Blundell TL (2000) A novel exhaustive search algorithm for predicting the conformation of polypeptide segments in proteins. Proteins: Structure, Function and Genetics. 40, 135-144.
- Rufino, SD., Donate, LE., Canard, LHJ. and Blundell, TL. (1997) Predicting the conformational class of short and medium size loops connecting regular secondary structures: Application to comparative modelling. J. Mol. Biol. 267, 352-367.
- N Srinivasan & Blundell TL (1993) An evaluation of the performance of an automated procedure for comparative modelling of protein tertiary structure. Protein Engineering 6:501-512
- Sibanda BL, Thornton JM and Blundell TL (1989) The conformation of B hairpins in protein structure: a systematic classification with applications to modelling by homology, electron density fitting and protein engineering J. Mol. Biol. 206,759 777
- Sutcliffe MJ, Hannef I, Carney D and Blundell TL (1987) Knowledge based modelling of homologous proteins, part I: three dimensional frameworks deÂrived from the simultaneous superposition of multiple structures Protein Engineering, 1, 377 384
- Sutcliffe M J, Hayes F and Blundell TL (1987) Knowledge based modelling of homologous proteins, part II: rules for replacement of sidechains. Protein Engineering 1:377-384
- Blundell TL, Carney D, Gardner S, Hayes F, Howlin B, Hubbard T, Overington J, Singh D, Sibanda BL, Sutcliffe M (1988) Knowledge based protein modelling and design; 18th Sir Hans Krebs Lecture Eur. J. Biochem. 173, 513 520
Analysis of Binding Sites
- Cheng, T., Blundell, T.L., Fernandez-Recio, J. (2007) pyDock: electrostatics and desolvation for effective scoring of rigid-body protein-protein docking Proteins 68, 503-515.
- Blundell TL, Fern‡ndez-Recio J. (2006) Cell biology: brief encounters bolster contacts. Nature. 444:279-80.
- Chelliah V, Blundell TL, Fern‡ndez-Recio, J. (2006) Efficient Restraints for Protein-Protein Docking by Comparison of Observed Amino Acid Substitution Patterns with those Predicted from Local Environment. J.Mol.Biol. 357, 1669-1682
- Chelliah V, Chen L, Blundell TL, Lovell SC (2004) Distinguishing Structural and Functional Restraints in Evolution in Order to Identify Interaction Sites. J. Mol. Biol. 342, 1487-504
Analysis of nsSNPs and Somatic Mutations
- Worth CL, Gong S, Blundell TL. (2009) Structural and functional constraints in the evolution of protein families. Nat Rev Mol Cell Biol. 2009 Oct;10(10):709-20. Epub 2009 Sep 16. Review.PMID: 19756040
- Worth CL, Blundell TL (2008) Satisfaction of hydrogen-bonding potential influences the conservation of polar sidechains Proteins: Structure, Function and Bioinformatics, 75, 413 - 429
- Worth CL, Blundell TL. (2010) On the evolutionary conservation of hydrogen bonds made by buried polar amino [Epub ahead of print] PMID: 20513243 [PubMed - as supplied by publisher]
- Gong S, Blundell TL. (2010) Structural and functional restraints on the occurrence of single amino acid variations in human proteins. PLoS One. 2010 Feb 12;5(2):e9186.
PMID: 20169194 [PubMed - in process]Free PMC ArticleFree text
- Forman, J.R., Worth, C.L., Bickerton, G.R.J., Eisen, T., & Blundell, T.L.(2009) Structural bioinformatics mutation analysis reveals genotype-phenotype correlations in von Hippel-Lindau disease and suggests molecu
- Worth CL and Blundell TL. (2007) Estimating the effects of SNPs on protein structure: loss of protein interactions and stability as indicators of mis-function and disease-association. Current Topics in Biochemical Research, 9, 53-62.
- lar mechanisms of tumourigenesis. Proteins: Structure, Function, and Bioinformatics 77, 84-96
- Worth C.L., Bickerton G.R.J, Schreyer A., Forman, J.R., Cheng T.M.K., Lee S., Gong S., Burke D.F. and Blundell T.L. (2007) A structural bioinformatics approach to the analysis of nonsynonymous single nucleotide polymorphisms (n SNPs) and their relation to disease. Journal Bioinformatics & Computational Biology 5, 1297 - 1318
- Burke DF, Worth CL, Prego EM, Cheng T, Smink LJ, Todd JA and Blundell TL (2007) Bioinformatic analysis of non-synonymous SNPs BMC Bioinformatics, 8:301
- Worth CL, Burke DF, Blundell TL. (2006) Estimating the effects of single nucleotide polymorphisms on protein structure: how good are we at identifying likely disease associated mutations? Proceedings of ÔÔMolecular Interactions—Bringing Chemistry to Life.ÕÕ. pp 11-26
- Topham, C.M., Srinivasan, N. and Blundell, T.L. Prediction of the stability of protein mutants based on structural environment-dependent amino acid substitution and propensity tables. (1997) Protein Engineering.10: 7-21.
- Lee S, Brown A, Pitt W, Higueruelo A, Gong S, Bickerton G, Schreyer A, Tanramluk D, Baylay A, Blundell T. (2009) Structural interactomics: informatics approaches to aid the interpretation of genetic variation and the development of novel therapeutics. Molecular BioSystems doi: 10.1039/B906402H
- Tanramluk D, Pitt WR, Schreyer A, Blundell TL (2009) On the Origins of Enzyme Inhibitor Selectivity and Promiscuity: A Case Study of Protein Kinase Binding to Staurosporine. Chem Biol Drug Des. 74(1):16-2
- Gore S, Burke DF and Blundell TL (2005) PROVAT: A tool for Voronoi Tesellation Analysis of Macromolecular structures. Bioinformatics 21(15):3316-7
- Blundell TL and Srinivasan N. Symmetry, stability, and dynamics of multidomain and multicomponent protein systems. (1996) Proc. Natl. Acad. Sci. USA. 93. 233-241