Publications by authors named "Stephen J Smerdon"

51 Publications

Uncoupling conformational states from activity in an allosteric enzyme.

Nat Commun 2017 08 7;8(1):203. Epub 2017 Aug 7.

Mycobacterial Metabolism and Antibiotic Research Laboratory, The Francis Crick Institute, 1 Midland Road, London, NW1 1AT, UK.

ATP-phosphoribosyltransferase (ATP-PRT) is a hexameric enzyme in conformational equilibrium between an open and seemingly active state and a closed and presumably inhibited form. The structure-function relationship of allosteric regulation in this system is still not fully understood. Here, we develop a screening strategy for modulators of ATP-PRT and identify 3-(2-thienyl)-L-alanine (TIH) as an allosteric activator of this enzyme. Kinetic analysis reveals co-occupancy of the allosteric sites by TIH and L-histidine. Crystallographic and native ion-mobility mass spectrometry data show that the TIH-bound activated form of the enzyme closely resembles the inhibited L-histidine-bound closed conformation, revealing the uncoupling between ATP-PRT open and closed conformations and its functional state. These findings suggest that dynamic processes are responsible for ATP-PRT allosteric regulation and that similar mechanisms might also be found in other enzymes bearing a ferredoxin-like allosteric domain.Active and inactive state ATP-phosphoribosyltransferases (ATP-PRTs) are believed to have different conformations. Here the authors show that in both states, ATP-PRT has a similar structural arrangement, suggesting that dynamic alterations are involved in ATP-PRT regulation by allosteric modulators.
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http://dx.doi.org/10.1038/s41467-017-00224-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5545217PMC
August 2017

Three-Dimensional Architecture of the Human BRCA1-A Histone Deubiquitinase Core Complex.

Cell Rep 2016 12;17(12):3099-3106

Structural Biology of DNA-Damage Signalling Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK. Electronic address:

BRCA1 is a tumor suppressor found to be mutated in hereditary breast and ovarian cancer and plays key roles in the maintenance of genomic stability by homologous recombination repair. It is recruited to damaged chromatin as a component of the BRCA1-A deubiquitinase, which cleaves K63-linked ubiquitin chains attached to histone H2A and H2AX. BRCA1-A contributes to checkpoint regulation, repair pathway choice, and HR repair efficiency through molecular mechanisms that remain largely obscure. The structure of an active core complex comprising two Abraxas/BRCC36/BRCC45/MERIT40 tetramers determined by negative-stain electron microscopy (EM) reveals a distorted V-shape architecture in which a dimer of Abraxas/BRCC36 heterodimers sits at the base, with BRCC45/Merit40 pairs occupying each arm. The location and ubiquitin-binding activity of BRCC45 suggest that it may provide accessory interactions with nucleosome-linked ubiquitin chains that contribute to their efficient processing. Our data also suggest how ataxia telangiectasia mutated (ATM)-dependent BRCA1 dimerization may stabilize self-association of the entire BRCA1-A complex.
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http://dx.doi.org/10.1016/j.celrep.2016.11.063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5199339PMC
December 2016

HF-Free Boc Synthesis of Peptide Thioesters for Ligation and Cyclization.

Angew Chem Int Ed Engl 2016 10;55(42):13174-13179

The Francis Crick Institute, 1 Midland road, London, NW1 1AT, UK.

We have developed a convenient method for the direct synthesis of peptide thioesters, versatile intermediates for peptide ligation and cyclic peptide synthesis. The technology uses a modified Boc SPPS strategy that avoids the use of anhydrous HF. Boc in situ neutralization protocols are used in combination with Merrifield hydroxymethyl resin and TFA/TMSBr cleavage. Avoiding HF extends the scope of Boc SPPS to post-translational modifications that are compatible with the milder cleavage conditions, demonstrated here with the synthesis of the phosphorylated protein CHK2. Peptide thioesters give easy, direct, access to cyclic peptides, illustrated by the synthesis of cyclorasin, a KRAS inhibitor.
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http://dx.doi.org/10.1002/anie.201607657DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5113665PMC
October 2016

Versatility in phospho-dependent molecular recognition of the XRCC1 and XRCC4 DNA-damage scaffolds by aprataxin-family FHA domains.

DNA Repair (Amst) 2015 Nov 23;35:116-25. Epub 2015 Oct 23.

Francis Crick Institute, Mill Hill Laboratory, The Ridgeway, Mill Hill, London NW7 1AA, UK. Electronic address:

Aprataxin, aprataxin and PNKP-like factor (APLF) and polynucleotide kinase phosphatase (PNKP) are key DNA-repair proteins with diverse functions but which all contain a homologous forkhead-associated (FHA) domain. Their primary binding targets are casein kinase 2-phosphorylated forms of the XRCC1 and XRCC4 scaffold molecules which respectively coordinate single-stranded and double-stranded DNA break repair pathways. Here, we present the high-resolution X-ray structure of a complex of phosphorylated XRCC4 with APLF, the most divergent of the three FHA domain family members. This, combined with NMR and biochemical analysis of aprataxin and APLF binding to singly and multiply-phosphorylated forms of XRCC1 and XRCC4, and comparison with PNKP reveals a pattern of distinct but overlapping binding specificities that are differentially modulated by multi-site phosphorylation. Together, our data illuminate important differences between activities of the three phospho-binding domains, in spite of a close evolutionary relationship between them.
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http://dx.doi.org/10.1016/j.dnarep.2015.10.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4655838PMC
November 2015

Synergic interplay of the La motif, RRM1 and the interdomain linker of LARP6 in the recognition of collagen mRNA expands the RNA binding repertoire of the La module.

Nucleic Acids Res 2015 Jan 8;43(1):645-60. Epub 2014 Dec 8.

Randall Division of Cell and Molecular Biophysics, King's College London, New Hunt's House, Guy's Campus, London SE1 1UL, UK

The La-related proteins (LARPs) form a diverse group of RNA-binding proteins characterized by the possession of a composite RNA binding unit, the La module. The La module comprises two domains, the La motif (LaM) and the RRM1, which together recognize and bind to a wide array of RNA substrates. Structural information regarding the La module is at present restricted to the prototypic La protein, which acts as an RNA chaperone binding to 3' UUUOH sequences of nascent RNA polymerase III transcripts. In contrast, LARP6 is implicated in the regulation of collagen synthesis and interacts with a specific stem-loop within the 5' UTR of the collagen mRNA. Here, we present the structure of the LaM and RRM1 of human LARP6 uncovering in both cases considerable structural variation in comparison to the equivalent domains in La and revealing an unprecedented fold for the RRM1. A mutagenic study guided by the structures revealed that RNA recognition requires synergy between the LaM and RRM1 as well as the participation of the interdomain linker, probably in realizing tandem domain configurations and dynamics required for substrate selectivity. Our study highlights a considerable complexity and plasticity in the architecture of the La module within LARPs.
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http://dx.doi.org/10.1093/nar/gku1287DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4288179PMC
January 2015

The NBS1-Treacle complex controls ribosomal RNA transcription in response to DNA damage.

Nat Cell Biol 2014 Aug 27;16(8):792-803. Epub 2014 Jul 27.

1] Department of Gynecology, University Hospital and University of Zurich, Wagistrasse 14, CH-8952 Schlieren, Switzerland [2] Institute of Veterinary Biochemistry and Molecular Biology, University of Zurich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.

Chromosome breakage elicits transient silencing of ribosomal RNA synthesis, but the mechanisms involved remained elusive. Here we discover an in trans signalling mechanism that triggers pan-nuclear silencing of rRNA transcription in response to DNA damage. This is associated with transient recruitment of the Nijmegen breakage syndrome protein 1 (NBS1), a central regulator of DNA damage responses, into the nucleoli. We further identify TCOF1 (also known as Treacle), a nucleolar factor implicated in ribosome biogenesis and mutated in Treacher Collins syndrome, as an interaction partner of NBS1, and demonstrate that NBS1 translocation and accumulation in the nucleoli is Treacle dependent. Finally, we provide evidence that Treacle-mediated NBS1 recruitment into the nucleoli regulates rRNA silencing in trans in the presence of distant chromosome breaks.
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http://dx.doi.org/10.1038/ncb3007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4962910PMC
August 2014

FAN1 activity on asymmetric repair intermediates is mediated by an atypical monomeric virus-type replication-repair nuclease domain.

Cell Rep 2014 Jul 26;8(1):84-93. Epub 2014 Jun 26.

Division of Molecular Structure, MRC National Institute for Medical Research, The Ridgeway, London NW7 1AA, UK.

FAN1 is a structure-selective DNA repair nuclease with 5' flap endonuclease activity, involved in the repair of interstrand DNA crosslinks. It is the only eukaryotic protein with a virus-type replication-repair nuclease ("VRR-Nuc") "module" that commonly occurs as a standalone domain in many bacteria and viruses. Crystal structures of three representatives show that they structurally resemble Holliday junction resolvases (HJRs), are dimeric in solution, and are able to cleave symmetric four-way junctions. In contrast, FAN1 orthologs are monomeric and cleave 5' flap structures in vitro, but not Holliday junctions. Modeling of the VRR-Nuc domain of FAN1 reveals that it has an insertion, which packs against the dimerization interface observed in the structures of the viral/bacterial VRR-Nuc proteins. We propose that these additional structural elements in FAN1 prevent dimerization and bias specificity toward flap structures.
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http://dx.doi.org/10.1016/j.celrep.2014.06.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4103454PMC
July 2014

IκB kinase-induced interaction of TPL-2 kinase with 14-3-3 is essential for Toll-like receptor activation of ERK-1 and -2 MAP kinases.

Proc Natl Acad Sci U S A 2014 Jun 27;111(23):E2394-403. Epub 2014 May 27.

Divisions of Immune Cell Biology,

The MEK-1/2 kinase TPL-2 is critical for Toll-like receptor activation of the ERK-1/2 MAP kinase pathway during inflammatory responses, but it can transform cells following C-terminal truncation. IκB kinase (IKK) complex phosphorylation of the TPL-2 C terminus regulates full-length TPL-2 activation of ERK-1/2 by a mechanism that has remained obscure. Here, we show that TPL-2 Ser-400 phosphorylation by IKK and TPL-2 Ser-443 autophosphorylation cooperated to trigger TPL-2 association with 14-3-3. Recruitment of 14-3-3 to the phosphorylated C terminus stimulated TPL-2 MEK-1 kinase activity, which was essential for TPL-2 activation of ERK-1/2. The binding of 14-3-3 to TPL-2 was also indispensible for lipopolysaccharide-induced production of tumor necrosis factor by macrophages, which is regulated by TPL-2 independently of ERK-1/2 activation. Our data identify a key step in the activation of TPL-2 signaling and provide a mechanistic insight into how C-terminal deletion triggers the oncogenic potential of TPL-2 by rendering its kinase activity independent of 14-3-3 binding.
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http://dx.doi.org/10.1073/pnas.1320440111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4060680PMC
June 2014

Phosphorylation-dependent PIH1D1 interactions define substrate specificity of the R2TP cochaperone complex.

Cell Rep 2014 Apr 20;7(1):19-26. Epub 2014 Mar 20.

DNA Damage Response Laboratory, London Research Institute, Clare Hall, South Mimms EN6 3LD, UK. Electronic address:

The R2TP cochaperone complex plays a critical role in the assembly of multisubunit machines, including small nucleolar ribonucleoproteins (snoRNPs), RNA polymerase II, and the mTORC1 and SMG1 kinase complexes, but the molecular basis of substrate recognition remains unclear. Here, we describe a phosphopeptide binding domain (PIH-N) in the PIH1D1 subunit of the R2TP complex that preferentially binds to highly acidic phosphorylated proteins. A cocrystal structure of a PIH-N domain/TEL2 phosphopeptide complex reveals a highly specific phosphopeptide recognition mechanism in which Lys57 and 64 in PIH1D1, along with a conserved DpSDD phosphopeptide motif within TEL2, are essential and sufficient for binding. Proteomic analysis of PIH1D1 interactors identified R2TP complex substrates that are recruited by the PIH-N domain in a sequence-specific and phosphorylation-dependent manner suggestive of a common mechanism of substrate recognition. We propose that protein complexes assembled by the R2TP complex are defined by phosphorylation of a specific motif and recognition by the PIH1D1 subunit.
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http://dx.doi.org/10.1016/j.celrep.2014.03.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3989777PMC
April 2014

A year in structural signaling: mTOR--the PIKK of the bunch?

Sci Signal 2014 Mar 4;7(315):pe6. Epub 2014 Mar 4.

Division of Molecular Structure, Medical Research Council National Institute for Medical Research, The Ridgeway, London NW7 1AA, UK.

The phosphatidylinositol 3-kinase-like protein kinases (PIKKs) are large, atypical serine-threonine kinases that function in many important cellular signaling processes. Despite their prominence, and due to the complexity of their architecture and interactions, PIKKs have long managed to evade high-resolution crystallographic analysis. Recent, near-atomic-resolution structures of nucleotide- and inhibitor-bound complexes of a large carboxyl-terminal fragment of mammalian target of rapamycin (mTOR) are set to transform our understanding of the expansive signaling functions of these fascinating molecules and inform efforts to design therapeutic agents against a broad spectrum of human diseases ranging from diabetes to cancer.
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http://dx.doi.org/10.1126/scisignal.2005174DOI Listing
March 2014

An attenuated mutant of the Rv1747 ATP-binding cassette transporter of Mycobacterium tuberculosis and a mutant of its cognate kinase, PknF, show increased expression of the efflux pump-related iniBAC operon.

FEMS Microbiol Lett 2013 Oct 23;347(2):107-15. Epub 2013 Aug 23.

Division of Mycobacterial Research, MRC National Institute for Medical Research, London, UK.

The ATP-binding cassette transporter Rv1747 is required for the growth of Mycobacterium tuberculosis in mice and in macrophages. Its structure suggests it is an exporter. Rv1747 forms a two-gene operon with pknF coding for the serine/threonine protein kinase PknF, which positively modulates the function of the transporter. We show that deletion of Rv1747 or pknF results in a number of transcriptional changes which could be complemented by the wild type allele, most significantly up-regulation of the iniBAC genes. This operon is inducible by isoniazid and ethambutol and by a broad range of inhibitors of cell wall biosynthesis and is required for efflux pump functioning. However, neither the Rv1747 or pknF mutant showed increased susceptibility to a range of drugs and cell wall stress reagents including isoniazid and ethambutol, cell wall structure and cell division appear normal by electron microscopy, and no differences in lipoarabinomannan were found. Transcription from the pknF promoter was not induced by a range of stress reagents. We conclude that the loss of Rv1747 affects cell wall biosynthesis leading to the production of intermediates that cause induction of iniBAC transcription and implicates it in exporting a component of the cell wall, which is necessary for virulence.
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http://dx.doi.org/10.1111/1574-6968.12230DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3908365PMC
October 2013

Activation of the yeast Hippo pathway by phosphorylation-dependent assembly of signaling complexes.

Science 2013 May 11;340(6134):871-5. Epub 2013 Apr 11.

David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

Scaffold-assisted signaling cascades guide cellular decision-making. In budding yeast, one such signal transduction pathway called the mitotic exit network (MEN) governs the transition from mitosis to the G1 phase of the cell cycle. The MEN is conserved and in metazoans is known as the Hippo tumor-suppressor pathway. We found that signaling through the MEN kinase cascade was mediated by an unusual two-step process. The MEN kinase Cdc15 first phosphorylated the scaffold Nud1. This created a phospho-docking site on Nud1, to which the effector kinase complex Dbf2-Mob1 bound through a phosphoserine-threonine binding domain, in order to be activated by Cdc15. This mechanism of pathway activation has implications for signal transmission through other kinase cascades and might represent a general principle in scaffold-assisted signaling.
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http://dx.doi.org/10.1126/science.1235822DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3884217PMC
May 2013

Substituted aminopyrimidine protein kinase B (PknB) inhibitors show activity against Mycobacterium tuberculosis.

Bioorg Med Chem Lett 2012 May 14;22(9):3349-53. Epub 2012 Mar 14.

Centre for Therapeutics Discovery, MRC Technology, 1-3 Burtonhole Lane, Mill Hill, London NW7 1AD, UK.

A high-throughput screen against PknB, an essential serine-threonine protein kinase present in Mycobacterium tuberculosis (M. tuberculosis), allowed the identification of an aminoquinazoline inhibitor which was used as a starting point for SAR investigations. Although a significant improvement in enzyme affinity was achieved, the aminoquinazolines showed little or no cellular activity against M. tuberculosis. However, switching to an aminopyrimidine core scaffold and the introduction of a basic amine side chain afforded compounds with nanomolar enzyme binding affinity and micromolar minimum inhibitory concentrations against M. tuberculosis. Replacement of the pyrazole head group with pyridine then allowed equipotent compounds with improved selectivity against a human kinase panel to be obtained.
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http://dx.doi.org/10.1016/j.bmcl.2012.02.107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3368261PMC
May 2012

Plk1 and CK2 act in concert to regulate Rad51 during DNA double strand break repair.

Mol Cell 2012 Feb;45(3):371-83

Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK.

Homologous recombination (HR) plays an important role in the maintenance of genome integrity. HR repairs broken DNA during S and G2 phases of the cell cycle but its regulatory mechanisms remain elusive. Here, we report that Polo-like kinase 1 (Plk1), which is vital for cell proliferation and is frequently upregulated in cancer cells, phosphorylates the essential Rad51 recombinase at serine 14 (S14) during the cell cycle and in response to DNA damage. Strikingly, S14 phosphorylation licenses subsequent Rad51 phosphorylation at threonine 13 (T13) by casein kinase 2 (CK2), which in turn triggers direct binding to the Nijmegen breakage syndrome gene product, Nbs1. This mechanism facilitates Rad51 recruitment to damage sites, thus enhancing cellular resistance to genotoxic stresses. Our results uncover a role of Plk1 in linking DNA damage recognition with HR repair and suggest a molecular mechanism for cancer development associated with elevated activity of Plk1.
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http://dx.doi.org/10.1016/j.molcel.2011.12.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3280358PMC
February 2012

The molecular basis of ATM-dependent dimerization of the Mdc1 DNA damage checkpoint mediator.

Nucleic Acids Res 2012 May 10;40(9):3913-28. Epub 2012 Jan 10.

Institute of Veterinary Biochemistry and Molecular Biology, University of Zürich - Irchel, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.

Mdc1 is a large modular phosphoprotein scaffold that maintains signaling and repair complexes at double-stranded DNA break sites. Mdc1 is anchored to damaged chromatin through interaction of its C-terminal BRCT-repeat domain with the tail of γH2AX following DNA damage, but the role of the N-terminal forkhead-associated (FHA) domain remains unclear. We show that a major binding target of the Mdc1 FHA domain is a previously unidentified DNA damage and ATM-dependent phosphorylation site near the N-terminus of Mdc1 itself. Binding to this motif stabilizes a weak self-association of the FHA domain to form a tight dimer. X-ray structures of free and complexed Mdc1 FHA domain reveal a 'head-to-tail' dimerization mechanism that is closely related to that seen in pre-activated forms of the Chk2 DNA damage kinase, and which both positively and negatively influences Mdc1 FHA domain-mediated interactions in human cells prior to and following DNA damage.
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http://dx.doi.org/10.1093/nar/gkr1300DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3351161PMC
May 2012

Analysis of the interaction with the hepatitis C virus mRNA reveals an alternative mode of RNA recognition by the human La protein.

Nucleic Acids Res 2012 Feb 18;40(3):1381-94. Epub 2011 Oct 18.

Randall Division of Cell and Molecular Biophysics, King's College London, New Hunt's House, Guy's Campus, London SE1 1UL, UK.

Human La protein is an essential factor in the biology of both coding and non-coding RNAs. In the nucleus, La binds primarily to 3' oligoU containing RNAs, while in the cytoplasm La interacts with an array of different mRNAs lacking a 3' UUU(OH) trailer. An example of the latter is the binding of La to the IRES domain IV of the hepatitis C virus (HCV) RNA, which is associated with viral translation stimulation. By systematic biophysical investigations, we have found that La binds to domain IV using an RNA recognition that is quite distinct from its mode of binding to RNAs with a 3' UUU(OH) trailer: although the La motif and first RNA recognition motif (RRM1) are sufficient for high-affinity binding to 3' oligoU, recognition of HCV domain IV requires the La motif and RRM1 to work in concert with the atypical RRM2 which has not previously been shown to have a significant role in RNA binding. This new mode of binding does not appear sequence specific, but recognizes structural features of the RNA, in particular a double-stranded stem flanked by single-stranded extensions. These findings pave the way for a better understanding of the role of La in viral translation initiation.
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http://dx.doi.org/10.1093/nar/gkr890DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3273827PMC
February 2012

Spatial exclusivity combined with positive and negative selection of phosphorylation motifs is the basis for context-dependent mitotic signaling.

Sci Signal 2011 Jun 28;4(179):ra42. Epub 2011 Jun 28.

Koch Institute for Integrative Cancer Research, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

The timing and localization of events during mitosis are controlled by the regulated phosphorylation of proteins by the mitotic kinases, which include Aurora A, Aurora B, Nek2 (never in mitosis kinase 2), Plk1 (Polo-like kinase 1), and the cyclin-dependent kinase complex Cdk1/cyclin B. Although mitotic kinases can have overlapping subcellular localizations, each kinase appears to phosphorylate its substrates on distinct sites. To gain insight into the relative importance of local sequence context in kinase selectivity, identify previously unknown substrates of these five mitotic kinases, and explore potential mechanisms for substrate discrimination, we determined the optimal substrate motifs of these major mitotic kinases by positional scanning oriented peptide library screening (PS-OPLS). We verified individual motifs with in vitro peptide kinetic studies and used structural modeling to rationalize the kinase-specific selection of key motif-determining residues at the molecular level. Cross comparisons among the phosphorylation site selectivity motifs of these kinases revealed an evolutionarily conserved mutual exclusion mechanism in which the positively and negatively selected portions of the phosphorylation motifs of mitotic kinases, together with their subcellular localizations, result in proper substrate targeting in a coordinated manner during mitosis.
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http://dx.doi.org/10.1126/scisignal.2001796DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3939359PMC
June 2011

Forkhead-associated (FHA) domain containing ABC transporter Rv1747 is positively regulated by Ser/Thr phosphorylation in Mycobacterium tuberculosis.

J Biol Chem 2011 Jul 26;286(29):26198-209. Epub 2011 May 26.

Division of Mycobacterial Research, Medical Research Council National Institute for Medical Research, Mill Hill, London NW7 1AA, UK.

One major signaling method employed by Mycobacterium tuberculosis, the causative agent of tuberculosis, is through reversible phosphorylation of proteins mediated by protein kinases and phosphatases. This study concerns one of these enzymes, the serine/threonine protein kinase PknF, that is encoded in an operon with Rv1747, an ABC transporter that is necessary for growth of M. tuberculosis in vivo and contains two forkhead-associated (FHA) domains. FHA domains are phosphopeptide recognition motifs that specifically recognize phosphothreonine-containing epitopes. Experiments to determine how PknF regulates the function of Rv1747 demonstrated that phosphorylation occurs on two specific threonine residues, Thr-150 and Thr-208. To determine the in vivo consequences of phosphorylation, infection experiments were performed in bone marrow-derived macrophages and in mice using threonine-to-alanine mutants of Rv1747 that prevent specific phosphorylation and revealed that phosphorylation positively modulates Rv1747 function in vivo. The role of the FHA domains in this regulation was further demonstrated by isothermal titration calorimetry, using peptides containing both phosphothreonine residues. FHA-1 domain mutation resulted in attenuation in macrophages highlighting the critical role of this domain in Rv1747 function. A mutant deleted for pknF did not, however, have a growth phenotype in an infection, suggesting that other kinases can fulfill its role when it is absent. This study provides the first information on the molecular mechanism(s) regulating Rv1747 through PknF-dependent phosphorylation but also indicates that phosphorylation activates Rv1747, which may have important consequences in regulating growth of M. tuberculosis.
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http://dx.doi.org/10.1074/jbc.M111.246132DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3138270PMC
July 2011

Effective inhibitors of the essential kinase PknB and their potential as anti-mycobacterial agents.

Tuberculosis (Edinb) 2011 Jul 11;91(4):277-86. Epub 2011 Apr 11.

Division of Mycobacterial Research, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA, United Kingdom.

PknB is an essential serine/threonine kinase of Mycobacterium tuberculosis with possible roles in a number of signalling pathways involved in cell division and metabolism. We screened a library of >50,000 compounds for inhibitors of the in vitro phosphorylation of GarA (Rv1827) by PknB and identified a number of inhibitors. A program of synthetic medicinal chemistry was subsequently conducted around one class of inhibitors and was successful in generating ATP competitive inhibitors with potency in the nanomolar range. Compounds in this class showed cross-reactivity with the related M. tuberculosis kinase, PknF, but not with PknG in an in vitro autophosphorylation assay. These synthesised inhibitors were able to prevent the growth of M. tuberculosis in an Alamar blue assay and in an intracellular model of infection, but only in the micromolar range. We attempted to determine if cell wall permeability was an explanation for the discrepancy between the potent in vitro compared with relatively poor in vivo activity, but found no evidence that the activity of the inhibitors could be improved by weakening the cell wall. Despite a number of drug discovery efforts attempting to develop inhibitors against PknB, it is yet to be reported that any such inhibitors prevent mycobacterial growth at submicromolar concentrations.
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http://dx.doi.org/10.1016/j.tube.2011.03.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3158675PMC
July 2011

Structural and functional analysis of phosphothreonine-dependent FHA domain interactions.

Structure 2010 Dec;18(12):1587-95

Division of Molecular Structure, MRC National Institute for Medical Research, The Ridgeway, London NW71AA, UK.

FHA domains are well established as phospho-dependent binding modules mediating signal transduction in Ser/Thr kinase signaling networks in both eukaryotic and prokaryotic species. Although they are unique in binding exclusively to phosphothreonine, the basis for this discrimination over phosphoserine has remained elusive. Here, we attempt to dissect overall binding specificity at the molecular level. We first determined the optimal peptide sequence for Rv0020c FHA domain binding by oriented peptide library screening. This served as a basis for systematic mutagenic and binding analyses, allowing us to derive relative thermodynamic contributions of conserved protein and peptide residues to binding and specificity. Structures of phosphopeptide-bound and uncomplexed Rv0020c FHA domain then directed molecular dynamics simulations which show how the extraordinary discrimination in favor of phosphothreonine occurs through formation of additional hydrogen-bonding networks that are ultimately stabilized by van der Waals interactions of the phosphothreonine γ-methyl group with a conserved pocket on the FHA domain surface.
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http://dx.doi.org/10.1016/j.str.2010.09.014DOI Listing
December 2010

Access to phosphoproteins and glycoproteins through semi-synthesis, Native Chemical Ligation and N→S acyl transfer.

Org Biomol Chem 2010 Nov 9;8(22):5113-9. Epub 2010 Sep 9.

Department of Chemistry, University College London, 20 Gordon Street, London, UKWC1H 0AJ.

Peptide thioesters are important tools for protein synthesis and semi-synthesis through their use in Native Chemical Ligation (NCL). NCL can be employed to assemble site-specifically modified proteins that can help elucidate the mechanisms of biomolecular processes. In this article we explore the compatibility of phosphopeptide synthesis and glycopeptide synthesis with thioester production through N→S acyl transfer.
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http://dx.doi.org/10.1039/c0ob00363hDOI Listing
November 2010

Structure of the amino-terminal domain from the cell-cycle regulator Swi6.

Proteins 2010 Oct;78(13):2861-5

Division of Molecular Structure, National Institute for Medical Research, Ridgeway, Mill Hill, London NW7 1AA, UK.

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http://dx.doi.org/10.1002/prot.22795DOI Listing
October 2010

A mitotic phosphorylation feedback network connects Cdk1, Plk1, 53BP1, and Chk2 to inactivate the G(2)/M DNA damage checkpoint.

PLoS Biol 2010 Jan 26;8(1):e1000287. Epub 2010 Jan 26.

David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

DNA damage checkpoints arrest cell cycle progression to facilitate DNA repair. The ability to survive genotoxic insults depends not only on the initiation of cell cycle checkpoints but also on checkpoint maintenance. While activation of DNA damage checkpoints has been studied extensively, molecular mechanisms involved in sustaining and ultimately inactivating cell cycle checkpoints are largely unknown. Here, we explored feedback mechanisms that control the maintenance and termination of checkpoint function by computationally identifying an evolutionary conserved mitotic phosphorylation network within the DNA damage response. We demonstrate that the non-enzymatic checkpoint adaptor protein 53BP1 is an in vivo target of the cell cycle kinases Cyclin-dependent kinase-1 and Polo-like kinase-1 (Plk1). We show that Plk1 binds 53BP1 during mitosis and that this interaction is required for proper inactivation of the DNA damage checkpoint. 53BP1 mutants that are unable to bind Plk1 fail to restart the cell cycle after ionizing radiation-mediated cell cycle arrest. Importantly, we show that Plk1 also phosphorylates the 53BP1-binding checkpoint kinase Chk2 to inactivate its FHA domain and inhibit its kinase activity in mammalian cells. Thus, a mitotic kinase-mediated negative feedback loop regulates the ATM-Chk2 branch of the DNA damage signaling network by phosphorylating conserved sites in 53BP1 and Chk2 to inactivate checkpoint signaling and control checkpoint duration.
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http://dx.doi.org/10.1371/journal.pbio.1000287DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2811157PMC
January 2010

Function of the nucleotide exchange activity of vav1 in T cell development and activation.

Sci Signal 2009 Dec 15;2(101):ra83. Epub 2009 Dec 15.

1Division of Immune Cell Biology, MRC National Institute for Medical Research, London NW7 1AA, UK.

The guanine nucleotide exchange factor (GEF) Vav1 is essential for transducing T cell antigen receptor (TCR) signals and therefore plays a critical role in the development and activation of T cells. It has been presumed that the GEF activity of Vav1 is important for its function; however, there has been no direct demonstration of this. Here, we generated mice expressing enzymatically inactive, but normally folded, Vav1 protein. Analysis of these mice showed that the GEF activity of Vav1 was necessary for the selection of thymocytes and for the optimal activation of T cells, including signal transduction to Rac1, Akt, and integrins. In contrast, the GEF activity of Vav1 was not required for TCR-induced calcium flux, activation of extracellular signal-regulated kinase and protein kinase D1, and cell polarization. Thus, in T cells, the GEF activity of Vav1 is essential for some, but not all, of its functions.
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http://dx.doi.org/10.1126/scisignal.2000420DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3434450PMC
December 2009

CK2 phosphorylation-dependent interaction between aprataxin and MDC1 in the DNA damage response.

Nucleic Acids Res 2010 Mar 14;38(5):1489-503. Epub 2009 Dec 14.

Queensland Institute of Medical Research, Radiation Biology and Oncology, Brisbane, QLD 4029, Australia.

Aprataxin, defective in the neurodegenerative disorder ataxia oculomotor apraxia type 1, resolves abortive DNA ligation intermediates during DNA repair. Here, we demonstrate that aprataxin localizes at sites of DNA damage induced by high LET radiation and binds to mediator of DNA-damage checkpoint protein 1 (MDC1/NFBD1) through a phosphorylation-dependent interaction. This interaction is mediated via the aprataxin FHA domain and multiple casein kinase 2 di-phosphorylated S-D-T-D motifs in MDC1. X-ray structural and mutagenic analysis of aprataxin FHA domain, combined with modelling of the pSDpTD peptide interaction suggest an unusual FHA binding mechanism mediated by a cluster of basic residues at and around the canonical pT-docking site. Mutation of aprataxin FHA Arg29 prevented its interaction with MDC1 and recruitment to sites of DNA damage. These results indicate that aprataxin is involved not only in single strand break repair but also in the processing of a subset of double strand breaks presumably through its interaction with MDC1.
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http://dx.doi.org/10.1093/nar/gkp1149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2836575PMC
March 2010

A supramodular FHA/BRCT-repeat architecture mediates Nbs1 adaptor function in response to DNA damage.

Cell 2009 Oct;139(1):100-11

Division of Molecular Structure, Medical Research Council National Institute for Medical Research, The Ridgeway, Mill Hill, London, UK.

The Mre11/Rad50/Nbs1 protein complex plays central enzymatic and signaling roles in the DNA-damage response. Nuclease (Mre11) and scaffolding (Rad50) components of MRN have been extensively characterized, but the molecular basis of Nbs1 function has remained elusive. Here, we present a 2.3A crystal structure of the N-terminal region of fission yeast Nbs1, revealing an unusual but conserved architecture in which the FHA- and BRCT-repeat domains structurally coalesce. We demonstrate that diphosphorylated pSer-Asp-pThr-Asp motifs, recently identified as multicopy docking sites within Mdc1, are evolutionarily conserved Nbs1 binding targets. Furthermore, we show that similar phosphomotifs within Ctp1, the fission yeast ortholog of human CtIP, promote interactions with the Nbs1 FHA domain that are necessary for Ctp1-dependent resistance to DNA damage. Finally, we establish that human Nbs1 interactions with Mdc1 occur through both its FHA- and BRCT-repeat domains, suggesting how their structural and functional interdependence underpins Nbs1 adaptor functions in the DNA-damage response.
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http://dx.doi.org/10.1016/j.cell.2009.07.043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2900601PMC
October 2009

An intramolecular switch regulates phosphoindependent FHA domain interactions in Mycobacterium tuberculosis.

Sci Signal 2009 Mar 24;2(63):ra12. Epub 2009 Mar 24.

Division of Molecular Structure, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.

Forkhead-associated (FHA) domains have gained considerable prominence as ubiquitous phosphothreonine-dependent binding modules; however, their precise roles in serine and threonine kinase (STK) pathways and mechanisms of regulation remain unclear. From experiments with Rv1827, an FHA domain-containing protein from Mycobacterium tuberculosis, we derived a complete molecular description of an FHA-mediated STK signaling process. First, binding of the FHA domain to each of three metabolic enzyme complexes regulated their catalytic activities but did not require priming phosphorylation. However, phosphorylation of a threonine residue within a conserved amino-terminal motif of Rv1827 triggered its intramolecular association with the FHA domain of Rv1827, thus blocking its interactions with each of the three enzymes. The solution structure of this inactivated form and further mutagenic studies showed how a previously unidentified intramolecular phosphoswitch blocked the access of the target enzymes to a common FHA interaction surface and how this shared surface accommodated three functionally related, but structurally diverse, binding partners. Thus, our data reveal an unsuspected versatility in the FHA domain that allows for the transformation of multiple kinase inputs into various downstream regulatory signals.
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http://dx.doi.org/10.1126/scisignal.2000212DOI Listing
March 2009

Pellino proteins splitting up the FHAmily!

Structure 2008 Dec;16(12):1752-4

Division of Molecular Structure, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.

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http://dx.doi.org/10.1016/j.str.2008.11.002DOI Listing
December 2008

Inhibition of Rac by the GAP activity of centralspindlin is essential for cytokinesis.

Science 2008 Dec;322(5907):1543-6

Institute for Molecular Biology, University of Oregon, Eugene, OR 97403, USA.

During cytokinesis, the guanosine triphosphatase (GTPase) RhoA orchestrates contractile ring assembly and constriction. RhoA signaling is controlled by the central spindle, a set of microtubule bundles that forms between the separating chromosomes. Centralspindlin, a protein complex consisting of the kinesin-6 ZEN-4 and the Rho family GTPase activating protein (GAP) CYK-4, is required for central spindle assembly and cytokinesis in Caenorhabditis elegans. However, the importance of the CYK-4 GAP activity and whether it regulates RhoA remain unclear. We found that two separation-of-function mutations in the GAP domain of CYK-4 lead to cytokinesis defects that mimic centralspindlin loss of function. These defects could be rescued by depletion of the GTPase Rac or its effectors, but not by depletion of RhoA. Thus, inactivation of Rac by centralspindlin functions in parallel with RhoA activation to drive contractile ring constriction during cytokinesis.
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http://dx.doi.org/10.1126/science.1163086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2736296PMC
December 2008

Chk2 oligomerization studied by phosphopeptide ligation: implications for regulation and phosphodependent interactions.

J Biol Chem 2008 Dec 23;283(51):36019-30. Epub 2008 Oct 23.

Division of Molecular Structure, MRC National Institute for Medical Research, The Ridgeway, London NW7 1AA, United Kingdom.

Chk2/CHEK2/hCds1 is a modular serine-threonine kinase involved in transducing DNA damage signals. Phosphorylation by ataxia telangiectasia-mutated kinase (ATM) promotes Chk2 self-association, autophosphorylation, and activation. Here we use expressed protein ligation to generate a Chk2 N-terminal regulatory region encompassing a fork-head-associated (FHA) domain, a stoichiometrically phosphorylated Thr-68 motif and intervening linker. Hydrodynamic analysis reveals that Thr-68 phosphorylation stabilizes weak FHA-FHA interactions that occur in the unphosphorylated species to form a high affinity dimer. Although clearly a prerequisite for Chk2 activation in vivo, we show that dimerization modulates potential phosphodependent interactions with effector proteins and substrates through either the pThr-68 site, or the canonical FHA phosphobinding surface with which it is tightly associated. We further show that the dimer-occluded pThr-68 motif is released by intra-dimer autophosphorylation of the FHA domain at the highly conserved Ser-140 position, a major pThr contact in all FHA-phosphopeptide complex structures, revealing a mechanism of Chk2 dimer dissociation following kinase domain activation.
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http://dx.doi.org/10.1074/jbc.M804075200DOI Listing
December 2008
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