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Eukaryotes have two paralogous developmentally regulated GTP-binding (DRG) proteins: DRG1 and DRG2, both of which have a conserved binding partner called DRG family regulatory protein 1 and 2 (DFRP1 and DFRP2), respectively. DFRPs are important for the function of DRGs and interact with their respective DRG via a conserved region called the DFRP domain. Despite being highly similar, DRG1 and DRG2 have strict binding specificity for their respective DFRP. Using AlphaFold generated structure models of the human DRG/DFRP complexes, we have biochemically characterized their interactions and identified interface residues involved in determining specificity. This analysis revealed that as few as five mutations in DRG1 can switch binding from DFRP1 to DFRP2. Moreover, while DFRP1 binding confers increased stability and GTPase activity to DRG1, DFRP2 binding only supports increased stability. Overall, this work provides new insight into the structural determinants responsible for the binding specificities of the DRG/DFRP complexes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Although protein hydroxylation is a relatively poorly characterized posttranslational modification, it has received significant recent attention following seminal work uncovering its role in oxygen sensing and hypoxia biology. Although the fundamental importance of protein hydroxylases in biology is becoming clear, the biochemical targets and cellular functions often remain enigmatic. JMJD5 is a "JmjC-only" protein hydroxylase that is essential for murine embryonic development and viability. However, no germline variants in JmjC-only hydroxylases, including JMJD5, have yet been described that are associated with any human pathology. Here we demonstrate that biallelic germline JMJD5 pathogenic variants are deleterious to JMJD5 mRNA splicing, protein stability, and hydroxylase activity, resulting in a human developmental disorder characterized by severe failure to thrive, intellectual disability, and facial dysmorphism. We show that the underlying cellular phenotype is associated with increased DNA replication stress and that this is critically dependent on the protein hydroxylase activity of JMJD5. This work contributes to our growing understanding of the role and importance of protein hydroxylases in human development and disease.

Despite being the first homolog of the bacterial RecQ helicase to be identified in humans, the function of RECQL1 remains poorly characterized. Furthermore, unlike other members of the human RECQ family of helicases, mutations in RECQL1 have not been associated with a genetic disease. Here, we identify 2 families with a genome instability disorder that we have named RECON (RECql ONe) syndrome, caused by biallelic mutations in the RECQL gene. The affected individuals had short stature, progeroid facial features, a hypoplastic nose, xeroderma, and skin photosensitivity and were homozygous for the same missense mutation in RECQL1 (p.Ala459Ser), located within its zinc binding domain. Biochemical analysis of the mutant RECQL1 protein revealed that the p.A459S missense mutation compromised its ATPase, helicase, and fork restoration activity, while its capacity to promote single-strand DNA annealing was largely unaffected. At the cellular level, this mutation in RECQL1 gave rise to a defect in the ability to repair DNA damage induced by exposure to topoisomerase poisons and a failure of DNA replication to progress efficiently in the presence of abortive topoisomerase lesions. Taken together, RECQL1 is the fourth member of the RecQ family of helicases to be associated with a human genome instability disorder.

The MRN (MRE11-RAD50-NBS1) complex is essential for repair of DNA double-strand breaks and stalled replication forks. Mutations of the MRN complex subunit MRE11 cause the hereditary cancer-susceptibility disease ataxia-telangiectasia-like disorder (ATLD). Here we show that MRE11 directly interacts with PIH1D1, a subunit of heat-shock protein 90 cochaperone R2TP complex, which is required for the assembly of large protein complexes, such as RNA polymerase II, small nucleolar ribonucleoproteins and mammalian target of rapamycin complex 1. The MRE11-PIH1D1 interaction is dependent on casein kinase 2 (CK2) phosphorylation of two acidic sequences within the MRE11 C terminus containing serines 558/561 and 688/689. Conversely, the PIH1D1 phospho-binding domain PIH-N is required for association with MRE11 phosphorylated by CK2. Consistent with these findings, depletion of PIH1D1 resulted in MRE11 destabilization and affected DNA-damage repair processes dependent on MRE11. Additionally, mutations of serines 688/689, which abolish PIH1D1 binding, also resulted in decreased MRE11 stability. As depletion of R2TP frequently leads to instability of its substrates and as truncation mutation of MRE11 lacking serines 688/689 leads to decreased levels of the MRN complex both in ATLD patients and an ATLD mouse model, our results suggest that the MRN complex is a novel R2TP complex substrate and that their interaction is regulated by CK2 phosphorylation.

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.

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., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., (© 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

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., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

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., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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.

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., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

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., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

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., (© 2013 The Authors. FEMS Microbiology Letters published by John Wiley & Sons Ltd on behalf of the Federation of European Microbiological Societies.)

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.

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., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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.

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., (Copyright © 2012 Elsevier Inc. All rights reserved.)

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.

Leucine zippers are oligomerization domains used in a wide range of proteins. Their structure is based on a highly conserved heptad repeat sequence in which two key positions are occupied by leucines. The leucine zipper of the cell cycle-regulated Nek2 kinase is important for its dimerization and activation. However, the sequence of this leucine zipper is most unusual in that leucines occupy only one of the two hydrophobic positions. The other position, depending on the register of the heptad repeat, is occupied by either acidic or basic residues. Using NMR spectroscopy, we show that this leucine zipper exists in two conformations of almost equal population that exchange with a rate of 17 s(-1). We propose that the two conformations correspond to the two possible registers of the heptad repeat. This hypothesis is supported by a cysteine mutant that locks the protein in one of the two conformations. NMR spectra of this mutant showed the predicted 2-fold reduction of peaks in the (15)N HSQC spectrum and the complete removal of cross peaks in exchange spectra. It is possible that interconversion of these two conformations may be triggered by external signals in a manner similar to that proposed recently for the microtubule binding domain of dynein and the HAMP domain. As a result, the leucine zipper of Nek2 kinase is the first example where the frameshift of coiled-coil heptad repeats has been directly observed experimentally.

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.

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., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

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.

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., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

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.

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.

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., Competing Interests: The authors have declared that no competing interests exist.

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.

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.

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.

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.

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.

The stimulatory RNA of the Visna-Maedi virus (VMV) -1 ribosomal frameshifting signal has not previously been characterized but can be modeled either as a two-stem helix, reminiscent of the HIV-1 frameshift-stimulatory RNA, or as an RNA pseudoknot. The pseudoknot is unusual in that it would include a 7 nucleotide loop (termed here an interstem element [ISE]) between the two stems. In almost all frameshift-promoting pseudoknots, ISEs are absent or comprise a single adenosine residue. Using a combination of RNA structure probing, site directed mutagenesis, NMR, and phylogenetic sequence comparisons, we show here that the VMV stimulatory RNA is indeed a pseudoknot, conforming closely to the modeled structure, and that the ISE is essential for frameshifting. Pseudoknot function was predictably sensitive to changes in the length of the ISE, yet altering its sequence to alternate pyrimidine/purine bases was also detrimental to frameshifting, perhaps through modulation of local tertiary interactions. How the ISE is placed in the context of an appropriate helical junction conformation is not known, but its presence impacts on other elements of the pseudoknot, for example, the necessity for a longer than expected loop 1. This may be required to accommodate an increased flexibility of the pseudoknot brought about by the ISE. In support of this, (1)H NMR analysis at increasing temperatures revealed that stem 2 of the VMV pseudoknot is more labile than stem 1, perhaps as a consequence of its connection to stem 1 solely via flexible single-stranded loops.

The malaria parasite proliferates in the bloodstream of its vertebrate host by invading and replicating within erythrocytes. To achieve successful invasion, a number of discrete and essential events need to take place at the parasite-host cell interface. Erythrocyte-binding antigen 175 (EBA-175) is a member of a family of Plasmodium falciparum erythrocyte-binding proteins involved in the formation of a tight junction, a necessary step in invasion. Here we present the crystal structure of EBA-175 region VI (rVI), a cysteine-rich domain that is highly conserved within the protein family and is essential for EBA-175 trafficking. The structure was solved by selenomethionine single-wavelength anomalous dispersion at 1.8 A resolution. It reveals a homodimer, containing in each subunit a compact five-alpha-helix core that is stabilized by four conserved disulfide bridges. rVI adopts a novel fold that is likely conserved across the protein family, indicating a conserved function. It shows no similarity to the Duffy-binding-like domains of EBA-175 involved in erythrocyte binding, indicating a distinct role. Remarkably, rVI possesses structural features related to the KIX-binding domain of the coactivator CREB-binding protein, supporting the binding and trafficking roles that have been ascribed to it and providing a rational basis for further experimental investigation of its function.

The dimeric Ser/Thr kinase Nek2 regulates centrosome cohesion and separation through phosphorylation of structural components of the centrosome, and aberrant regulation of Nek2 activity can lead to aneuploid defects characteristic of cancer cells. Mutational analysis of autophosphorylation sites within the kinase domain identified by mass spectrometry shows a complex pattern of positive and negative regulatory effects on kinase activity that are correlated with effects on centrosomal splitting efficiency in vivo. The 2.2-A resolution x-ray structure of the Nek2 kinase domain in complex with a pyrrole-indolinone inhibitor reveals an inhibitory helical motif within the activation loop. This helix presents a steric barrier to formation of the active enzyme and generates a surface that may be exploitable in the design of specific inhibitors that selectively target the inactive state. Comparison of this "auto-inhibitory" conformation with similar arrangements in cyclin-dependent kinase 2 and epidermal growth factor receptor kinase suggests a role for dimerization-dependent allosteric regulation that combines with autophosphorylation and protein phosphatase 1c phosphatase activity to generate the precise spatial and temporal control required for Nek2 function in centrosomal maturation.

UvrD is a helicase that is widely conserved in gram-negative bacteria. A uvrD homologue was identified in Mycobacterium tuberculosis on the basis of the homology of its encoded protein with Escherichia coli UvrD, with which it shares 39% amino acid identity, distributed throughout the protein. The gene was cloned, and a histidine-tagged form of the protein was expressed and purified to homogeneity. The purified protein had in vitro ATPase activity that was dependent upon the presence of DNA. Oligonucleotides as short as four nucleotides were sufficient to promote the ATPase activity. The DNA helicase activity of the enzyme was only fueled by ATP and dATP. UvrD preferentially unwound 3'-single-stranded tailed duplex substrates over 5'-single-stranded ones, indicating that the protein had a duplex-unwinding activity with 3'-to-5' polarity. A 3' single-stranded DNA tail of 18 nucleotides was required for effective unwinding. By using a series of synthetic oligonucleotide substrates, we demonstrated that M. tuberculosis UvrD has an unwinding preference towards nicked DNA duplexes and stalled replication forks, representing the likely sites of action in vivo. The potential role of M. tuberculosis UvrD in maintenance of bacterial genomic integrity makes it a promising target for drug design against M. tuberculosis.

14-3-3 proteins are a ubiquitous class of regulatory proteins found in all eukaryotic cells and were the first class of molecules to be recognized as discrete phosphoserine/threonine binding modules. 14-3-3 proteins bind a large number of different substrates to regulate a wide array of cellular signaling events including cell cycle progression and DNA damage responses, programmed cell death, cytoskeletal dynamics, transcriptional control of gene expression, as well as processes directly related to cancer progression. In this review, the structural basis of phosphorylation-dependent binding of 14-3-3 to peptide and protein ligands is discussed along with mechanisms that govern how 14-3-3 regulates the function of its bound ligands. The X-ray crystal structures of all human 14-3-3 proteins bound to peptides have now been solved. Here, we use structural comparisons between isoforms as a framework for discussion of ligand binding by 14-3-3 as well as the mechanisms through which post-translational modification of the different isoforms alters their function.

Eukaryotic initiation factor 5 (eIF5) plays multiple roles in translation initiation. Its N-terminal domain functions as a GTPase-activator protein (GAP) for GTP bound to eIF2, while its C-terminal region nucleates the interactions between multiple translation factors, including eIF1, which acts to inhibit GTP hydrolysis or P(i) release, and the beta subunit of eIF2. These proteins and the events in which they participate are critical for the accurate recognition of the correct start codon during translation initiation. Here, we report the three-dimensional solution structure of the N-terminal domain of human eIF5, comprising two subdomains, both reminiscent of nucleic-acid-binding modules. The N-terminal subdomain contains the "arginine finger" motif that is essential for GAP function but which, unusually, resides in a partially disordered region of the molecule. This implies that a conformational reordering of this portion of eIF5 is likely to occur upon formation of a competent complex for GTP hydrolysis, following the appropriate activation signal. Interestingly, the N-terminal subdomain of eIF5 reveals an alpha/beta fold structurally similar to both the archaeal orthologue of the beta subunit of eIF2 and, unexpectedly, to eIF1. These results reveal a novel protein fold common to several factors involved in related steps of translation initiation. The implications of these observations are discussed in terms of the mechanism of translation initiation.

Histone variant H2AX phosphorylation in response to DNA damage is the major signal for recruitment of DNA-damage-response proteins to regions of damaged chromatin. Loss of H2AX causes radiosensitivity, genome instability, and DNA double-strand-break repair defects, yet the mechanisms underlying these phenotypes remain obscure. Here, we demonstrate that mammalian MDC1/NFBD1 directly binds to phospho-H2AX (gammaH2AX) by specifically interacting with the phosphoepitope at the gammaH2AX carboxyl terminus. Moreover, through a combination of biochemical, cell-biological, and X-ray crystallographic approaches, we reveal the molecular details of the MDC1/NFBD1-gammaH2AX complex. These data provide compelling evidence that the MDC1/NFBD1 BRCT repeat domain is the major mediator of gammaH2AX recognition following DNA damage. We further show that MDC1/NFBD1-gammaH2AX complex formation regulates H2AX phosphorylation and is required for normal radioresistance and efficient accumulation of DNA-damage-response proteins on damaged chromatin. Thus, binding of MDC1/NFBD1 to gammaH2AX plays a central role in the mammalian response to DNA damage.

Forkhead-associated (FHA) domains are modular phosphopeptide recognition motifs with a striking preference for phosphothreonine-containing epitopes. FHA domains have been best characterized in eukaryotic signaling pathways but have been identified in six proteins in Mycobacterium tuberculosis, the causative organism of tuberculosis. One of these, coded by gene Rv1747, is an ABC transporter and the only one to contain two such modules. A deletion mutant of Rv1747 is attenuated in a mouse intravenous injection model of tuberculosis where the bacterial load of the mutant is 10-fold lower than that of the wild type in both lungs and spleen. In addition, growth of the mutant in mouse bone marrow-derived macrophages and dendritic cells is significantly impaired. In contrast, growth of this mutant in vitro was indistinguishable from that of the wild type. The mutant phenotype was lost when the mutation was complemented by the wild-type allele, confirming that it was due to mutation of Rv1747. Using yeast two-hybrid analysis, we have shown that the Rv1747 protein interacts with the serine-threonine protein kinase PknF. This interaction appears to be phospho-dependent since it is abrogated in a kinase-dead mutant and by mutations in the presumed activation loop of PknF and in the first FHA domain of Rv1747. These results demonstrate that the protein coded by Rv1747 is required for normal virulent infection by M. tuberculosis in mice and, since it interacts with a serine-threonine protein kinase in a kinase-dependent manner, indicate that it forms part of an important phospho-dependent signaling pathway.

The C-terminal domain (CTD) of the large subunit of RNA polymerase II is a platform for mRNA processing factors and links gene transcription to mRNA capping, splicing and polyadenylation. Pcf11, an essential component of the mRNA cleavage factor IA, contains a CTD-interaction domain that binds in a phospho-dependent manner to the heptad repeats within the RNA polymerase II CTD. We show here that the phosphorylated CTD exists as a dynamic disordered ensemble in solution and, by induced fit, it assumes a structured conformation when bound to Pcf11. In addition, we detected cis-trans populations for the CTD prolines, and found that only the all-trans form is selected for binding. These data suggest that the recognition of the CTD is regulated by independent site-specific modifications (phosphorylation and proline cis-trans isomerization) and, probably, by the local concentration of suitable binding sites.

Retroviruses are the aetiological agents of a range of human diseases including AIDS and T-cell leukaemias. They follow complex life cycles, which are still only partly understood at the molecular level. Maturation of newly formed retroviral particles is an essential step in production of infectious virions, and requires proteolytic cleavage of Gag polyproteins in the immature particle to form the matrix, capsid and nucleocapsid proteins present in the mature virion. Capsid proteins associate to form a dense viral core that may be spherical, cylindrical or conical depending on the genus of the virus. Nonetheless, these assemblies all appear to be composed of a lattice formed from hexagonal rings, each containing six capsid monomers. Here, we describe the X-ray structure of an individual hexagonal assembly from N-tropic murine leukaemia virus (N-MLV). The interface between capsid monomers is generally polar, consistent with weak interactions within the hexamer. Similar architectures are probably crucial for the regulation of capsid assembly and disassembly in all retroviruses. Together, these observations provide new insights into retroviral uncoating and how cellular restriction factors may interfere with viral replication.

Germline mutations in the BRCA1 tumor suppressor gene often result in a significant increase in susceptibility to breast and ovarian cancers. Although the molecular basis of their effects remains largely obscure, many mutations are known to target the highly conserved C-terminal BRCT repeats that function as a phosphoserine/phosphothreonine-binding module. We report the X-ray crystal structure at a resolution of 1.85 A of the BRCA1 tandem BRCT domains in complex with a phosphorylated peptide representing the minimal interacting region of the DEAH-box helicase BACH1. The structure reveals the determinants of this novel class of BRCA1 binding events. We show that a subset of disease-linked mutations act through specific disruption of phospho-dependent BRCA1 interactions rather than through gross structural perturbation of the tandem BRCT domains.

Nuclear export of the transcription factor Swi6 during the budding yeast Saccharomyces cerevisiae cell cycle is known to require phosphorylation of the Swi6 serine 160 residue. We show that Clb6/Cdc28 kinase is required for this nuclear export. Furthermore, Cdc28 combined with the S-phase cyclin Clb6 specifically phosphorylates serine 160 of Swi6 in vitro. Nuclear import of Swi6 occurs concomitantly with dephosphorylation of serine 160 in late M phase. We show that Cdc14 phosphatase, the principal effector of the mitotic exit network, can trigger nuclear import of Swi6 in vivo and that Cdc14 dephosphorylates Swi6 at serine 160 in vitro. Taken together, these observations show how Swi6 dephosphorylation and phosphorylation are integrated into changes of Cdc28 activity governing entry and exit from the G1 phase of the cell cycle.