Phosphopeptide binding domains mediate the directed and localized assembly of protein complexes essential to intracellular kinase signaling. To identify phosphopeptide binding proteins, we developed a proteomic screening method using immobilized partially-degenerate phosphopeptide mixtures combined with SILAC and microcapillary LC/MS/MS. The method was used to identify proteins that specifically bound to phosphorylated peptide library affinity matrices, including pTyr, and the motifs pSer/pThr-Pro, pSer/pThr-X-X-X-pSer/pThr, pSer/pThr-Glu/Asp or pSer/pThr-pSer/pThr in degenerate sequence contexts. Heavy and light SILAC lysates were flowed over columns containing these phosphorylated and non-phosphorylated (control) peptide libraries respectively, and bound proteins were eluted, combined, digested and analyzed by LC/MS/MS using a hybrid quadrupole-TOF mass spectrometer. Heavy:light peptide ion ratios were calculated, and peptides that yielded ratios greater than ~3:1 were considered as being from potential phosphopeptide binding proteins since this ratio represents the lowest ratio from a known positive control. Many of those identified were known phosphopeptide-binding proteins, including the SH2 domain containing p85 subunit of PI3K bound to pTyr, 14-3-3 bound to pSer/pThr-Asp/Glu, polo-box domain containing PLK1 and Pin1 bound to pSer/pThr-Pro and pyruvate kinase M2 binding to pTyr. Approximately half of the hits identified by the peptide library screens were novel. Protein domain enrichment analysis revealed that most pTyr hits contain SH2 domains, as expected and to lesser extent SH3, C1, STAT, Tyr phosphatase, Pkinase, C2 and PH domains, however, pSer/pThr motifs did not reveal enriched domains across hits.
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.
ABC Transporter; Bacterial Protein Kinases; Post-translational Modification; Prokaryotic Signal Transduction; Serine/Threonine Protein Kinase; Mycobacterium tuberculosis
The Mre11/Rad50/Nbs1 (MRN) 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 2.3Å 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 di-phosphorylated pSer-Asp-pThr-Asp-like motifs, recently identified as multi-copy docking sites within human Mdc1, are evolutionarily conserved Nbs1 binding targets. Furthermore, we identify similar phospho-motifs within Ctp1, the fission yeast orthologue of the human tumor-suppressor, CtIP, and show that their interactions with the Nbs1 FHA domain are necessary for Ctp1-dependent resistance to DNA-damage. Finally, we establish that human Nbs1 interactions with Mdc1 can occur through both its FHA and BRCT-repeat domains, suggesting how their structural and functional inter-dependence may underpin Nbs1 adaptor functions in the DNA-damage response.
FHA domains are protein modules that switch signals in diverse biological pathways by monitoring the phosphorylation of threonine residues of target proteins. As part of the effort to gain insight into cellular avoidance of cancer, FHA domains involved in the cellular response to DNA damage have been especially well characterized. The complete protein where the FHA domain resides and the interaction partners determine the nature of the signaling. Thus, a key biochemical question is: how do FHA domains pick out their partners from among thousands of alternatives in the cell? This Account discusses the structure, affinity, and specificity of FHA domains and the formation of their functional structure.
Although FHA domains share sequence identity at only five loop residues, they all fold into a β-sandwich of two β-sheets. The conserved Arg and Ser of the recognition loops recognize the phosphorylation of the Thr targeted. Side chains emanating from loops that join β-strand 4 with 5, 6 with 7, or 10 with 11 make specific contacts with amino acids of the ligand that tailor sequence preferences. Many FHA domains choose a partner in extended conformation, somewhat according to the residue three after the phosphoThr in sequence (pT+3 position). One group of FHA domains chooses a short carboxylate-containing side chain at pT+3. Another group chooses a long, branched aliphatic side chain. A third group prefers other hydrophobic or uncharged, polar side chains at pT+3. However, another FHA domain instead chooses on the basis of pT−2, pT−3, and pT+1 positions. An FHA domain from a marker of human cancer instead chooses a much longer protein fragment that adds a β-strand to its β-sheet and that presents hydrophobic residues from a novel helix to the usual recognition surface. This novel recognition site and more remote sites for the binding of other types of protein partners were predicted for the entire family of FHA domains by a bioinformatics approach.
The phosphopeptide-dependent dynamics of an FHA domain, SH2 domain, and PTB domain suggest a common theme: rigid, preformed binding surfaces support van der Waals contacts that provide favorable binding enthalpy. Despite the lack of pronounced conformational changes in FHA domains linked to binding events, more subtle adjustments may be possible. In the one FHA domain tested, phosphoThr peptide binding is accompanied by increased flexibility just outside the binding site and increased rigidity across the β-sandwich. The folding of the same FHA domain progresses through near-native intermediates that stabilize the recognition loops in the center of the phosphoprotein-binding surface; this may promote rigidity in the interface and affinity for targets phosphorylated on threonine.
FHA domain; phosphorylation-dependent signaling; protein structure; molecular recognition; protein dynamics; protein folding
Phosphorylation and dephosphorylation of the C-terminal domain (CTD) of RNA polymerase II (Pol II) represent a critical regulatory checkpoint for transcription. Transcription initiation requires Fcp1/Scp1-mediated dephosphorylation of phospho-CTD. Fcp1 and Scp1 belong to a family of Mg2+-dependent phosphoserine (P.Ser)/phosphothreonine (P.Thr)-specific phosphatases. We recently showed that Scp1 is an evolutionarily conserved regulator of neuronal gene silencing. Here, we present the X-ray crystal structures of a dominant-negative form of human Scp1 (D96N mutant) bound to mono- and diphosphorylated peptides encompassing the CTD heptad repeat (Y1S2P3T4S5P6S7). Moreover, kinetic and thermodynamic analyses of Scp1-phospho-CTD peptide complexes support the structures determined. This combined structure-function analysis discloses the residues in Scp1 involved in CTD binding and its preferential dephosphorylation of P.Ser5 of the CTD heptad repeat. Moreover, these results provide a template for the design of specific inhibitors of Scp1 for the study of neuronal stem cell development.
The DNA damage response depends on the concerted activity of protein serine/threonine kinases and modular phosphoserine/threonine binding domains to relay the damage signal and recruit repair proteins. The PIKK family of protein kinases, which includes ATM/ATR/DNA-PK, preferentially phosphorylate Ser-Gln sites, while their basophilic downstream effecter kinases, Chk1/Chk2/MK2 preferentially phosphorylate hydrophobic-X-Arg-X-X-Ser/Thr-hydrophobic sites. A subset of tandem BRCT domains act as phosphopeptide binding modules that bind to ATM/ATR/DNA-PK substrates after DNA damage. Conversely, 14-3-3 proteins interact with substrates of Chk1/Chk2/MK2. FHA domains have been shown to interact with substrates of ATM/ATR/DNA-PK and CK2. In this review we consider how substrate phsophorylation together with BRCT domains, FHA domains and 14-3-3 proteins function to regulate ionizing radiation-induced nuclear foci and help to establish the G2/M checkpoint. We discuss the role of MDC1 a molecular scaffold that recruits early proteins to foci, such as NBS1 and RNF8, through distinct phosphodependent interactions. In addition, we consider the role of 14-3-3 proteins and the Chk2 FHA domain in initiating and maintaining cell cycle arrest.
Mutants of the highly promiscuous lantipeptide synthetase ProcM phosphorylate a wide range of peptides attached to ProcA leader peptides, both in vitro and in E. coli. As such, the ProcM mutants are useful enzymes for the enzymatic preparation of phosphorylated peptides.
Phosphorylation is an abundant post-translational modification involved in a myriad of cell signaling pathways. Herein, we have engineered the class II lantipeptide synthetase ProcM to generate a variety of peptides containing O-phosphoserine (pSer) and O-phosphothreonine (pThr) residues, either in vitro or in vivo.
Peptidyl prolyl cis-trans isomerase (PPIase) interacting with NIMA-1 (Pin1) catalyzes the cis-trans isomerization of pSer/pThr–Pro amide bonds. Pin1 is a two-domain protein that represents a promising target for the treatment of cancer. Both domains of Pin1 bind the pSer/pThr–Pro motif; PPIase enzymatic activity occurs in the catalytic domain, and the WW domain acts as a recognition module for the pSer/pThr–Pro motif. An assay we call an Enzyme-Linked Enzyme Binding Assay (ELEBA), was developed to measure the Kd of ligands that bind selectively to the WW domain. A ligand specific for the WW domain of Pin1 was covalently immobilized in a 96-well plate. Commercially available Pin1 conjugated to horseradish peroxidase was used for chemiluminescent detection of ligands that block the association of the WW domain with immobilized ligand. The peptide ligands were derived from the cell cycle regulatory phosphatase, Cdc25c, residues 45-50. The Kd values for Fmoc–VPRpTPVGGGK–NH2 and Ac–VPRpTPV–NH2 were determined to be 36 ± 4 μM and 110 ± 30 μM respectively. The ELEBA offers a selective approach to detect ligands that bind to the Pin1 WW domain, even in the presence of the catalytic domain. This method may be applied to any dual specificity, multi-domain protein.
Pin1; WW domain; PPIase; HRP; chemiluminescent binding assay; Pin1 ligands
The forkhead-associated (FHA) domain recognizes phosphothreonine (pT) with high specificity and functional diversity. TIFA (TRAF-interacting protein with an FHA domain) is the smallest FHA-containing human protein. Its overexpression was previously suggested to provoke NF-κB activation, yet its exact roles in this signaling pathway and the underlying molecular mechanism remain unclear. Here we identify a novel threonine phosphorylation site on TIFA and show that this phosphorylated threonine (pT) binds with the FHA domain of TIFA, leading to TIFA oligomerization and TIFA-mediated NF-κB activation. Detailed analysis indicated that unphosphorylated TIFA exists as an intrinsic dimer and that the FHA-pT9 binding occurs between different dimers of TIFA. In addition, silencing of endogenous TIFA resulted in attenuation of tumor necrosis factor alpha (TNF-α)-mediated downstream signaling. We therefore propose that the TIFA FHA-pT9 binding provides a previously unidentified link between TNF-α stimulation and NF-κB activation. The intermolecular FHA-pT9 binding between dimers also represents a new mechanism for the FHA domain.
Prokaryotic cell wall biosynthesis is coordinated with cell growth and division, but the mechanisms regulating this dynamic process remain obscure. Here, we describe a phosphorylation-dependent regulatory complex that controls peptidoglycan (PG) biosynthesis in Mycobacterium tuberculosis. We found that PknB, a PG-responsive Ser-Thr protein kinase (STPK), initiates complex assembly by phosphorylating a kinase-like domain in the essential PG biosynthetic protein, MviN. This domain was structurally diverged from active kinases and did not mediate phosphotransfer. Threonine phosphorylation of the pseudokinase domain recruited the FhaA protein through its forkhead-associated (FHA) domain. The crystal structure of this phosphorylated pseudokinase–FHA domain complex revealed the basis of FHA domain recognition, which included unexpected contacts distal to the phosphorylated threonine. Conditional degradation of these proteins in mycobacteria demonstrated that MviN was essential for growth and PG biosynthesis and that FhaA regulated these processes at the cell poles and septum. Controlling this spatially localized PG regulatory complex is only one of several cellular roles ascribed to PknB, suggesting that the capacity to coordinate signaling across multiple processes is an important feature conserved between eukaryotic and prokaryotic STPK networks.
Members of the LanL family of lanthionine synthetases consist of three catalytic domains, an N-terminal pSer/pThr lyase domain, a central Ser/Thr kinase domain, and a C-terminal lanthionine cyclase domain. The N-terminal lyase domain has sequence homology with members of the OspF family of effector proteins. In this study, the residues in the lyase domain of VenL that are conserved in the active site of OspF proteins were mutated to evaluate their importance for catalysis. In addition, residues that are fully conserved in the LanL family but not in the OspF family were mutated. Activity assays with these mutant proteins are consistent with a model in which Lys80 in VenL deprotonates the α-proton of pSer/pThr residues to initiate the elimination reaction. Lys51 is proposed to activate this proton by coordination to the carbonyl of the pSer/pThr, and His53 is believed to protonate the phosphate leaving group. These functions are very similar to the corresponding homologous residues in OspF proteins. On the other hand, recognition of the phosphate group of pSer/pThr appears to be achieved differently in VenL than in the OspF proteins. Arg156 and Lys103 are thought to interact with the phosphate group on the basis of a structural homology model.
The activity of protein phosphatase-1 (PP1) inhibitor-1 (I-1) is antithetically modulated by the cAMP-protein kinase A (PKA) and Ca2+-protein kinase C (PKC) signaling axes. β-adrenergic (β-AR) stimulation results in PKA-phosphorylation of I-1 at threonine 35 (Thr35) and depressed PP1 activity, while PKC phosphorylation at serine 67 (Ser67) and/or Thr75 increases PP1 activity. In heart failure, pThr35 is decreased while pSer67 and pThr75 are elevated. However, the role of Ser67/Thr75 phosphorylation in vivo and its effects on Ca2+-cycling are not known. Thus, our aim was to investigate the functional significance of Ser67 and Thr75 phosphorylation in intact hearts. We generated transgenic mice (TG) with cardiac-specific overexpression of constitutively phosphorylated I-1 at Ser67 and Thr75 (S67D/T75D) and evaluated cardiac function. The S67D/T75D cardiomyocytes exhibited significantly depressed Ca2+-kinetics and contractile parameters, compared with wild-type (WT) cells. The decreased Ca2+-cycling was associated with a 27 % increase in PP1 activity, no alterations in PP2 activity and impaired phosphorylation of myosin-binding protein-C (MyBPC). Upon aging, there was cardiac remodeling associated with increases in systolic and diastolic left ventricular internal diameter dimensions (at 16 months), compared with WTs. The results indicate that phosphorylation of I-1 at Ser67 and Thr75 is associated with increased PP1 activity and depressed cardiomyocyte Ca2+-cycling, which manifests in geometrical alterations over the long term. Thus, hyper-phosphorylation of these sites in failing hearts may contribute to deteriorative remodeling.
Protein phosphatase 1; Inhibitor-1; Calcium cycling; Cardiac function
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.
We screened growth differentiation factor 9 (GDF9) coding regions for mutations in a Chinese sample of 100 women with premature ovarian failure (POF) and discovered 4 novel SNPs: c.436C>T (p.Arg146Cys), c.588A>C (silent), c.712A>G (p.Thr238Ala) and c.1283G>C (p.Ser428Thr). Non-synonymous SNPs c.436C>T and c.1283G>C were also detected in the control population. The c.712A>G perturbation results in a missense mutation (p.Thr238Ala) and was not present in any of 96 controls. Substitution of the hydrophobic amino acid residue alanine for hydrophilic threonine may disrupt GDF9 function.
Premature Ovarian Failure; Growth Differentiation Factor 9; Mutation; Single Nucleotide Polymorphism
The BRCA1 BRCT domain binds pSer-x-x-Phe motifs in partner proteins to regulate the cellular response to DNA damage. Approximately 120 distinct missense variants have been identified in the BRCA1 BRCT through breast cancer screening, and several of these have been linked to an increased cancer risk. Here we probe the structures and peptide-binding activities of variants that affect the BRCA1 BRCT phosphopeptide-binding groove. The results obtained from the G1656D and T1700A variants illustrate the role of Ser1655 in pSer recognition. Mutations at Arg1699 (R1699W and R1699Q) significantly reduce peptide binding through loss of contacts to the main chain of the Phe(+3) residue and, in the case of R1699W, to a destabilization of the BRCT fold. The R1835P and E1836K variants do not dramatically reduce peptide binding, in spite of the fact that these mutations significantly alter the structure of the walls of the Phe(+3) pocket.
Protein phosphorylation plays an important role in cell signaling. However, in the Archaea, little is known about which proteins are phosphorylated and which kinases are involved. In this study, we identified, for the first time, a typical eukaryote-like Ser/Thr protein kinase and its protein partner, a forkhead-associated (FHA)-domain-containing protein, from the archaeon Sulfolobus tokodaii strain 7. This protein kinase, ST1565, physically interacted with the FHA-domain-containing protein, ST0829, both in vivo and in vitro. ST1565 preferred Mn2+ as a cofactor for autophosphorylation and for substrate phosphorylation; the optimal temperature for this was 45°C, and the optimal pH was 5.5 to 7.5. The critical amino acid residues of the conserved FHA and kinase domain sites were identified by performing a series of mutation assays. Thr329 was part of a major activation site in the kinase, while Thr326 was a negative regulation site. Several mutants with amino acid substitutions in the conserved FHA domain sites of ST0829 did not physically interact with ST1565. A structural model for the FHA domain demonstrated that the mutation sites were located at the edge of the protein and thus were in the domain that potentially interacts with ST1565. This report describes pioneering work on the third domain of life, the Archaea, showing that a protein kinase interacts with and phosphorylates an FHA-domain-containing protein. Our data provide critical information on the structural or functional characteristics of archaeal proteins and could help increase our understanding of fundamental signaling mechanisms in all three domains of life.
Phosphosignaling through pSer/pThr/pTyr is emerging as a common signaling mechanism in prokaryotes. The human pathogen Staphylococcus aureus (S. aureus) produces two low-molecular weight protein tyrosine phosphatases, PtpA and PtpB, with unknown functions. To provide the structural context for understanding PtpA function and substrate recognition, establish PtpA’s structural relations within the protein tyrosine phosphatase family, and to provide a framework for the design of specific inhibitors, we solved the crystal structure of PtpA at 1 Å resolution. While PtpA adopts the common, conserved PTP fold and shows close overall similarity to eukaryotic PTPs, several features in the active site and surface organization are unique and can be explored to design selective inhibitors. A peptide bound in the active site mimics a phosphotyrosine substrate, affords insight into substrate recognition, and provides a testable substrate prediction. Genetic deletion of ptpA or ptpB does not affect in vitro growth or cell wall integrity, raising the possibility that PtpA and PtpB have specialized functions during infection.
Protein Tyrosine Phosphatase; crystal structure; S. aureus; phosphosignaling; substrate
MxiG is a single-pass membrane protein that oligomerizes within the inner membrane ring of the Shigella flexneri type III secretion system (T3SS). The MxiG N-terminal domain (MxiG-N) is the predominant cytoplasmic structure; however, its role in T3SS assembly and secretion is largely uncharacterized. We have determined the solution structure of MxiG-N residues 6–112 (MxiG-N(6–112)), representing the first published structure of this T3SS domain. The structure shows strong structural homology to forkhead-associated (FHA) domains. Canonically, these cell-signaling modules bind phosphothreonine (Thr(P)) via highly conserved residues. However, the putative phosphate-binding pocket of MxiG-N(6–112) does not align with other FHA domain structures or interact with Thr(P). Furthermore, mutagenesis of potential phosphate-binding residues has no effect on S. flexneri T3SS assembly and function. Therefore, MxiG-N has a novel function for an FHA domain. Positioning of MxiG-N(6–112) within the EM density of the S. flexneri needle complex gives insight into the ambiguous stoichiometry of the T3SS, supporting models with 24 MxiG subunits in the inner membrane ring.
Bacteria; NMR; Protein Assembly; Protein Secretion; Signal Transduction; Shigella flexneri; Type Three Secretion System
The Rad53 kinase plays a central role in yeast DNA damage checkpoints. Rad53 contains two FHA phosphothreonine-binding domains that are required for Rad53 activation and possibly downstream signaling. Here we show that the N-terminal Rad53 FHA1 domain interacts with the RNA recognition motif, coiled-coil, and SQ/TQ cluster domain-containing protein Mdt1 (YBl051C). The interaction of Rad53 and Mdt1 depends on the structural integrity of the FHA1 phosphothreonine-binding site as well as threonine-305 of Mdt1. Mdt1 is constitutively threonine phosphorylated and hyperphosphorylated in response to DNA damage in vivo. DNA damage-dependent Mdt1 hyperphosphorylation depends on the Mec1 and Tel1 checkpoint kinases, and Mec1 can directly phosphorylate a recombinant Mdt1 SQ/TQ domain fragment. MDT1 overexpression is synthetically lethal with a rad53 deletion, whereas mdt1 deletion partially suppresses the DNA damage hypersensitivity of checkpoint-compromised strains and generally improves DNA damage tolerance. In the absence of DNA damage, mdt1 deletion leads to delayed anaphase completion, with an elongated cell morphology reminiscent of that of G2/M cell cycle mutants. mdt1-dependent and DNA damage-dependent cell cycle delays are not additive, suggesting that they act in the same pathway. The data indicate that Mdt1 is involved in normal G2/M cell cycle progression and is a novel target of checkpoint-dependent cell cycle arrest pathways.
Tandem breast cancer C-terminal (BRCT) domains, present in many DNA repair and cell cycle checkpoint signaling proteins, are phosphoprotein binding modules. The best-characterized tandem BRCT domains to date are from the protein BRCA1 (BRCA1-BRCT), an E3 ubiquitin ligase that has been linked to breast and ovarian cancer. While X-ray crystallography and NMR spectroscopy studies have uncovered the structural determinants of specificity of BRCA1-BRCT for phosphorylated peptides, a detailed kinetic and thermodynamic characterization of the interaction is also required to understand how structure and dynamics are connected and therefore better probe the mechanism of phosphopeptide recognition by BRCT domains. Through a global analysis of binding kinetics data obtained from surface plasmon resonance (SPR) and stopped-flow fluorescence spectroscopy, we show that the recognition mechanism is complex and best modeled by two equilibrium conformations of BRCA1-BRCT in the free state that both interact with a phosphopeptide, with dissociation constants (Kd) in the micromolar range. We show that the apparent global dissociation constant derived from this kinetic analysis is similar to the Kd values measured using steady-state SPR, isothermal titration calorimetry, and fluorescence anisotropy. The dynamic nature of BRCA1-BRCT may facilitate the binding of BRCA1 to different phosphorylated protein targets.
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.
Peptidyl-prolyl isomerase Pin1 regulates the function and/or stability of phosphoproteins by altering the conformation of specific pSer/pThr-Pro peptide bonds. In this work, a cyclic peptide library was synthesized and screened against the catalytic domain of human Pin1. The selected inhibitors contained a consensus motif of D-pThr-Pip-Nal (where Pip is L-piperidine-2-carboxylic acid and Nal is L-2-naphthylalanine). Representative compounds were tested for binding to Pin1 by isothermal titration calorimetry and inhibition of Pin1 activity and the most potent inhibitors had KD (and KI) values in the low nanomolar range. Treatment of breast cancer cells with the inhibitors, which were rendered membrane permeable by attachment of an octaarginine sequence, inhibited cell proliferation and increased the protein levels of two previously established Pin1 substrates, PML and SMRT. Finally, a second generation of cell permeable Pin1 inhibitors was designed by replacing the noncritical residues within the cyclic peptide ring with arginine residues and shown to have anti-proliferative activity against the cancer cells.
Peptidyl-prolyl cis-trans isomerization; specificity; phosphothreonine; kinetics; peptide library; cell permeability
MDC1 is a key mediator of the DNA-damage response in mammals with several phosphorylation-dependent protein interaction domains. The function of its N-terminal forkhead-associated (FHA) domain remains elusive. Here, we show with structural, biochemical and cellular data that the FHA domain mediates phosphorylation-dependent dimerization of MDC1 in response to DNA damage. Crystal structures of the FHA domain reveal a face-to-face dimer with pseudo-dyad symmetry. We found that the FHA domain recognizes phosphothreonine 4 (pT4) at the N-terminus of MDC1 and determined its crystal structure in complex with a pT4 peptide. Biochemical analysis further revealed that in the dimer, the FHA domain binds in trans to pT4 from the other subunit, which greatly stabilizes the otherwise unstable dimer. We show that T4 is phosphorylated primarily by ATM upon DNA damage. MDC1 mutants with the FHA domain deleted or impaired in its ability to dimerize formed fewer foci at DNA-damage sites, but the localization defect was largely rescued by an artificial dimerization module, suggesting that dimerization is the primary function of the MDC1 FHA domain. Our results suggest a novel mechanism for the regulation of MDC1 function through T4 phosphorylation and FHA-mediated dimerization.
Oxidation of membrane phospholipids is associated with
neurodegenerative disease, and cancer. Oxyradical damage to phospholipids
results in the production of reactive aldehydes that adduct proteins
and modulate their function. 4-Hydroxynonenal (HNE), a common product
of oxidative damage to lipids, adducts proteins at exposed Cys, His,
or Lys residues. Here, we demonstrate that peptidyl-prolyl cis/trans-isomerase A1 (Pin1), an enzyme
that catalyzes the conversion of the peptide bond of pSer/pThr-Pro
moieties in signaling proteins from cis to trans, is highly susceptible
to HNE modification. Incubation of purified Pin1 with HNE followed
by MALDI-TOF/TOF mass spectrometry resulted in detection of Michael
adducts at the active site residues His-157 and Cys-113. Time and
concentration dependencies indicate that Cys-113 is the primary site
of HNE modification. Pin1 was adducted in MDA-MB-231 breast cancer
cells treated with 8-alkynyl-HNE as judged by click chemistry conjugation
with biotin followed by streptavidin-based pulldown and Western blotting
with anti-Pin1 antibody. Furthermore, orbitrap MS data support the
adduction of Cys-113 in the Pin1 active site upon HNE treatment of
MDA-MB-231 cells. siRNA knockdown of Pin1 in MDA-MB-231 cells partially
protected the cells from HNE-induced toxicity. Recent studies indicate
that Pin1 is an important molecular target for the chemopreventive
effects of green tea polyphenols. The present study establishes that
it is also a target for electrophilic modification by products of
A net increase in the backbone rigidity of the kinase-interacting FHA domain (KI-FHA) from the Arabidopsis receptor kinase-associated protein phosphatase (KAPP) accompanies the binding of a phosphoThr peptide from its CLV1 receptor-like kinase partner, according to 15N NMR relaxation at 11.7 and 14.1 T. All of the loops of free KI-FHA display evidence of nsec-scale motions. Many of these same residues have residual dipolar couplings that deviate from structural predictions. Binding of the CLV1 pT868 peptide seems to reduce nsec-scale fluctuations of all loops, including half of the residues of recognition loops. Residues important for affinity are found to be rigid, i.e. conserved residues and residues of the subsite for the key pT+3 peptide position. –This behavior parallels SH2 and PTB domain recognition of pTyr peptides. PhosphoThr peptide binding increases KI-FHA backbone rigidity (S2) of three recognition loops, a loop nearby, seven strands from the β-sandwich, and a distal loop. Compensating the trend of increased rigidity, binding enhances fast mobility at a few sites in four loops on the periphery of the recognition surface and in two loops on the far side of the β-sandwich. Line broadening evidence of µsec to msec-scale fluctuations occurs across the six-stranded β-sheet and nearby edges of the β-sandwich; this forms a network connected by packing of interior side chains and H-bonding. A patch of the slowly fluctuating residues coincides with the site of segment-swapped dimerization in crystals of the FHA domain of human Chfr. Phosphopeptide binding introduces µsec to msec-scale fluctuations to more residues of the long 8/9 recognition loop of KI-FHA. The rigidity of this FHA domain appears to couple as a whole to pThr peptide binding.
FHA domain; phosphoThr-binding module; protein-protein interactions; NMR relaxation; relaxation dispersion; dynamics; residual dipolar couplings