High affinity anchoring of the decoration protein pb10 onto the bacteriophage T5 capsid.

Bacteriophage capsids constitute icosahedral shells of exceptional stability that protect the viral genome. Many capsids display on their surface decoration proteins whose structure and function remain largely unknown. The decoration protein pb10 of phage T5 binds at the centre of the 120 hexamers formed by the major capsid protein. Here we determined the 3D structure of pb10 and investigated its capsid-binding properties using NMR, SAXS, cryoEM and SPR. Pb10 consists of an α-helical capsid-binding domain and an Ig-like domain exposed to the solvent. It binds to the T5 capsid with a remarkably high affinity and its binding kinetics is characterized by a very slow dissociation rate. We propose that the conformational exchange events observed in the capsid-binding domain enable rearrangements upon binding that contribute to the quasi-irreversibility of the pb10-capsid interaction. Moreover we show that pb10 binding is a highly cooperative process, which favours immediate rebinding of newly dissociated pb10 to the 120 hexamers of the capsid protein. In extreme conditions, pb10 protects the phage from releasing its genome. We conclude that pb10 may function to reinforce the capsid thus favouring phage survival in harsh environments.

Bacteriophage SPP1 tail tube protein self-assembles into ß-structure-rich tubes.

The majority of known bacteriophages have long tails that serve for bacterial target recognition and viral DNA delivery into the host. These structures form a tube from the viral capsid to the bacterial cell. The tube is formed primarily by a helical array of tail tube protein (TTP) subunits. In phages with a contractile tail, the TTP tube is surrounded by a sheath structure. Here, we report the first evidence that a phage TTP, gp17.1 of siphophage SPP1, self-assembles into long tubes in the absence of other viral proteins. gp17.1 does not exhibit a stable globular structure when monomeric in solution, even if it was confidently predicted to adopt the ß-sandwich fold of phage λ TTP. However, Fourier transform infrared and nuclear magnetic resonance spectroscopy analyses showed that its ß-sheet content increases significantly during tube assembly, suggesting that gp17.1 acquires a stable ß-sandwich fold only after self-assembly. EM analyses revealed that the tube is formed by hexameric rings stacked helicoidally with the same organization and helical parameters found for the tail of SPP1 virions. These parameters were used to build a pseudo-atomic model of the TTP tube. The large loop spanning residues 40-56 is located on the inner surface of the tube, at the interface between adjacent monomers and hexamers. In line with our structural predictions, deletion of this loop hinders gp17.1 tube assembly in vitro and interferes with SPP1 tail assembly during phage particle morphogenesis in bacteria.

Molecular exploration of the α(1A)-adrenoceptor orthosteric site: binding site definition for epinephrine, HEAT and prazosin.

Despite the physiological and pharmacological importance of the α1A-adrenoreceptor, the mode of interactions of classical agonists and radioactive ligands with this receptor is not yet clearly defined. Here, we used mutagenesis studies and binding experiments to evaluate the importance of 11 receptor sites for the binding of (125)I-HEAT, (3)H-prazosin and epinephrine. Only one residue (F312) commonly interacts with the three molecules, and, surprisingly, D106 interacts only with epinephrine in a moderate way. Our docking model shows that prazosin and HEAT are almost superimposed into the orthosteric pocket with their tetralone and quinazoline rings close to the phenyl ring of the agonist.

Regulation of the catalytic activity of the human phosphatase PTPN4 by its PDZ domain.

The human protein tyrosine phosphatase non-receptor type 4 (PTPN4) prevents cells death. Targeting its PDZ domain abrogates this protection and triggers apoptosis. We demonstrate here that the PDZ domain inhibits the phosphatase activity of PTPN4. The mere binding of a PDZ ligand is sufficient to release the catalytic inhibition. We combined analytical ultracentrifugation, small angle X-ray scattering and NMR to understand how the PDZ domain controls PTPN4 activity. We show that the physiologically active PTPN4 two-domain, encompassing the PDZ and the phosphatase domains, adopts a predominant compact conformation in solution. The PDZ ligand binding restores the catalytic competence of PTPN4 disrupting the transient interdomain communication. This study strengthens the emerging notion that PDZ domains can act as regulators of enzyme activity and therefore are active players in the dynamic regulation of signaling pathways.

Orthosteric binding of ρ-Da1a, a natural peptide of snake venom interacting selectively with the α1A-adrenoceptor.

ρ-Da1a is a three-finger fold toxin from green mamba venom that is highly selective for the α1A-adrenoceptor. This toxin has atypical pharmacological properties, including incomplete inhibition of (3)H-prazosin or (125)I-HEAT binding and insurmountable antagonist action. We aimed to clarify its mode of action at the α1A-adrenoceptor. The affinity (pKi 9.26) and selectivity of ρ-Da1a for the α1A-adrenoceptor were confirmed by comparing binding to human adrenoceptors expressed in eukaryotic cells. Equilibrium and kinetic binding experiments were used to demonstrate that ρ-Da1a, prazosin and HEAT compete at the α1A-adrenoceptor. ρ-Da1a did not affect the dissociation kinetics of (3)H-prazosin or (125)I-HEAT, and the IC50 of ρ-Da1a, determined by competition experiments, increased linearly with the concentration of radioligands used, while the residual binding by ρ-Da1a remained stable. The effect of ρ-Da1a on agonist-stimulated Ca(2+) release was insurmountable in the presence of phenethylamine- or imidazoline-type agonists. Ten mutations in the orthosteric binding pocket of the α1A-adrenoceptor were evaluated for alterations in ρ-Da1a affinity. The D106(3.32)A and the S188(5.42)A/S192(5.46)A receptor mutations reduced toxin affinity moderately (6 and 7.6 times, respectively), while the F86(2.64)A, F288(6.51)A and F312(7.39)A mutations diminished it dramatically by 18- to 93-fold. In addition, residue F86(2.64) was identified as a key interaction point for (125)I-HEAT, as the variant F86(2.64)A induced a 23-fold reduction in HEAT affinity. Unlike the M1 muscarinic acetylcholine receptor toxin MT7, ρ-Da1a interacts with the human α1A-adrenoceptor orthosteric pocket and shares receptor interaction points with antagonist (F86(2.64), F288(6.51) and F312(7.39)) and agonist (F288(6.51) and F312(7.39)) ligands. Its selectivity for the α1A-adrenoceptor may result, at least partly, from its interaction with the residue F86(2.64), which appears to be important also for HEAT binding.

Inhibition of TGF-ß signaling at the nuclear envelope: characterization of interactions between MAN1, Smad2 and Smad3, and PPM1A.

Signaling by transforming growth factor-ß (TGF-ß) is critical for various developmental processes and culminates in the activation of the transcription factors Smad2 and Smad3. MAN1, an integral protein of the inner nuclear membrane, inhibits TGF-ß signaling by binding to Smad2 and Smad3. Depletion of the gene LEMD3 encoding MAN1 leads to developmental anomalies in mice, and heterozygous loss-of-function mutations in LEMD3 in humans cause sclerosing bone dysplasia. We modeled the three-dimensional structure of the MAN1-Smad2 complex from nuclear magnetic resonance and small-angle x-ray scattering data. As predicted by this model, we found that MAN1 competed in vitro and in cells with the transcription factor FAST1 (forkhead activin signal transducer 1) for binding to Smad2. The model further predicted that MAN1 bound to activated Smad2-Smad4 or Smad3-Smad4 complexes, which was confirmed by in vitro experiments; however, in cells, MAN1 bound only to Smad2 and Smad3 and not to the Smad4-containing complexes. Overexpression of MAN1 led to dephosphorylation of Smad2 and Smad3, thus hindering their recognition by Smad4, and MAN1 bound directly in vitro to the phosphatase PPM1A, which catalyzes the dephosphorylation of Smad2/3. These results demonstrate a nuclear envelope-localized mechanism of inactivating TGF-ß signaling in which MAN1 competes with transcription factors for binding to Smad2 and Smad3 and facilitates their dephosphorylation by PPM1A.

New α-adrenergic property for synthetic MTß and CM-3 three-finger fold toxins from black mamba.

Despite their isolation more than fifteen years ago from the venom of the African mamba Dendroaspis polylepis, very few data are known on the functional activity of MTß and CM-3 toxins. MTß was initially classified as a muscarinic toxin interacting non-selectively and with low affinity with the five muscarinic receptor subtypes while no biological function was determined for CM-3. Recent results highlight the multifunctional activity of three-finger fold toxins for muscarinic and adrenergic receptors and reveal some discrepancies in the pharmacological profiles of their venom-purified and synthetic forms. Here, we report the pharmacological characterization of chemically-synthesized MTß and CM-3 toxins on nine subtypes of muscarinic and adrenergic receptors and demonstrate their high potency for α-adrenoceptors and in particular a sub-nanomolar affinity for the α1A-subtype. Strikingly, no or very weak affinity were found for muscarinic receptors, highlighting that pharmacological characterizations of venom-purified peptides may be risky due to possible contaminations. The biological profile of these two homologous toxins looks like that one previously reported for the Dendroaspis angusticeps ρ-Da1a toxin. Nevertheless, MTß and CM-3 interact more potently than ρ-Da1a with α1B- and α1D-AR subtypes. A computational analysis of the stability of the MTß structure suggests that mutation S38I, could be involved in this gain in function.

Structure of the MutLα C-terminal domain reveals how Mlh1 contributes to Pms1 endonuclease site.

Mismatch-repair factors have a prominent role in surveying eukaryotic DNA-replication fidelity and in ensuring correct meiotic recombination. These functions depend on MutL-homolog heterodimers with Mlh1. In humans, MLH1 mutations underlie half of hereditary nonpolyposis colorectal cancers (HNPCCs). Here we report crystal structures of the MutLα (Mlh1-Pms1 heterodimer) C-terminal domain (CTD) from Saccharomyces cerevisiae, alone and in complex with fragments derived from Mlh1 partners. These structures reveal structural rearrangements and additional domains in MutLα as compared to the bacterial MutL counterparts and show that the strictly conserved C terminus of Mlh1 forms part of the Pms1 endonuclease site. The structures of the ternary complexes between MutLα(CTD) and Exo1 or Ntg2 fragments reveal the binding mode of the MIP-box motif shared by several Mlh1 partners. Finally, the structures provide a rationale for the deleterious impact of MLH1 mutations in HNPCCs.

Structural model of ligand-G protein-coupled receptor (GPCR) complex based on experimental double mutant cycle data: MT7 snake toxin bound to dimeric hM1 muscarinic receptor.

The snake toxin MT7 is a potent and specific allosteric modulator of the human M1 muscarinic receptor (hM1). We previously characterized by mutagenesis experiments the functional determinants of the MT7-hM1 receptor interaction (Fruchart-Gaillard, C., Mourier, G., Marquer, C., Stura, E., Birdsall, N. J., and Servent, D. (2008) Mol. Pharmacol. 74, 1554-1563) and more recently collected evidence indicating that MT7 may bind to a dimeric form of hM1 (Marquer, C., Fruchart-Gaillard, C., Mourier, G., Grandjean, O., Girard, E., le Maire, M., Brown, S., and Servent, D. (2010) Biol. Cell 102, 409-420). To structurally characterize the MT7-hM1 complex, we adopted a strategy combining double mutant cycle experiments and molecular modeling calculations. First, thirty-three ligand-receptor proximities were identified from the analysis of sixty-one double mutant binding affinities. Several toxin residues that are more than 25 Å apart still contact the same residues on the receptor. As a consequence, attempts to satisfy all the restraints by docking the toxin onto a single receptor failed. The toxin was then positioned onto two receptors during five independent flexible docking simulations. The different possible ligand and receptor extracellular loop conformations were described by performing simulations in explicit solvent. All the docking calculations converged to the same conformation of the MT7-hM1 dimer complex, satisfying the experimental restraints and in which (i) the toxin interacts with the extracellular side of the receptor, (ii) the tips of MT7 loops II and III contact one hM1 protomer, whereas the tip of loop I binds to the other protomer, and (iii) the hM1 dimeric interface involves the transmembrane helices TM6 and TM7. These results structurally support the high affinity and selectivity of the MT7-hM1 interaction and highlight the atypical mode of interaction of this allosteric ligand on its G protein-coupled receptor target.

Structural analysis of the Smad2-MAN1 interaction that regulates transforming growth factor-ß signaling at the inner nuclear membrane.

MAN1, an integral protein of the inner nuclear membrane, influences transforming growth factor-ß (TGF-ß) signaling by directly interacting with R-Smads. Heterozygous loss of function mutations in the gene encoding MAN1 cause sclerosing bone dysplasias and an increased level of TGF-ß signaling in cells. As a first step in elucidating the mechanism of TGF-ß pathway regulation by MAN1, we characterized the structure of the MAN1 C-terminal region that binds Smad2. Using nuclear magnetic resonance spectroscopy, we observed that this region is comprised of a winged helix domain, a structurally heterogeneous linker, a U2AF homology motif (UHM) domain, and a disordered C-terminus. From nuclear magnetic resonance and small-angle X-ray scattering data, we calculated a family of models for this MAN1 region. Our data indicate that the linker plays the role of an intramolecular UHM ligand motif (ULM) interacting with the UHM domain. We mapped the Smad2 binding site onto the MAN1 structure by combining GST pull-down, fluorescence, and yeast two-hybrid approaches. The linker region, the UHM domain, and the C-terminus are necessary for Smad2 binding with a micromolar affinity. Moreover, the intramolecular interaction between the linker and the UHM domain is critical for Smad2 binding. On the basis of the structural heterogeneity and binding properties of the linker, we suggest that it can interact with other UHM domains, thus regulating the MAN1-Smad2 interaction.

Structure of bacteriophage SPP1 head-to-tail connection reveals mechanism for viral DNA gating.

In many bacterial viruses and in certain animal viruses, the double-stranded DNA genome enters and exits the capsid through a portal gatekeeper. We report a pseudoatomic structure of a complete portal system. The bacteriophage SPP1 gatekeeper is composed of dodecamers of the portal protein gp6, the adaptor gp15, and the stopper gp16. The solution structures of gp15 and gp16 were determined by NMR. They were then docked together with the X-ray structure of gp6 into the electron density of the approximately 1-MDa SPP1 portal complex purified from DNA-filled capsids. The resulting structure reveals that gatekeeper assembly is accompanied by a large rearrangement of the gp15 structure and by folding of a flexible loop of gp16 to form an intersubunit parallel beta-sheet that closes the portal channel. This stopper system prevents release of packaged DNA. Disulfide cross-linking between beta-strands of the stopper blocks the key conformational changes that control genome ejection from the virus at the beginning of host infection.

Characterization of a highly conserved binding site of Mlh1 required for exonuclease I-dependent mismatch repair.

Mlh1 is an essential factor of mismatch repair (MMR) and meiotic recombination. It interacts through its C-terminal region with MutL homologs and proteins involved in DNA repair and replication. In this study, we identified the site of yeast Mlh1 critical for the interaction with Exo1, Ntg2, and Sgs1 proteins, designated as site S2 by reference to the Mlh1/Pms1 heterodimerization site S1. We show that site S2 is also involved in the interaction between human MLH1 and EXO1 or BLM. Binding at this site involves a common motif on Mlh1 partners that we called the MIP-box for the Mlh1 interacting protein box. Direct and specific interactions between yeast Mlh1 and peptides derived from Exo1, Ntg2, and Sgs1 and between human MLH1 and peptide derived from EXO1 and BLM were measured with K(d) values ranging from 8.1 to 17.4 microM. In Saccharomyces cerevisiae, a mutant of Mlh1 targeted at site S2 (Mlh1-E682A) behaves as a hypomorphic form of Exo1. The site S2 in Mlh1 mediates Exo1 recruitment in order to optimize MMR-dependent mutation avoidance. Given the conservation of Mlh1 and Exo1 interaction, it may readily impact Mlh1-dependent functions such as cancer prevention in higher eukaryotes.

Human mismatch repair protein MSH6 contains a PWWP domain that targets double stranded DNA.

The eukaryotic mismatch repair (MMR) protein MSH6 exhibits a core region structurally and functionally similar to bacterial MutS. However, it possesses an additional N-terminal region (NTR), comprising a PCNA binding motif, a large region of unknown function and a nonspecific DNA binding fragment. Yeast NTR was recently described as an extended tether between PCNA and the core of MSH6 . In contrast, we show that human NTR presents a globular PWWP domain in the region of unknown function. We demonstrate that this PWWP domain binds double-stranded DNA, without any preference for mismatches or nicks, whereas its apparent affinity for single-stranded DNA is about 20 times lower. The S144I mutation, which in human MSH6 causes inherited somatic defects in MMR resulting in increased development of hereditary non polyposis colorectal cancer , is located in the DNA binding surface of the PWWP domain. However, it only moderately affects domain stability, and it does not perturb DNA binding in vitro.

Solution structure of the region 51-160 of human KIN17 reveals an atypical winged helix domain.

Human KIN17 is a 45-kDa eukaryotic DNA- and RNA-binding protein that plays an important role in nuclear metabolism and in particular in the general response to genotoxics. Its amino acids sequence contains a zinc finger motif (residues 28-50) within a 30-kDa N-terminal region conserved from yeast to human, and a 15-kDa C-terminal tandem of SH3-like subdomains (residues 268-393) only found in higher eukaryotes. Here we report the solution structure of the region 51-160 of human KIN17. We show that this fragment folds into a three-alpha-helix bundle packed against a three-stranded beta-sheet. It belongs to the winged helix (WH) family. Structural comparison with analogous WH domains reveals that KIN17 WH module presents an additional and highly conserved 3(10)-helix. Moreover, KIN17 WH helix H3 is not positively charged as in classical DNA-binding WH domains. Thus, human KIN17 region 51-160 might rather be involved in protein-protein interaction through its conserved surface centered on the 3(10)-helix.

The checkpoint Saccharomyces cerevisiae Rad9 protein contains a tandem tudor domain that recognizes DNA.

DNA damage checkpoints are signal transduction pathways that are activated after genotoxic insults to protect genomic integrity. At the site of DNA damage, 'mediator' proteins are in charge of recruiting 'signal transducers' to molecules 'sensing' the damage. Budding yeast Rad9, fission yeast Crb2 and metazoan 53BP1 are presented as mediators involved in the activation of checkpoint kinases. Here we show that, despite low sequence conservation, Rad9 exhibits a tandem tudor domain structurally close to those found in human/mouse 53BP1 and fission yeast Crb2. Moreover, this region is important for the resistance of Saccharomyces cerevisiae to different genotoxic stresses. It does not mediate direct binding to a histone H3 peptide dimethylated on K79, nor to a histone H4 peptide dimethylated on lysine 20, as was demonstrated for 53BP1. However, the tandem tudor region of Rad9 directly interacts with single-stranded DNA and double-stranded DNAs of various lengths and sequences through a positively charged region absent from 53BP1 and Crb2 but present in several yeast Rad9 homologs. Our results argue that the tandem tudor domains of Rad9, Crb2 and 53BP1 mediate chromatin binding next to double-strand breaks. However, their modes of chromatin recognition are different, suggesting that the corresponding interactions are differently regulated.

The carboxyl-terminal nucleoplasmic region of MAN1 exhibits a DNA binding winged helix domain.

MAN1 is an integral protein of the inner nuclear membrane that interacts with nuclear lamins and emerin, thus playing a role in nuclear organization. It also binds to chromatin-associated proteins and transcriptional regulators, including the R-Smads, Smad1, Smad2, and Smad3. Mutations in the human gene encoding MAN1 cause sclerosing bone dysplasias, which sometimes have associated skin abnormalities. At the molecular level, these mutations lead to loss of the MAN1-R-Smads interaction, thus perturbing transforming growth factor beta superfamily signaling pathway. As a first step to understanding the physical basis of MAN1 interaction with R-Smads, we here report the structural characterization of the carboxyl-terminal nucleoplasmic region of MAN1, which is responsible for Smad binding. This region exhibits an amino-terminal globular domain adopting a winged helix fold, as found in several Smad-associated sequence-specific DNA binding factors. Consistently, it binds to DNA through the positively charged recognition helix H3 of its winged helix motif. However, it does not show the predicted carboxyl-terminal U2AF homology domain in solution, suggesting that the folding and stability of such a domain in MAN1 depend upon binding to an unidentified partner. Modeling the complex between DNA and the winged helix domain shows that the regions involved in DNA binding are essentially distinct from those reported to be involved in Smad binding. This suggests that MAN1 binds simultaneously to R-Smads and their targeted DNA sequences.