Michael Weyand

Structural and mechanistic insights into the interaction between Rho and mammalian Dia.

Formins are involved in a variety of cellular processes that require the remodelling of the cytoskeleton. They contain formin homology domains FH1 and FH2, which initiate actin assembly. The Diaphanous-related formins form a subgroup that is characterized by an amino-terminal Rho GTPase-binding domain (GBD) and an FH3 domain, which bind somehow to the carboxy-terminal Diaphanous autoregulatory domain (DAD) to keep the protein in an inactive conformation. Upon binding of activated Rho proteins, the DAD is released and the ability of the formin to nucleate and elongate unbranched actin filaments is induced. Here we present the crystal structure of RhoC in complex with the regulatory N terminus of mammalian Diaphanous 1 (mDia1) containing the GBD/FH3 region, an all-helical structure with armadillo repeats. Rho uses its ‘switch’ regions for interacting with two subdomains of GBD/FH3. We show that the FH3 domain of mDia1 forms a stable dimer and we also identify the DAD-binding site. Although binding of Rho and DAD on the N-terminal fragment of mDia1 are mutually exclusive, their binding sites are only partially overlapping. On the basis of our results, we propose a structural model for the regulation of mDia1 by Rho and DAD.

The GTPase activating protein Rap1GAP uses a catalytic asparagine

Rap1 is a Ras-like guanine-nucleotide-binding protein (GNBP) that is involved in a variety of signal-transduction processes. It regulates integrin-mediated cell adhesion and might activate extracellular signal-regulated kinase. Like other Ras-like GNBPs, Rap1 is regulated by guanine-nucleotide-exchange factors (GEFs) and GTPase-activating proteins (GAPs). These GAPs increase the slow intrinsic GTPase reaction of Ras-like GNBPs by many orders of magnitude and allow tight regulation of signalling. The activation mechanism involves stabilization of the catalytic glutamine of the GNBP and, in most cases, the insertion of a catalytic arginine of GAP into the active site. Rap1 is a close homologue of Ras but does not possess the catalytic glutamine essential for GTP hydrolysis in all other Ras-like and Gα proteins. Furthermore, RapGAPs are not related to other GAPs and apparently do not use a catalytic arginine residue. Here we present the crystal structure of the catalytic domain of the Rap1-specific Rap1GAP at 2.9 Å. By mutational analysis, fluorescence titration and stopped-flow kinetic assay, we demonstrate that Rap1GAP provides a catalytic asparagine to stimulate GTP hydrolysis. Implications for the disease tuberous sclerosis are discussed.

Structure of the Roc-COR domain tandem of C. tepidum, a prokaryotic homologue of the human LRRK2 Parkinson kinase

Ras of complex proteins (Roc) belongs to the superfamily of Ras‐related small G‐proteins that always occurs in tandem with the C‐terminal of Roc (COR) domain. This Roc–COR tandem is found in the bacterial and eukaryotic world. Its most prominent member is the leucine‐rich repeat kinase LRRK2, which is mutated and activated in Parkinson patients. Here, we investigated biochemically and structurally the Roco protein from Chlorobium tepidum. We show that Roc is highly homologous to Ras, whereas the COR domain is a dimerisation device. The juxtaposition of the G‐domains and mutational analysis suggest that the Roc GTPase reaction is stimulated and/or regulated by dimerisation in a nucleotide‐dependent manner. The region most conserved between bacteria and man is the interface between Roc and COR, where single‐point Parkinson mutations of the Roc and COR domains are in close proximity. The analogous mutations in C. tepidum Roc–COR decrease the GTPase reaction rate, most likely due to a modification of the interaction between the Roc and COR domains.

Phosphorylation-independent interaction between 14-3-3 and exoenzyme S: from structure to pathogenesis

14‐3‐3 proteins are phosphoserine/phosphothreonine‐recognizing adapter proteins that regulate the activity of a vast array of targets. There are also examples of 14‐3‐3 proteins binding their targets via unphosphorylated motifs. Here we present a structural and biological investigation of the phosphorylation‐independent interaction between 14‐3‐3 and exoenzyme S (ExoS), an ADP‐ribosyltransferase toxin of Pseudomonas aeruginosa. ExoS binds to 14‐3‐3 in a novel binding mode mostly relying on hydrophobic contacts. The 1.5 Å crystal structure is supported by cytotoxicity analysis, which reveals that substitution of the corresponding hydrophobic residues significantly weakens the ability of ExoS to modify the endogenous targets RAS/RAP1 and to induce cell death. Furthermore, mutation of key residues within the ExoS binding site for 14‐3‐3 impairs virulence in a mouse pneumonia model. In conclusion, we show that ExoS binds 14‐3‐3 in a novel reversed orientation that is primarily dependent on hydrophobic residues. This interaction is phosphorylation independent and is required for the function of ExoS.

Impaired Binding of 14-3-3 to C-RAF in Noonan Syndrome Suggests New Approaches in Diseases with Increased Ras Signaling

ABSTRACT The Ras-RAF-mitogen-activated protein kinase (Ras-RAF-MAPK) pathway is overactive in many cancers and in some developmental disorders. In one of those disorders, namely, Noonan syndrome, nine activating C-RAF mutations cluster around Ser259, a regulatory site for inhibition by 14-3-3 proteins. We show that these mutations impair binding of 14-3-3 proteins to C-RAF and alter its subcellular localization by promoting Ras-mediated plasma membrane recruitment of C-RAF. By presenting biophysical binding data, the 14-3-3/C-RAFpS259 crystal structure, and cellular analyses, we indicate a mechanistic link between a well-described human developmental disorder and the impairment of a 14-3-3/target protein interaction. As a broader implication of these findings, modulating the C-RAFSer259/14-3-3 protein-protein interaction with a stabilizing small molecule may yield a novel potential approach for treatment of diseases resulting from an overactive Ras-RAF-MAPK pathway.

Structure of the p53 C-terminus bound to 14-3-3: implications for stabilization of the p53 tetramer.

MINT‐7711931: 14‐3‐3 sigma (uniprotkb:P31947) and p53 (uniprotkb:P04637) bind (MI:0407) by isothermal titration calorimetry (MI:0065)

Crystal Structures of a New Class of Allosteric Effectors Complexed to Tryptophan Synthase

Tryptophan synthase is a bifunctional α2β2 complex catalyzing the last two steps of l-tryptophan biosynthesis. The natural substrates of the α-subunit indole- 3-glycerolphosphate and glyceraldehyde-3-phosphate, and the substrate analogs indole-3-propanolphosphate anddl-α-glycerol-3-phosphate are allosteric effectors of the β-subunit activity. It has been shown recently, that the indole-3-acetyl amino acids indole-3-acetylglycine and indole-3-acetyl-l-aspartic acid are both α-subunit inhibitors and β-subunit allosteric effectors, whereas indole-3-acetyl-l-valine is only an α-subunit inhibitor (Marabotti, A., Cozzini, P., and Mozzarelli, A. (2000) Biochim. Biophys. Acta 1476, 287–299). The crystal structures of tryptophan synthase complexed with indole-3-acetylglycine and indole-3-acetyl-l-aspartic acid show that both ligands bind to the active site such that the carboxylate moiety is positioned similarly as the phosphate group of the natural substrates. As a consequence, the residues of the α-active site that interact with the ligands are the same as observed in the indole 3-glycerolphosphate-enzyme complex. Ligand binding leads to closure of loop αL6 of the α-subunit, a key structural element of intersubunit communication. This is in keeping with the allosteric role played by these compounds. The structure of the enzyme complex with indole-3-acetyl-l-valine is quite different. Due to the hydrophobic lateral chain, this molecule adopts a new orientation in the α-active site. In this case, closure of loop αL6 is no longer observed, in agreement with its functioning only as an inhibitor of the α-subunit reaction.

Crystal Structure of the βSer178 → Pro Mutant of Tryptophan Synthase A “KNOCK-OUT” ALLOSTERIC ENZYME

The catalytic activity of the pyridoxal 5′-phosphate-dependent tryptophan synthase α2β2 complex is allosterically regulated. The hydrogen bond between the helix βH6 residue βSer178 and the loop αL6 residue Gly181 was shown to be critical in ligand-induced intersubunit signaling, with the α-β communication being completely lost in the mutant βSer178 → Pro (Marabotti, A., De Biase, D., Tramonti, A., Bettati, S., and Mozzarelli, A. (2001) J. Biol. Chem. 276, 17747–17753). The structural basis of the impaired allosteric regulation was investigated by determining the crystal structures of the mutant βSer178 → Pro in the absence and presence of the α-subunit ligands indole-3-acetylglycine and glycerol 3-phosphate. The mutation causes local and distant conformational changes especially in the β-subunit. The ligand-free structure exhibits larger differences at the N-terminal part of helix βH6, whereas the enzyme ligand complexes show differences at the C-terminal side. In contrast to the wild-type enzyme loop αL6 remains in an open conformation even in the presence of α-ligands. This effects the equilibrium between active and inactive conformations of the α-active site, altering k cat andK m , and forms the structural basis for the missing allosteric communication between the α- and β-subunits.

Conformation of the Dileucine-Based Sorting Motif in HIV-1 Nef Revealed by Intermolecular Domain Assembly

The human immunodeficiency virus 1 (HIV‐1) Nef protein is a pathogenicity factor required for effective progression to AIDS, which modulates host cell signaling pathways and T‐cell receptor internalization. We have determined the crystal structure of Nef, allele SF2, in complex with an engineered SH3 domain of human Hck showing unnaturally tight binding and inhibitory potential toward Nef. This complex provides the most complete Nef structure described today, and explains the structural basis of the high affinity of this interaction. Intriguingly, the 33‐residue C‐terminal flexible loop is resolved in the structure by its interactions with a highly conserved hydrophobic groove on the core domain of an adjacent Nef molecule. The loop mediates the interaction of Nef with the cellular adaptor protein machinery for the stimulated internalization of surface receptors. The endocytic dileucine‐based sorting motif is exposed at the tip of the acidic loop, giving the myristoylated Nef protein a distinctly dipolar character. The intermolecular domain assembly of Nef provides insights into a possible regulation mechanism for cargo trafficking.

Structural insights of the MLF1/14-3-3 interaction.

Myeloid leukaemia factor 1 (MLF1) binds to 14‐3‐3 adapter proteins by a sequence surrounding Ser34 with the functional consequences of this interaction largely unknown. We present here the high‐resolution crystal structure of this binding motif [MLF1(29–42)pSer34] in complex with 14‐3‐3ε and analyse the interaction with isothermal titration calorimetry. Fragment‐based ligand discovery employing crystals of the binary 14‐3‐3ε/MLF1(29–42)pSer34 complex was used to identify a molecule that binds to the interface rim of the two proteins, potentially representing the starting point for the development of a small molecule that stabilizes the MLF1/14‐3‐3 protein–protein interaction. Such a compound might be used as a chemical biology tool to further analyse the 14‐3‐3/MLF1 interaction without the use of genetic methods.