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.

Supramolecular chemistry has recently emerged as a promising way to modulate protein functions, but devising molecules that will interact with a protein in the desired manner is difficult as many competing interactions exist in a biological environment (with solvents, salts or different sites for the target biomolecule). We now show that lysine-specific molecular tweezers bind to a 14-3-3 adapter protein and modulate its interaction with partner proteins. The tweezers inhibit binding between the 14-3-3 protein and two partner proteins--a phosphorylated (C-Raf) protein and an unphosphorylated one (ExoS)--in a concentration-dependent manner. Protein crystallography shows that this effect arises from the binding of the tweezers to a single surface-exposed lysine (Lys214) of the 14-3-3 protein in the proximity of its central channel, which normally binds the partner proteins. A combination of structural analysis and computer simulations provides rules for the tweezers' binding preferences, thus allowing us to predict their influence on this type of protein-protein interactions.

Formins induce the nucleation and polymerisation of unbranched actin filaments via the formin-homology domains 1 and 2. Diaphanous-related formins (Drfs) are regulated by a RhoGTPase-binding domain situated in the amino-terminal (N-terminal) region and a carboxyterminal Diaphanous-autoregulatory domain (DAD), whose interaction stabilises an autoinhibited inactive conformation. Binding of active Rho releases DAD and activates the catalytic activity of mDia. Here, we report on the interaction of DAD with the regulatory N-terminus of mDia1 (mDia N ) and its release by RhoKGTP. We have defined the elements required for tight binding and solved the three-dimensional structure of a complex between an mDia N construct and DAD by X-ray crystallography. The core DAD region is an a-helical peptide, which binds in the most highly conserved region of mDia N using mainly hydrophobic interactions. The structure suggests a twostep mechanism for release of autoinhibition whereby RhoKGTP, although having a partially nonoverlapping binding site, displaces DAD by ionic repulsion and steric clashes. We show that RhoKGTP accelerates the dissociation of DAD from the mDia N KDAD complex.

Subtilases are serine proteases found in Archae, Bacteria, yeasts, and higher eukaryotes. Plants possess many more of these subtilisin-like endopeptidases than animals, e.g., 56 identified genes in Arabidopsis compared with only 9 in humans, indicating important roles for subtilases in plant biology. We report the first structure of a plant subtilase, SBT3 from tomato, in the active apo form and complexed with a chloromethylketone (cmk) inhibitor. The domain architecture comprises an N-terminal protease domain displaying a 132 aa protease-associated (PA) domain insertion and a C-terminal sevenstranded jelly-roll fibronectin (Fn) III-like domain. We present the first structural evidence for an explicit function of PA domains in proteases revealing a vital role in the homo-dimerization of SBT3 and in enzyme activation. Although Ca 2؉ -binding sites are conserved and critical for stability in other subtilases, SBT3 was found to be Ca 2؉ -free and its thermo stability is Ca 2؉ -independent.calcium ͉ proprotein convertase ͉ protease-associated domain ͉ subtilisin ͉ thermostability S ubtilases constitute the S8 family in clan SB of serine proteases (http://merops.sanger.ac.uk). They are characterized by a catalytic triad of Asp, His, and Ser residues in an arrangement shared with subtilisins from Bacillus species (1). The first eukaryotic subtilase to be identified was kexin. It is involved in the maturation of ␣-mating factor and killer toxin from their respective precursor proteins in yeast (2). Nine subtilisin-like endopeptidases have since been discovered in mammals, seven of which are related to kexin and also involved in the highly specific processing of precursor proteins. Their substrates include polypeptide hormone precursors, growth factors, receptors, enzymes, and viral surface glycoproteins, which are typically cleaved on the carboxyl side of paired basic residues (3). The remaining two subtilases, PCSK9 and S1P, belong to the proteinase K and pyrolysin subfamilies of subtilases (3). The discovery of mammalian proprotein convertases (PCs), characterized by their exquisite substrate specificity compared with bacterial subtilisins, further stimulated interest in this class of serine proteases.Plants appear to lack kexin-related PCs but they possess a largely expanded pyrolysin family with 56 genes identified in Arabidopsis thaliana (4). They have been implicated in general protein turnover (5), the regulation of plant development (6), biotic and abiotic stress responses (7,8), and the processing of precursors of peptide growth factors in plants (9). It therefore seems that the majority of plant subtilases assumed plant-specific functions in the course of evolution. With the physiological roles of plant subtilases beginning to emerge, it will now be interesting to see whether or not the adoption of specific roles in plant physiology is reflected in unique structural or biochemical features that distinguish subtilases in plants from those in other organisms.To address this question we recently purified and cha...

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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 Ser 259 , 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-RAFpS 259 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-RAFSer 259 /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.

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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 pSer34] in complex with 14-3-3e and analyse the interaction with isothermal titration calorimetry. Fragment-based ligand discovery employing crystals of the binary 14-3-3e ⁄ 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. Structured digital abstract l 14-3-3 epsilon and MLF1 bind by x-ray crystallography (View interaction) l 14-3-3 epsilon and MLF1 bind by isothermal titration calorimetry (View Interaction: 1, 2)