ABSTRACT
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.
Subject(s)
14-3-3 Proteins/chemistry , 14-3-3 Proteins/metabolism , Biomimetic Materials/chemistry , Biomimetic Materials/metabolism , 14-3-3 Proteins/genetics , ADP Ribose Transferases/chemistry , ADP Ribose Transferases/metabolism , Bacterial Toxins/chemistry , Bacterial Toxins/metabolism , Humans , Models, Molecular , Molecular Conformation , Phosphorylation , Protein Binding , Proto-Oncogene Proteins c-raf/chemistry , Proto-Oncogene Proteins c-raf/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolismABSTRACT
Nuclear receptor binding to coactivator proteins is an obligate first step in the regulation of gene transcription. Nuclear receptors preferentially bind to an LXXLL peptide motif which is highly conserved throughout the 300 or so natural coactivator proteins. This knowledge has shaped current understanding of this fundamental protein-protein interaction, and continues to inspire the search for new drug therapies. However, sequence specificity beyond the LXXLL motif and the molecular functioning of flanking residues still requires urgent addressing. Here, ribosome display has been used to reassess the estrogen receptor for new and enlarged peptide recognition motifs, leading to the discovery of a potent and highly evolved PXLXXLLXXP binding consensus. Molecular modeling and X-ray crystallography studies have provided the molecular insights on the role of the flanking prolines in priming the length of the α-helix and enabling optimal interactions of the α-helix dipole and its surrounding amino acids with the surface charge clamp and the receptor activation function 2. These findings represent new structural parameters for modulating the nuclear receptor-coactivator interaction based on linear sequences of proteinogenic amino acids and for the design of chemically modified inhibitors.
Subject(s)
Peptides/chemistry , Peptides/metabolism , Proline/chemistry , Receptors, Estrogen/metabolism , Amino Acid Motifs , Amino Acid Sequence , Consensus Sequence , Crystallography, X-Ray , Gene Library , Humans , Models, Molecular , Molecular Sequence Data , Peptide Library , Proline/metabolism , Protein Binding , Protein Structure, Secondary , Receptors, Estrogen/chemistryABSTRACT
The regulation and function of peptidylarginine deiminase isoform VI (PAD6), which is a highly abundant protein associated with the cytoplasmic lattices in mammalian oocytes, is poorly understood so far. It has been shown previously, that 14-3-3 proteins, a class of regulatory adapter proteins ubiquitous in eukaryotes, bind to PAD6 in vivo in a phosphorylation dependent manner. Here we identify possible 14-3-3 binding sites in human PAD6 by in silico methods, looking for conserved, surface exposed serine residues. Two of these sites were confirmed as 14-3-3 binding sites by fluorescence polarization competition and X-ray crystallography. We furthermore suggest a role of RSK-type kinases in the phosphorylation of one of these two binding sites and provide evidence in the form of in vitro kinase assays with p70S6 kinase and RSK1.
Subject(s)
14-3-3 Proteins/chemistry , Biomarkers, Tumor/chemistry , Exonucleases/chemistry , Hydrolases/chemistry , Amino Acid Sequence , Binding Sites , Crystallography, X-Ray , Exoribonucleases , Humans , Hydrophobic and Hydrophilic Interactions , Models, Molecular , Molecular Sequence Data , Peptide Fragments/chemistry , Phosphoproteins/chemistry , Phosphorylation , Protein Binding , Protein Interaction Domains and Motifs , Protein Processing, Post-Translational , Protein Structure, Secondary , Protein-Arginine Deiminase Type 6 , Protein-Arginine Deiminases , Ribosomal Protein S6 Kinases, 90-kDa/chemistryABSTRACT
Modulation of protein-protein interactions (PPIs) is a highly demanding, but also a very promising approach in chemical biology and targeted drug discovery. In contrast to inhibiting PPIs with small, chemically tractable molecules, stabilisation of these interactions can only be achieved with complex natural products, like rapamycin, FK506, taxol, forskolin, brefeldin and fusicoccin. Fusicoccin stabilises the activatory complex of the plant H(+)-ATPase PMA2 and 14-3-3 proteins. Recently, we have shown that the stabilising effect of fusicoccin could be mimicked by a trisubstituted pyrrolinone (pyrrolidone1, 1). Here, we report the synthesis, functional activity and crystal structure of derivatives of 1 that stabilise the 14-3-3-PMA2 complex. With a limited compound collection three modifications that are important for activity enhancement could be determined: 1) conversion of the pyrrolinone scaffold into a pyrazole, 2) introduction of a tetrazole moiety to the phenyl ring that contacts PMA2, and 3) addition of a bromine to the phenyl ring that exclusively contacts the 14-3-3 protein. The crystal structure of a pyrazole derivative of 1 in complex with 14-3-3 and PMA2 revealed that the more rigid core of this molecule positions the stabiliser deeper into the rim of the interface, enlarging especially the contact surface to PMA2. Combination of the aforementioned features gave rise to a molecule (37) that displays a threefold increase in stabilising the 14-3-3-PMA2 complex over 1. Compound 37 and the other active derivatives show no effect on two other important 14-3-3 protein-protein interactions, that is, with CRaf and p53. This is the first study that describes the successful optimisation of a PPI stabiliser identified by screening.
Subject(s)
14-3-3 Proteins/chemistry , Pyrroles/chemical synthesis , 14-3-3 Proteins/metabolism , Crystallography, X-Ray , Molecular Structure , Plants/chemistry , Protein Binding , Pyrazoles/chemical synthesis , Pyrazoles/chemistry , Pyrroles/chemistry , Structure-Activity RelationshipABSTRACT
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. Database Structural data are available in the Protein Data Bank under the accession number(s) 3UAL [14-3-3ε/MLF1(29-42)pSer34 complex] and 3UBW [14-3-3ε/MLF1(29-42)pSer34/3-pyrrolidinol complex] Structured digital abstract ⢠14-3-3 epsilon and MLF1 bind by x-ray crystallography (View interaction) ⢠14-3-3 epsilon and MLF1 bind by isothermal titration calorimetry (View Interaction: 1, 2).
Subject(s)
14-3-3 Proteins/chemistry , Models, Molecular , Protein Structure, Tertiary , Proteins/chemistry , 14-3-3 Proteins/genetics , 14-3-3 Proteins/metabolism , Amino Acid Motifs/genetics , Amino Acid Sequence , Binding Sites/genetics , Calorimetry/methods , Cell Cycle Proteins , Crystallography, X-Ray , DNA-Binding Proteins , Humans , Hydrogen Bonding , Hydrophobic and Hydrophilic Interactions , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Proteins/genetics , Proteins/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Serine/chemistry , Serine/genetics , Serine/metabolismABSTRACT
A library of small tetrahydroisoquinoline ligands, previously identified via structure- and chemistry-based hierarchical organization of library scaffolds in tree-like arrangements, has been generated as novel estrogen receptor agonistic fragments via traditional medicinal chemistry exploration. The approach described has allowed for the rapid evaluation of a structure-activity relationship of the ligands concerning estrogen receptor affinity and estrogen receptor ß subtype selectivity. The structural biological insights obtained from the fragments aid the understanding of larger analogues and constitute attractive starting points for further optimization.
Subject(s)
Drug Design , Estrogen Receptor alpha/metabolism , Estrogen Receptor beta/metabolism , Tetrahydroisoquinolines/chemistry , Tetrahydroisoquinolines/metabolism , Estrogen Receptor alpha/agonists , Estrogen Receptor alpha/antagonists & inhibitors , Estrogen Receptor alpha/chemistry , Estrogen Receptor beta/agonists , Estrogen Receptor beta/antagonists & inhibitors , Estrogen Receptor beta/chemistry , Ligands , Models, Molecular , Protein Structure, Tertiary , Small Molecule Libraries/chemistry , Small Molecule Libraries/metabolism , Small Molecule Libraries/pharmacology , Substrate Specificity , Tetrahydroisoquinolines/pharmacologySubject(s)
Estrogen Receptor alpha/chemistry , Estrogen Receptor beta/chemistry , Ligands , Amino Acid Sequence , Crystallography, X-Ray , Estrogen Receptor alpha/metabolism , Estrogen Receptor beta/metabolism , Molecular Sequence Data , Peptides/chemical synthesis , Peptides/chemistry , Phosphorylation , Protein Binding , Protein Structure, TertiaryABSTRACT
The 14-3-3 proteins are a class of eukaryotic acidic adapter proteins, with seven isoforms in humans. 14-3-3 proteins mediate their biological function by binding to target proteins and influencing their activity. They are involved in pivotal pathways in the cell such as signal transduction, gene expression, enzyme activation, cell division and apoptosis. The Yes-associated protein (YAP) is a WW-domain protein that exists in two transcript variants of 48 and 54 kDa in humans. By transducing signals from the cytoplasm to the nucleus, YAP is important for transcriptional regulation. In both variants, interaction with 14-3-3 proteins after phosphorylation of Ser127 is important for nucleocytoplasmic trafficking, via which the localization of YAP is controlled. In this study, 14-3-3σ has been cloned, purified and crystallized in complex with a phosphopeptide from the YAP 14-3-3-binding domain, which led to a crystal that diffracted to 1.15 A resolution. The crystals belonged to space group C222(1), with unit-cell parameters a=82.3, b=112.1, c=62.9 A.
Subject(s)
14-3-3 Proteins/chemistry , Adaptor Proteins, Signal Transducing/chemistry , Phosphoproteins/chemistry , 14-3-3 Proteins/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Crystallography, X-Ray , Humans , Models, Molecular , Phosphoproteins/metabolism , Protein Binding , Protein Structure, Quaternary , Structural Homology, Protein , Transcription Factors , YAP-Signaling ProteinsABSTRACT
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.
Subject(s)
14-3-3 Proteins/metabolism , Proto-Oncogene Proteins c-raf/metabolism , Signal Transduction , ras Proteins/metabolism , 14-3-3 Proteins/chemistry , 14-3-3 Proteins/genetics , Animals , Binding Sites/genetics , Cell Line , Chlorocebus aethiops , Crystallization , Crystallography, X-Ray , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Kinetics , Microscopy, Confocal , Models, Molecular , Mutation , Noonan Syndrome/genetics , Noonan Syndrome/metabolism , Phosphorylation , Protein Binding , Protein Structure, Tertiary , Proto-Oncogene Proteins c-raf/chemistry , Proto-Oncogene Proteins c-raf/genetics , Serine/genetics , Serine/metabolism , Transfection , ras Proteins/geneticsABSTRACT
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 seven-stranded 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.
Subject(s)
Calcium/metabolism , Protein Structure, Tertiary , Solanum lycopersicum/enzymology , Subtilisins/chemistry , Catalytic Domain/genetics , Crystallization , Enzyme Activation , Enzyme Stability , Solanum lycopersicum/genetics , Models, Molecular , Mutagenesis, Site-Directed , Protein Multimerization , Protein Structure, Quaternary , Protein Structure, Secondary , Subtilisins/genetics , Subtilisins/metabolismABSTRACT
The subtilase SBT3 from Solanum lycopersicum (tomato) was purified from a tomato cell culture and crystallized using the sitting-drop vapour-diffusion method. A native data set was collected to 2.5 A resolution at 100 K using synchrotron radiation. For experimental phasing, CsCl-derivative and tetrakis(acetoxymercuri)methane (TAMM) derivative crystals were employed for MIRAS phasing. Three caesium sites and one TAMM site were identified, which allowed solution of the structure.
Subject(s)
Solanum lycopersicum/enzymology , Subtilisins/analysis , Subtilisins/chemistry , Crystallization , Crystallography, X-Ray , Solanum lycopersicum/genetics , Subtilisins/isolation & purification , Subtilisins/metabolismABSTRACT
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 carboxy-terminal 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 Rho*GTP. 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 alpha-helical peptide, which binds in the most highly conserved region of mDia(N) using mainly hydrophobic interactions. The structure suggests a two-step mechanism for release of autoinhibition whereby Rho*GTP, although having a partially nonoverlapping binding site, displaces DAD by ionic repulsion and steric clashes. We show that Rho*GTP accelerates the dissociation of DAD from the mDia(N)*DAD complex.
Subject(s)
Carrier Proteins/antagonists & inhibitors , Carrier Proteins/metabolism , Guanosine Triphosphate/physiology , rho GTP-Binding Proteins/physiology , Amino Acid Sequence , Animals , Binding Sites , Carrier Proteins/genetics , Crystallography, X-Ray , DNA Mutational Analysis , Formins , Mice , Molecular Sequence Data , Mutation , Protein Structure, TertiaryABSTRACT
An N-terminal construct of mouse mDia1 was recombinantly expressed in Escherichia coli, purified and crystallized in complex with truncated human RhoC using the hanging-drop vapour-diffusion method. Crystals were obtained using PEG 2K MME and MgSO4 as a precipitating agent and belong to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 148.4, b = 85.2, c = 123.2 A. Complete native and SeMet-derivative data sets were collected at 100 K to 3.0 and 3.4 A resolution, respectively, using synchrotron radiation.