Crystal structure of the cytoplasmic domain of the type I TGF beta receptor in complex with FKBP12

Activation of the type I TGFbeta receptor (TbetaR-I) requires phosphorylation of a regulatory segment known as the GS region, located upstream of the serine/threonine kinase domain in the cytoplasmic portion of the receptor. The crystal structure of a fragment of unphosphorylated TbetaR-I, containing both the GS region and the catalytic domain, has been determined in complex with the FK506-binding protein FKBP12. TbetaR-I adopts an inactive conformation that is maintained by the unphosphorylated GS region. FKBP12 binds to the GS region of the receptor, capping the TbetaR-II phosphorylation sites and further stabilizing the inactive conformation of TbetaR-I. Certain structural features at the catalytic center of TbetaR-I are characteristic of tyrosine kinases rather than Ser/Thr kinases.     

Crystal structure of the tyrosine kinase domain of colony-stimulating factor-1 receptor (cFMS) in complex with two inhibitors

The cFMS proto-oncogene encodes for the colony-stimulating factor-1 receptor, a receptor-tyrosine kinase responsible for the differentiation and maturation of certain macrophages. Upon binding its ligand colony-stimulating factor-1 cFMS autophosphorylates, dimerizes, and induces phosphorylation of downstream targets. We report the novel crystal structure of unphosphorylated cFMS in complex with two members of different classes of drug-like protein kinase inhibitors. cFMS exhibits a typical bi-lobal kinase fold, and its activation loop and DFG motif are found to be in the canonical inactive conformation. Both ATP competitive inhibitors are bound in the active site and demonstrate a binding mode similar to that of STI-571 bound to cABL. The DFG motif is prevented from switching into the catalytically competent conformation through interactions with the inhibitors. Activation of cFMS is also inhibited by the juxtamembrane domain, which interacts with residues of the active site and prevents formation of the activated kinase. Together the structures of cFMS provide further insight into the autoinhibition of receptor-tyrosine kinases via their respective juxtamembrane domains; additionally the binding mode of two novel classes of kinase inhibitors will guide the design of novel molecules targeting macrophage-related diseases.     

Crystal structure of the NEMO ubiquitin-binding domain in complex with Lys 63-linked di-ubiquitin

NEMO is essential for activation of the NF-κB signaling pathway, which is regulated by ubiquitination of proteins. The C-terminal leucine zipper of NEMO and its adjacent coiled-coil region (CC2-LZ) reportedly bind to linear ubiquitin chains with 1 μM affinity and to Lys 63-linked chains with 100 μM affinity. Here we report the crystal structure of the CC2-LZ region of mouse NEMO in complex with Lys 63-linked di-ubiquitin (K63-Ub₂) at 2.7 Å resolution. The ubiquitin-binding region consists of a 130 Å-long helix and forms a parallel coiled-coil dimer. The Ile 44-centered hydrophobic patch of ubiquitin is recognized in the middle of the NEMO ubiquitin-binding region. NEMO interacts with each K63-Ub₂via a single ubiquitin-binding site, consistent with low affinity binding with K63-Ub₂.

Structured summary

MINT-7262681: NEMO (uniprotkb:O88522) binds (MI:0407) to Ubiquitin (uniprotkb:P62991) by pull down (MI:0096)MINT-7262667: Ubiquitin (uniprotkb:P62991) and NEMO (uniprotkb:O88522) bind (MI:0407) by X-ray crystallography (MI:0114)     

Crystal structure of EMS16 in complex with the integrin alpha2-I domain

Snake venoms contain a number of heterodimeric C-type lectin-like proteins (CLPs) that interact specifically with components of the haemostatic system. EMS16 from the venom of Echis multisquamatus binds to the collagen receptor, integrin alpha2beta1, also known as glycoprotein (GP) Ia/IIa, and specifically inhibits collagen binding. Here we report the crystal structure of EMS16 in complex with recombinant integrin alpha2-I domain that plays a central role in collagen binding. The structure of the complex at 1.9 Angstrom resolution reveals that the collagen-binding site of the alpha2-I domain is covered completely by the bound EMS16. This blockage by EMS16 appears to spatially inhibit collagen binding to the alpha2-I domain. The bound alpha2-I domain adopts a closed conformation, which is seen in the absence of ligand, suggesting that EMS16 stabilizes a closed conformation corresponding to the less active structure of the alpha2-I domain. EMS16 does not directly bind to the manganese ion and residues of the metal ion-dependent adhesion site (MIDAS) of the alpha2-I domain, suggesting that EMS16 may have the potential to bind specifically to the alpha2-I domain in a metal ion-independent fashion.     

Crystal structure of the glutamate receptor GluA1 N-terminal domain

The AMPA (α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) subfamily of iGluRs (ionotropic glutamate receptors) is essential for fast excitatory neurotransmission in the central nervous system. The malfunction of AMPARs (AMPA receptors) has been implicated in many neurological diseases, including Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis. The active channels of AMPARs and other iGluR subfamilies are tetramers formed exclusively by assembly of subunits within the same subfamily. It has been proposed that the assembly process is controlled mainly by the extracellular ATD (N-terminal domain) of iGluR. In addition, ATD has also been implicated in synaptogenesis, iGluR trafficking and trans-synaptic signalling, through unknown mechanisms. We report in the present study a 2.5 ? (1 ?=0.1 nm) resolution crystal structure of the ATD of GluA1. Comparative analyses of the structure of GluA1-ATD and other subunits sheds light on our understanding of how ATD drives subfamily-specific assembly of AMPARs. In addition, analysis of the crystal lattice of GluA1-ATD suggests a novel mechanism by which the ATD might participate in inter-tetramer AMPAR clustering, as well as in trans-synaptic protein-protein interactions.     

Insights into function of PSI domains from structure of the Met receptor PSI domain

PSI domains are cysteine-rich modules found in extracellular fragments of hundreds of signaling proteins, including plexins, semaphorins, integrins, and attractins. Here, we report the solution structure of the PSI domain from the human Met receptor, a receptor tyrosine kinase critical for proliferation, motility, and differentiation. The structure represents a cysteine knot with short regions of secondary structure including a three-stranded antiparallel beta-sheet and two alpha-helices. All eight cysteines are involved in disulfide bonds with the pattern consistent with that for the PSI domain from Sema4D. Comparison with the Sema4D structure identifies a structurally conserved core comprising the N-terminal half of the PSI domain. Interestingly, this part links adjacent SEMA and immunoglobulin domains in the Sema4D structure, suggesting that the PSI domain serves as a wedge between propeller and immunoglobulin domains and is responsible for the correct positioning of the ligand-binding site of the receptor.     

Crystal structure of the vitamin D nuclear receptor ligand binding domain in complex with a locked side chain analog of calcitriol

The crystal structures of vitamin D nuclear receptor (VDR) have revealed that all compounds are anchored by the same residues to the ligand binding pocket (LBP). Based on this observation, a synthetic analog with a locked side chain (21-nor-calcitriol-20(22),23-diyne) has been synthesized in order to gain in entropy energy with a predefined active side chain conformation. The crystal structure of VDR LBD bound to this locked side chain analogue while confirming the docking provides a structural basis for the activity of this compound.     

Crystal structure of the carboxyltransferase domain of acetyl-coenzyme A carboxylase in complex with CP-640186

Acetyl-coenzyme A carboxylases (ACCs) are important targets for the development of therapeutic agents against obesity, diabetes, and other diseases. CP-640186 is a potent inhibitor of mammalian ACCs and can reduce body weight and improve insulin sensitivity in test animals. It is believed to target the carboxyltransferase (CT) domain of these enzymes. Here we report the crystal structure of the yeast CT domain in complex with CP-640186. The inhibitor is bound in the active site at the interface of a dimer of the CT domain. CP-640186 has tight interactions with the putative biotin binding site in the CT domain and demonstrates a distinct mode of inhibiting the CT activity as compared to the herbicides that inhibit plant ACCs. The affinity of inhibitors for the CT domain has been assessed using kinetic and fluorescence anisotropy binding studies. The structural information identifies three regions for drug binding in the active site of CT.     

Crystal structure of the human receptor activity-modifying protein 1 extracellular domain

Receptor activity-modifying protein (RAMP) 1 forms a heterodimer with calcitonin receptor-like receptor (CRLR) and regulates its transport to the cell surface. The CRLR.RAMP1 heterodimer functions as a specific receptor for calcitonin gene-related peptide (CGRP). Here, we report the crystal structure of the human RAMP1 extracellular domain. The RAMP1 structure is a three-helix bundle that is stabilized by three disulfide bonds. The RAMP1 residues important for cell-surface expression of the CRLR.RAMP1 heterodimer are clustered to form a hydrophobic patch on the molecular surface. The hydrophobic patch is located near the tryptophan residue essential for binding of the CGRP antagonist, BIBN4096BS. These results suggest that the hydrophobic patch participates in the interaction with CRLR and the formation of the ligand-binding pocket when it forms the CRLR.RAMP1 heterodimer.     

Crystal structure of the ligand-bound glucagon-like peptide-1 receptor extracellular domain

The glucagon-like peptide-1 receptor (GLP-1R) belongs to Family B1 of the seven-transmembrane G protein-coupled receptors, and its natural agonist ligand is the peptide hormone glucagon-like peptide-1 (GLP-1). GLP-1 is involved in glucose homeostasis, and activation of GLP-1R in the plasma membrane of pancreatic beta-cells potentiates glucose-dependent insulin secretion. The N-terminal extracellular domain (nGLP-1R) is an important ligand binding domain that binds GLP-1 and the homologous peptide Exendin-4 with differential affinity. Exendin-4 has a C-terminal extension of nine amino acid residues known as the "Trp cage", which is absent in GLP-1. The Trp cage was believed to interact with nGLP-1R and thereby explain the superior affinity of Exendin-4. However, the molecular details that govern ligand binding and specificity of nGLP-1R remain undefined. Here we report the crystal structure of human nGLP-1R in complex with the antagonist Exendin-4(9-39) solved by the multiwavelength anomalous dispersion method to 2.2A resolution. The structure reveals that Exendin-4(9-39) is an amphipathic alpha-helix forming both hydrophobic and hydrophilic interactions with nGLP-1R. The Trp cage of Exendin-4 is not involved in binding to nGLP-1R. The hydrophobic binding site of nGLP-1R is defined by discontinuous segments including primarily a well defined alpha-helix in the N terminus of nGLP-1R and a loop between two antiparallel beta-strands. The structure provides for the first time detailed molecular insight into ligand binding of the human GLP-1 receptor, an established target for treatment of type 2 diabetes.     

Inhibition by copper(II) binding of hepatocyte growth factor (HGF) interaction with its receptor Met and blockade of HGF/Met function

Overexpression of hepatocyte growth factor (HGF) and its receptor Met often occurs in carcinoma cells, leading to establishment of an HGF/Met autocrine loop. Therefore, disruption of the HGF/Met autocrine loop may lead to down-regulation of tumorigenesis. To study the HGF/Met interaction, we have developed a cell-free system to detect HGF binding to a Met fusion protein, Met-IgG, using a modified enzyme-linked immunosorbent assay methodology. Since we previously showed that HGF can be purified by copper(II) affinity chromatography, we further explored the effect of copper(II) on the HGF/Met interaction. The divalent metal cations copper(II) and zinc(II) significantly inhibited HGF binding to immobilized Met-IgG with IC(50) values of 230-270 microM, respectively, whereas manganese(II) and magnesium(II) were less inhibitory with 20-60-fold higher IC(50) values. Incubation of 1 mM copper(II) with HGF resulted in nondenaturing and denaturing gel-mobility shifts, indicating that copper(II) binds directly to HGF. This interaction occurs at the N terminus of HGF, as incubation of 1 mM copper(II) with both HGF and the HGF derivative NK1 yielded similar results on SDS-PAGE. HGF-induced activation of Met and cell scattering were inhibited upon addition of HGF in the presence of 1 mM and 500 microM copper(II), respectively. Chemical protonation with diethyl pyrocarbonate of HGF histidine residues impeded the ability of 500 microM copper(II) to inhibit the binding of HGF to immobilized Met-IgG. Based on the NK1 domain structure, we propose that copper(II) may interact with HGF via the histidine residues in either N-terminal or kringle domains. The inhibition of HGF/Met interaction and subsequent downstream cellular functions may be through direct interference by copper(II), such as a change in charge or an induced local conformational change. This putative copper(II) binding domain may be the basis for developing potential inhibitors of HGF/Met binding and downstream functions and could lead to novel strategies for anti-cancer treatment.     

Production of the constant domain of murine T-cell receptor beta-chain in Escherichia coli

T-cell antigen receptor is a heterodimer of disulfide-linked alpha- and beta-chains. Although the essential features of T-cell receptor seem to be rather similar to those of immunoglobulin, the amount of T-cell receptor expressed on the surface of a T-cell is not large enough to be analyzed physcio-chemically. In this study, the DNA fragment encoding 120 amino acids from the 116th to the 235th of the murine beta-chain which corresponds to the presumed constant domain was inserted into an expression vector in E. coli. A large amount of this 18 kDa protein was observed to be synthesized in E. coli, and might be a good source for the three dimensional analysis of the T-cell receptor molecule.     

A mechanism of cell survival: sequestration of Fas by the HGF receptor Met

Death receptors such as Fas are present in a variety of organs including liver and play an important role in homeostasis. What prevents these harmful receptors from forming homooligomers, clustering, and initiating the apoptotic pathway is not known. Here, we report the discovery of a cell survival mechanism by which Met, a growth factor receptor tyrosine kinase, directly binds to and sequesters the death receptor Fas in hepatocytes. This interaction prevents Fas self-aggregation and Fas ligand binding, thus inhibiting Fas activation and apoptosis. Our results describe a direct link between growth factor tyrosine kinase receptors and death receptors to establish a novel paradigm in growth regulation.     

Crystal structure of soluble domain of malaria sporozoite protein UIS3 in complex with lipid

Malaria parasite UIS3 (up-regulated in infective sporozoites gene 3) is essential for sporozoite development in infected hepatocytes. UIS3 encodes for a membrane protein that is localized to the parasite parasitophorous vacuolar membrane in infected hepatocytes. We describe here 2.5-A resolution crystal structure of Plasmodium falciparum UIS3 soluble domain (PfUIS3(130-229)) in complex with the lipid phosphatidylethanolamine (PE). PfUIS3(130-229) is a novel, compact, and all alpha-helical structure bound to one molecule of PE. The PfUIS3(130-229)-PE complex structure reveals a novel binding site with specific interactions between PfUIS3(130-229) and the PE head group. One acyl chain of PE wraps around part of PfUIS3(130-229) and docks onto a hydrophobic channel. We additionally provide new structural and biochemical evidence of PfUIS3(130-229) interactions with lipids (phosphatidylethanolamine), with phospholipid liposomes, and with the human liver fatty acid-binding protein. The direct interaction of PfUIS3(130-229) with liver fatty acid-binding protein most likely provides the parasite with a conduit for importing essential fatty acids/lipids. Therefore, our analyses have implications for lipid transport into the parasite during the rapid growth phases of sporozoites. Given that PfUIS3 is essential for establishment of liver stage infection by P. falciparum, our data provide a new target for abrogating parasite development within liver cells before typical symptoms of malaria can manifest.     

Crystal structure of the FK506 binding domain of human FKBP25 in complex with FK506

Human FKBP25 (hFKBP25) is a nuclear immunophilin and interacts with several nuclear proteins, hence involving in many nuclear events. Similar to other FKBPs, FK506 binding domain (FKBD) of hFKBP25 also binds to immunosuppressive drugs such as rapamycin and FK506, albeit with a lower affinity for the latter. The molecular basis underlying this difference in affinity could not be addressed due to the lack of the crystal structure of hFKBD25 in complex with FK506. Here, we report the crystal structure of hFKBD25 in complex with FK506 determined at 1.8 Å resolution and its comparison with the hFKBD25–rapamycin complex, bringing out the microheterogeneity in the mode of interaction of these drugs, which could possibly explain the lower affinity for FK506.     

Crystal structure of the second PDZ domain of SAP97 in complex with a GluR-A C-terminal peptide

Synaptic targeting of GluR-A subunit-containing glutamate receptors involves an interaction with synapse-associated protein 97 (SAP97). The C-terminus of GluR-A, which contains a class I PDZ ligand motif (-x-Ser/Thr-x-phi-COOH where phi is an aliphatic amino acid) associates preferentially with the second PDZ domain of SAP97 (SAP97(PDZ2)). To understand the structural basis of this interaction, we have determined the crystal structures of wild-type and a SAP97(PDZ2) variant in complex with an 18-mer C-terminal peptide (residues 890-907) of GluR-A and of two variant PDZ2 domains in unliganded state at 1.8-2.44 A resolutions. SAP97(PDZ2) folds to a compact globular domain comprising six beta-strands and two alpha-helices, a typical architecture for PDZ domains. In the structure of the peptide complex, only the last four C-terminal residues of the GluR-A are visible, and align as an antiparallel beta-strand in the binding groove of SAP97(PDZ2). The free carboxylate group and the aliphatic side chain of the C-terminal leucine (Leu907), and the hydroxyl group of Thr905 of the GluR-A peptide are engaged in essential class I PDZ interactions. Comparison between the free and complexed structures reveals conformational changes which take place upon peptide binding. The betaAlpha-betaBeta loop moves away from the C-terminal end of alphaB leading to a slight opening of the binding groove, which may better accommodate the peptide ligand. The two conformational states are stabilized by alternative hydrogen bond and coulombic interactions of Lys324 in betaAlpha-betaBeta loop with Asp396 or Thr394 in betaBeta. Results of in vitro binding and immunoprecipitation experiments using a PDZ motif-destroying L907A mutation as well as the insertion of an extra alanine residue between the C-terminal Leu907 and the stop codon are also consistent with a 'classical' type I PDZ interaction between SAP97 and GluR-A C-terminus.