It is remarkable that the thyroid-stimulating autoantibody shows almost identical receptor-binding features to TSH although the structures and origins of these two ligands are very different. Furthermore, our structure of the TSHR and its complex with M22 provide foundations for developing new strategies to understand and control both glycoprotein hormone receptor activation and the autoimmune response to the TSHR.

The polypeptide growth factor, hepatocyte growth factor͞scatter factor (HGF͞SF), shares the multidomain structure and proteolytic mechanism of activation of plasminogen and other complex serine proteinases. HGF͞SF, however, has no enzymatic activity. Instead, it controls the growth, morphogenesis, or migration of epithelial, endothelial, and muscle progenitor cells through the receptor tyrosine kinase MET. Using small-angle x-ray scattering and cryoelectron microscopy, we show that conversion of pro(single-chain)-HGF͞SF into the active two-chain form is associated with a major structural transition from a compact, closed conformation to an elongated, open one. We also report the structure of a complex between two-chain HGF͞SF and the MET ectodomain (MET928) with 1:1 stoichiometry in which the N-terminal and first kringle domain of HGF͞SF contact the face of the seven-blade ␤-propeller domain of MET harboring the loops connecting the ␤-strands b-c and d-a, whereas the C-terminal serine proteinase homology domain binds the opposite ''b'' face. Finally, we describe a complex with 2:2 stoichiometry between two-chain HGF͞SF and a truncated form of the MET ectodomain (MET567), which is assembled around the dimerization interface seen in the crystal structure of the NK1 fragment of HGF͞SF and displays the features of a functional, signaling unit. The study shows how the proteolytic mechanism of activation of the complex proteinases has been adapted to cell signaling in vertebrate organisms, offers a description of monomeric and dimeric ligand-receptor complexes, and provides a foundation to the structural basis of HGF͞SF-MET signaling.cell signaling ͉ plasminogen ͉ serine proteinases ͉ kringle ͉ x-ray scattering H epatocyte growth factor͞scatter factor (HGF͞SF) (1-6) are vertebrate-specific polypeptide growth factors with a domain structure related to that of plasminogen (7). Interest in HGF͞SF and its receptor MET (8) stems from unique biological roles in embryogenesis (9-11), tissue regeneration (12, 13), and cancer (14). These activities have led to a strong interest in the structure of the molecules as this knowledge may underpin the development of MET-based therapeutics.HGF͞SF consists of six domains: an N-terminal domain (n), four copies of the kringle domain (k1-k4), and a C-terminal domain (sp) structurally related to the catalytic domain of serine proteinases (Fig. 1A). The factor is synthesized as a precursor protein (pro-or single-chain HGF͞SF) and is proteolytically processed to a two-chain form by cleavage of the linker connecting the k4 and sp domains ( Fig. 1 A and B). Single-chain HGF͞SF binds MET (15, 16) but is unable to induce biological responses, for example, dispersion of MDCK cell colonies, even at concentrations 100-fold higher than two-chain HGF͞SF (Fig. 1 C-E).MET is also synthesized as a single-chain precursor that is cleaved by furin yielding an N-terminal ␣-chain and a C-terminal ␤-chain. The MET ectodomain consists of two moieties: the large, N-terminal sema domain, which is responsible f...

A complex of the TSH receptor extracellular domain (amino acids 22-260; TSHR260) bound to a blocking-type human monoclonal autoantibody (K1-70) was purified, crystallised and the structure solved at 1 . 9 Å resolution. complexes show a root mean square deviation on all C a atoms of only 0 . 51 Å . These high-resolution crystal structures provide a foundation for developing new strategies to understand and control TSHR activation and the autoimmune response to the TSHR.

Little is known about the large ectodomain of MET, the product of the c-met protooncogene and receptor for hepatocyte growth factor͞scatter factor (HGF͞SF). Here, we establish by deletion mutagenesis that the HGF͞SF and heparin-binding sites of MET are contained within a large N-terminal domain spanning the ␣-chain (amino acids 25-307) and the first 212 amino acids of the ␤-chain (amino acids 308 -519). Neither the cystine-rich domain (amino acids 520 -561) nor the C-terminal half of MET (amino acids 562-932) bind HGF͞SF or heparin directly. The MET ectodomain, which behaves as a rod-shaped monomer with a large Stokes radius in solution, binds HGF͞SF in the absence or presence of heparin, and forms a stable HGF͞SF-heparin-MET complex with 1:1:1 stoichiometry. We also show that the ligand-binding domain adopts a ␤-propeller fold, which is similar to the N-terminal domain of ␣V integrin, and that the C-terminal half contains four Ig domains (amino acids 563-654, 657-738, 742-836, and 839 -924) of the unusual structural E set, which could be modeled on bacterial enzymes. Our studies provide 3D models and a functional map of the MET ectodomain. They have broad implications for structurefunction of the MET receptor and the related semaphorin and plexin proteins.Ig domain ͉ sema domain ͉ integrin ␣-chain ͉ hidden Markov models ͉ semaphorins R eceptor tyrosine kinases (RTKs) mediate intercellular signals, which are essential for the development and maintenance of the cells of multicellular organisms. The minimal domain structure of RTKs consists of an extracellular ligandbinding domain, a single transmembrane helix, and a cytoplasmic kinase domain. This minimal structure, however, is very rare, and, typically, the extracellular moiety of RTKs, the ectodomain, consists of complex and distinctive domain sets, which enable classification of the RTKs in different families (1).There is a strong preference for certain domains to occur in the ectodomain of RTKs. The fibronectin type-3 (FN-3) domain, for example, is present as two copies in the large Eph receptor family, three copies in the insulin and IGF-1 receptors, and at least seven copies in the rod outer segment receptor (1). Cystine-rich domains of variable length are also commonly found in RTKs.A large number of RTKs contain Ig domains and the ectodomain of certain families consists solely of Ig domains: the fibroblast growth factor (FGF) receptors contain two or three, depending on RNA splicing, the platelet-derived growth factor (PDGF), colony-stimulating factor 1 (CSF1), KIT, and FLT kinase͞serine-threonine kinase 1 (FLK2͞STK1) receptors contain five, and the FMS-like (FLT1), FLK1, FLT4, and cholecystokinin 4 (CCK4) receptors contain seven (1). Ig domains can also be present in combination with FN-3, cystine-rich, or other domains (1). Interestingly, most Ig domains present in RTKs and cell adhesion molecules belong to a distinct structural set known as the I set, with architecture intermediate between the V and C1 sets (2).MET, the RTK encoded by the c-met protoon...

We describe a general mass spectrometry approach to determine subunit stoichiometry and lipid binding in intact membrane protein complexes. By exploring conditions for preserving interactions during transmission into the gas phase and for optimally stripping away detergent, by subjecting the complex to multiple collisions, we release the intact complex largely devoid of detergent This enabled us to characterize both subunit stoichiometry and lipid binding in 4 membrane protein complexes.Membrane proteins perform a wide range of biological functions including respiration, signal transduction and molecular transport. Despite their obvious importance, these proteins and their complexes remain notoriously difficult to study. High-resolution methods, such as X-ray crystallography, require recombinant expression and crystallization of proteins, both of which are difficult for membrane subunits. Low-resolution methods to characterize membrane protein complexes include blue native gel electrophoresis 1 , sedimentation equilibrium or sedimentation velocity measurements via analytical ultracentrifugation 2 and small-angle X-ray scattering 3 . Although excellent results regarding the size of membrane proteins can be obtained, large quantities of protein are usually required, and it is essential to account for the contribution of the micelle to the overall mass. It can also be difficult to obtain definitive data on large or unstable complexes that do not form single oligomeric states in detergent solutions. Moreover the low accuracy of ultracentrifugation or X-ray scattering data does not normally reveal lipid binding or allow observation of posttranslational modifications.

The Toll-like receptor 4 (TLR4) is a class I transmembrane receptor expressed on the surface of immune system cells. TLR4 is activated by exposure to lipopolysaccharides derived from the outer membrane of Gram negative bacteria and forms part of the innate immune response in mammals. Like other class 1 receptors, TLR4 is activated by ligand induced dimerization, and recent studies suggest that this causes concerted conformational changes in the receptor leading to self association of the cytoplasmic Toll/Interleukin 1 receptor (TIR) signalling domain. This homodimerization event is proposed to provide a new scaffold that is able to bind downstream signalling adaptor proteins. TLR4 uses two different sets of adaptors; TRAM and TRIF, and Mal and MyD88. These adaptor pairs couple two distinct signalling pathways leading to the activation of interferon response factor 3 (IRF-3) and nuclear factor κB (NFκB) respectively. In this paper we have generated a structural model of the TLR4 TIR dimer and used molecular docking to probe for potential sites of interaction between the receptor homodimer and the adaptor molecules. Remarkably, both the Mal and TRAM adaptors are strongly predicted to bind at two symmetry-related sites at the homodimer interface. This model of TLR4 activation is supported by extensive functional studies involving site directed mutagenesis, inhibition by cell permeable peptides and stable protein phosphorylation of receptor and adaptor TIR domains. Our results also suggest a molecular mechanism for two recent findings, the caspase 1 dependence of Mal signalling and the protective effects conferred by the Mal polymorphism Ser180Leu.

The properties of a human monoclonal antibody to the thyrotropin receptor (TSHR) (M22) with the characteristics of patient sera thyroid stimulating autoantibodies is described. Similar concentrations (pmol/L) of M22 Fab and porcine TSH had similar stimulating effects on cyclic adenosine monophosphate (cAMP) production in TSHR-transfected Chinese hamster ovary cells whereas higher doses of intact M22 immunoglobulin G (IgG) were required to cause the same level of stimulation. Patient sera containing TSHR autoantibodies with TSH antagonist (blocking) activity inhibited M22 Fab and IgG stimulation in a similar way to their ability to block TSH stimulation. Thyroid-stimulating monoclonal antibodies (TSmAbs) produced in mice inhibited 125I-TSH binding and 125I-M22 Fab binding to the TSHR but the mouse TSmAbs were less effective inhibitors than M22. These competition studies emphasized the close relationship between the binding sites on the TSHR for TSH, TSHR autoantibodies with TSH agonist activity, and TSHR autoantibodies with TSH antagonist activity. Recombinant M22 Fab could be produced in Escherichia coli and the recombinant and hybridoma produced Fabs were similarly active in terms of inhibition of TSH binding and cAMP stimulation. The crystal structure of M22 Fab was determined to 1.65 A resolution and is that of a standard Fab although the hypervariable region of the heavy chain protrudes further from the framework than the hypervariable region of the light chain. The M22 antigen binding site is rich in aromatic residues and its surface is dominated by acidic patches on one side and basic patches on the other in agreement with an important role for charge-charge interactions in the TSHR-autoantibody interaction.

In Gram-negative bacteria, drug resistance is due in part to the activity of transmembrane efflux-pumps, which are composed of three types of proteins. A representative pump from Escherichia coli is an assembly of the trimeric outer-membrane protein TolC, which is an allosteric channel, the trimeric innermembrane proton-antiporter AcrB, and the periplasmic protein, AcrA. The pump displaces drugs vectorially from the bacterium using proton electrochemical force. Crystal structures are available for TolC and AcrB from E. coli, and for the AcrA homologue MexA from Pseudomonas aeruginosa. Based on homology modelling and molecular docking, we show how AcrA, AcrB and TolC might assemble to form a tripartite pump, and how allostery may occur during transport.