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  • Structural basis of hepatocyte growth factorscatterfactor and MET signallingErmanno Gherardi*, Sara Sandin, Maxim V. Petoukhov, John Finch, Mark E. Youles*, Lars-Goran Ofverstedt,Ricardo N. Miguel**, Tom L. Blundell**, George F. Vande Woude, Ulf Skoglund, and Dmitri I. Svergun

    *Medical Research Council Centre and Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, United Kingdom; Department of Cell andMolecular Biology, Medical Nobel Institute, Karolinska Institutet, Box 285, 171 77 Stockholm, Sweden; European Molecular Biology Laboratory, HamburgOutstation, Notkestrasse 85, D-22603 Hamburg, Germany; Institute of Crystallography, Russian Academy of Sciences, Leninsky Prospect 59, Moscow 117333,Russia; **Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1GA, United Kingdom; and Van Andel ResearchInstitute, 333 Bostwick Avenue Northeast, Grand Rapids, MI 49503

    Edited by Joseph Schlessinger, Yale University School of Medicine, New Haven, CT, and approved January 10, 2006 (received for review October 15, 2005)

    The polypeptide growth factor, hepatocyte growth factorscatterfactor (HGFSF), shares the multidomain structure and proteolyticmechanism of activation of plasminogen and other complex serineproteinases. HGFSF, however, has no enzymatic activity. Instead,it controls the growth, morphogenesis, or migration of epithelial,endothelial, and muscle progenitor cells through the receptortyrosine kinase MET. Using small-angle x-ray scattering and cryo-electron microscopy, we show that conversion of pro(single-chain)-HGFSF into the active two-chain form is associated with a majorstructural transition from a compact, closed conformation to anelongated, open one. We also report the structure of a complexbetween two-chain HGFSF and the MET ectodomain (MET928)with 1:1 stoichiometry in which the N-terminal and first kringledomain of HGFSF contact the face of the seven-blade -propellerdomain of MET harboring the loops connecting the -strands bcand da, whereas the C-terminal serine proteinase homologydomain binds the opposite b face. Finally, we describe a complexwith 2:2 stoichiometry between two-chain HGFSF and a truncatedform of the MET ectodomain (MET567), which is assembled aroundthe dimerization interface seen in the crystal structure of the NK1fragment of HGFSF and displays the features of a functional,signaling unit. The study shows how the proteolytic mechanism ofactivation of the complex proteinases has been adapted to cellsignaling in vertebrate organisms, offers a description of mono-meric and dimeric ligand-receptor complexes, and provides a foun-dation to the structural basis of HGFSF-MET signaling.

    cell signaling plasminogen serine proteinases kringle x-ray scattering

    Hepatocyte growth factorscatter factor (HGFSF) (16) arevertebrate-specific polypeptide growth factors with a do-main structure related to that of plasminogen (7). Interest inHGFSF and its receptor MET (8) stems from unique biologicalroles in embryogenesis (911), tissue regeneration (12, 13), andcancer (14). These activities have led to a strong interest in thestructure of the molecules as this knowledge may underpin thedevelopment of MET-based therapeutics.

    HGFSF consists of six domains: an N-terminal domain (n),four copies of the kringle domain (k1k4), and a C-terminaldomain (sp) structurally related to the catalytic domain of serineproteinases (Fig. 1A). The factor is synthesized as a precursorprotein (pro- or single-chain HGFSF) and is proteolyticallyprocessed to a two-chain form by cleavage of the linker con-necting the k4 and sp domains (Fig. 1 A and B). Single-chainHGFSF binds MET (15, 16) but is unable to induce biologicalresponses, for example, dispersion of MDCK cell colonies, evenat concentrations 100-fold higher than two-chain HGFSF (Fig.1 CE).

    MET is also synthesized as a single-chain precursor that iscleaved by furin yielding an N-terminal -chain and a C-terminal-chain. The MET ectodomain consists of two moieties: thelarge, N-terminal sema domain, which is responsible for ligandbinding and adopts a -propeller fold, and a stalk structure,

    which consists of four copies of Ig-like domains (ig) (15) (Fig. 1A and B). The sema domain and the stalk structure are joinedby a small cystine-rich domain (cr) (Fig. 1 A).

    The structural basis of the conversion of single-chain totwo-chain HGFSF and MET activation by two-chain HGFSFare unknown. Several nuclear magnetic resonance and crystalstructures of HGFSF fragments corresponding to the n domain(17), the n and k1 domains (nk1) (1820), and the sp domain(21), and a crystal structure of the sema and cr domains of METin complex with the sp domain of HGFSF (22) have providedimportant insights but failed to show the interdomain interac-tions and the relevant changes which underlie biological activity.

    The objective of this study is to define structures, by cryo-electron microscopy (cryo-EM) and small-angle x-ray scattering(SAXS), for single-chain and two-chain HGFSF, the METectodomain, and their complexes. Unlike x-ray crystallographyand nuclear magnetic resonance, cryo-EM and SAXS can pro-

    Conflict of interest statement: No conflicts declared.

    This paper was submitted directly (Track II) to the PNAS office.

    Abbreviations: CET, cryo-electron tomography; EM, electron microscopy; HGFSF, hepato-cyte growth factorscatter factor; SAXS, small-angle x-ray scattering; MM, molecular mass.

    Data deposition: The atomic coordinates of 3D models of kringles 2, 3, and 4 of HGFSF andig2ig4 of MET have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes2CED, 2CEE, 2CEG, and 2CEW).

    To whom correspondence should be addressed. E-mail: egherard@mrc-lmb.cam.ac.uk.

    2006 by The National Academy of Sciences of the USA

    Fig. 1. Domain structure and biological activity of the three main proteinsused in this study. (A) Domain structure. (B) SDSPAGE under reducing con-ditions. (CE) Typical appearance of colonies of MDCK cells under standardculture conditions (C) or after addition of single-chain (D) or two-chain (E)HGFSF at the concentrations indicated. sc-SF, single HGFSF; tc-SF, two-chainHGFSF.

    40464051 PNAS March 14, 2006 vol. 103 no. 11 www.pnas.orgcgidoi10.1073pnas.0509040103

  • vide critical information on the overall architecture of large andflexible multidomain proteins. For 3D reconstruction of EMpreparations, we have used cryo-electron tomography (CET), amethod recently and successfully used in protein structure (23)as a result of the development of effective reconstruction anddenoising procedures (24). CET offers major advantages oversingle particle reconstruction in cases, such as HGFSF andMET, in which the proteins under study display structuralheterogeneity (see Results). Using novel ab initio and rigid bodymodeling methods (25), 3D structures were also, and indepen-dently, reconstructed by SAXS, a method that yields trulyaveraged solution structures over 1015 molecules in the illu-minated volume. The structures obtained by cryo-EM and SAXSare reported here.

    ResultsSingle-Chain and Two-Chain HGFSF. The binding affinity of single-chain HGFSF for MET is high (Kd 109 M) (16, 26). Whydoes single-chain HGFSF fail to activate MET? Under trans-mission EM, the structures of single-chain and two-chainHGFSF stained with uranyl acetate differed markedly. Themajority of single-chain particles appeared as ring-shaped,closed structures in which the larger sp domain makes contactwith the chain (Fig. 2A), and only a few particles displayed anelongated, open conformation (data not shown). In contrast,two-chain HGFSF, consistently appeared as an elongatedmolecule (Fig. 2B).

    Single-chain HGFSF particles reconstructed by CET(130 125 85 ) consisted of two distinct moieties, shapedas a rod and a cone and folded against each other (Fig. 2C).The volume, shape, and dimensions of the two substructuressuggested that the rod contains domains n to k3 (nk3) and thatthe cone contains the k4 and sp domains, as confirmed by thefact that the available crystal structure of a fragment ofthrombin (27) (Fig. 2G) corresponding to the k4-sp fragmentof HGFSF could be readily accommodated into the electrondensity of the cone (Fig. 2E). Thus, the short 7-aa linkerbetween k3 and k4, and not the long linker between k4 and sp,acts as a primary molecular hinge in HGFSF. Unlike single-chain HGFSF, the majority of two-chain particles recon-structed by CET had an extended structure similar to the oneshown in Fig. 2 D and F and in agreement with the results ofnegatively stained EM preparations.

    SAXS analysis established that both single-chain and two-chain HGFSF are monomeric in solution and that the Rg andDmax of two-chain HGFSF are larger than those of single-chain(Table 1). Fig. 2H presents the processed x-ray scatteringpatterns and ab initio low resolution bead models by DAMMIN(28). These models and all ab initio models (Figs. 2H, 3F, 4A, and5B), fitted neatly the experimental data with 1.0. The p(r)functions in Fig. 2I demonstrated that, although two-chainHGFSF is more elongated, the two functions are nearly iden-tical up to distances of 6 nm, suggesting that the differencebetween the two proteins is because of the movement of one endof the molecule. Rigid body modeling (29) yielded the solutionsin Fig. 2 J, which converge to a common structure for the nk3segment and differ by a translation of the sp and, to a lesserextent, the k4 domain. Consequently, the receptor binding sitesof the k1 (30) and sp (21, 22) domains, shown as blue and redpatches in Fig. 2 J, are very close to each other in the model ofsingle-chain HGFSF but become separated by 100 intwo-chain HGFSF.

    Movies 14, displaying representative 3D reconstructions, are available as supportinginformation on the PNAS web site.

    Fig. 2. Structure of single-chain and two-chain HG