Crystal structure of human epidermal growth factor and its dimerization.

Epidermal growth factor (EGF) is a typical growth-stimulating peptide and functions by binding to specific cell-surface receptors and inducing dimerization of the receptors. Little is known about the molecular mechanism of EGF-induced dimerization of EGF receptors. The crystal structure of human EGF has been determined at pH 8.1. There are two human EGF molecules A and B in the asymmetric unit of the crystals, which form a potential dimer. Importantly, a number of residues known to be indispensable for EGF binding to its receptor are involved in the interface between the two EGF molecules, suggesting a crucial role of EGF dimerization in the EGF-induced dimerization of receptors. In addition, the crystal structure of EGF shares the main features of the NMR structure of mouse EGF determined at pH 2.0, but structural comparisons between different models have revealed new detailed features and properties of the EGF structure.     

Probing fibroblast growth factor dimerization and role of heparin-like glycosaminoglycans in modulating dimerization and signaling

For a number of growth factors and cytokines, ligand dimerization is believed to be central to the formation of an active signaling complex. In the case of fibroblast growth factor-2 (FGF2) signaling, heparin/heparan sulfate-like glycosaminoglycans (HLGAGs) are involved through interaction with both FGF2 and its receptors (FGFRs) in assembling a tertiary complex and modulating FGF2 activity. Biochemical data have suggested different modes of HLGAG-induced FGF2 dimerization involving specific protein-protein contacts. In addition, several recent x-ray crystallography studies of FGF.FGFR and FGF.FGFR.HLGAG complexes have revealed other modes of molecular assemblage, with no FGF-FGF contacts. All these different biochemical and structural findings have clarified less and in fact raised more questions as to which mode of FGF2 dimerization, if any, is essential for signaling. In this study, we address the issue of FGF2 dimerization in signaling using a combination of biochemical, biophysical, and site-directed mutagenesis approaches. Our findings presented here provide direct evidence of FGF2 dimerization in mediating FGF2 signaling.     

Male-to-female sex reversal in mice lacking fibroblast growth factor 9

Fgfs direct embryogenesis of several organs, including the lung, limb, and anterior pituitary. Here we report male-to-female sex reversal in mice lacking Fibroblast growth factor 9 (Fgf9), demonstrating a novel role for FGF signaling in testicular embryogenesis. Fgf9(-/-) mice also exhibit lung hypoplasia and die at birth. Reproductive system phenotypes range from testicular hypoplasia to complete sex reversal, with most Fgf9(-/-) XY reproductive systems appearing grossly female at birth. Fgf9 appears to act downstream of Sry to stimulate mesenchymal proliferation, mesonephric cell migration, and Sertoli cell differentiation in the embryonic testis. While Sry is found only in some mammals, Fgfs are highly conserved. Thus, Fgfs may function in sex determination and reproductive system development in many species.     

Crystal structure of the ternary complex of a NaV C-terminal domain, a fibroblast growth factor homologous factor, and calmodulin

Voltage-gated Na? (Na(V)) channels initiate neuronal action potentials. Na(V) channels are composed of a transmembrane domain responsible for voltage-dependent Na? conduction and a cytosolic C-terminal domain (CTD) that regulates channel function through interactions with many auxiliary proteins, including fibroblast growth factor homologous factors (FHFs) and calmodulin (CaM). Most ion channel structural studies have focused on mechanisms of permeation and voltage-dependent gating but less is known about how intracellular domains modulate channel function. Here we report the crystal structure of the ternary complex of a human Na(V) CTD, an FHF, and Ca2?-free CaM at 2.2 ?. Combined with functional experiments based on structural insights, we present a platform for understanding the roles of these auxiliary proteins in Na(V) channel regulation and the molecular basis of mutations that lead to neuronal and cardiac diseases. Furthermore, we identify a critical interaction that contributes to the specificity of individual Na(V) CTD isoforms for distinctive FHFs.     

Crystal structure of a ternary FGF-FGFR-heparin complex reveals a dual role for heparin in FGFR binding and dimerization

The crystal structure of a dimeric 2:2:2 FGF:FGFR:heparin ternary complex at 3 A resolution has been determined. Within each 1:1 FGF:FGFR complex, heparin makes numerous contacts with both FGF and FGFR, thereby augmenting FGF-FGFR binding. Heparin also interacts with FGFR in the adjoining 1:1 FGF:FGFR complex to promote FGFR dimerization. The 6-O-sulfate group of heparin plays a pivotal role in mediating both interactions. The unexpected stoichiometry of heparin binding in the structure led us to propose a revised model for FGFR dimerization. Biochemical data in support of this model are also presented. This model provides a structural basis for FGFR activation by small molecule heparin analogs and may facilitate the design of heparin mimetics capable of modulating FGF signaling.     

Crystal structure of inhibitor of growth 4 (ING4) dimerization domain reveals functional organization of ING family of chromatin-binding proteins

The protein ING4 binds to histone H3 trimethylated at Lys-4 (H3K4me3) through its C-terminal plant homeodomain, thus recruiting the HBO1 histone acetyltransferase complex to target promoters. The structure of the plant homeodomain finger bound to an H3K4me3 peptide has been described, as well as the disorder and flexibility in the ING4 central region. We report the crystal structure of the ING4 N-terminal domain, which shows an antiparallel coiled-coil homodimer with each protomer folded into a helix-loop-helix structure. This arrangement suggests that ING4 can bind simultaneously two histone tails on the same or different nucleosomes. Dimerization has a direct impact on ING4 tumor suppressor activity because monomeric mutants lose the ability to induce apoptosis after genotoxic stress. Homology modeling based on the ING4 structure suggests that other ING dimers may also exist.     

Functional phylogeny relates LET-756 to fibroblast growth factor 9

Fibroblast growth factors (FGFs) are secreted regulatory proteins involved in various developmental processes. In vertebrates, the FGF superfamily comprises 22 members. In non-vertebrates, six FGF genes have been identified in Ciona intestinalis, three in Drosophila melanogaster, and two (let-756 and egl-17) in Caenorhabditis elegans. The core of LET-756 shares a 30-50% sequence identity with the various members of the superfamily. The relationships between vertebrate and non-vertebrate FGFs are not clear. We made chimeric FGFs by replacing the core region of LET-756 by the cores of various mammalian, fly, and worm FGFs. LET-756 deleted in its core region was no longer able to rescue the lethal phenotype of a let-756 null mutant, and only chimeras containing the cores of FGFs 9, 16, and 20 showed rescue capacity. This core contains an internal motif of six amino acid residues (EFISIA) whose deletion or mutation abolished both the rescue activity and FGF secretion in the supernatant of transfected COS-1 cells. Chimera containing the core of C. intestinalis FGF9/16/20, a potential ortholog of FGF9 lacking the complete EFISIA motif, was not able to rescue the lethal phenotype or be secreted. However, the introduction of the EFISIA motif restored both activities. The data show that the EFISIA motif in the core of LET-756 is essential for its biological activity and that FGFs 9, 16, and 20, which contain that motif, are functionally close to LET-756 and may be evolutionary related. This non-classical mode of secretion using an internal motif is conserved throughout evolution.     

Crystal structure of the Shank PDZ-ligand complex reveals a class I PDZ interaction and a novel PDZ-PDZ dimerization

The Shank/proline-rich synapse-associated protein family of multidomain proteins is known to play an important role in the organization of synaptic multiprotein complexes. For instance, the Shank PDZ domain binds to the C termini of guanylate kinase-associated proteins, which in turn interact with the guanylate kinase domain of postsynaptic density-95 scaffolding proteins. Here we describe the crystal structures of Shank1 PDZ in its peptide free form and in complex with the C-terminal hexapeptide (EAQTRL) of guanylate kinase-associated protein (GKAP1a) determined at 1.8- and 2.25-A resolutions, respectively. The structure shows the typical class I PDZ interaction of PDZ-peptide complex with the consensus sequence -X-(Thr/Ser)-X-Leu. In addition, Asp-634 within the Shank1 PDZ domain recognizes the positively charged Arg at -1 position and hydrogen bonds, and salt bridges between Arg-607 and the side chains of the ligand at -3 and -5 positions contribute further to the recognition of the peptide ligand. Remarkably, whether free or complexed, Shank1 PDZ domains form dimers with a conserved beta B/beta C loop and N-terminal beta A strands, suggesting a novel model of PDZ-PDZ homodimerization. This implies that antiparallel dimerization through the N-terminal beta A strands could be a common configuration among PDZ dimers. Within the dimeric structure, the two-peptide binding sites are arranged so that the N termini of the bound peptide ligands are in close proximity and oriented toward the 2-fold axis of the dimer. This configuration may provide a means of facilitating dimeric organization of PDZ-target assemblies.     

Crystal structure of the human protein kinase CK2 regulatory subunit reveals its zinc finger-mediated dimerization.

Protein kinase CK2 is a tetramer composed of two alpha catalytic subunits and two beta regulatory subunits. The structure of a C-terminal truncated form of the human beta subunit has been determined by X-ray crystallography to 1.7 A resolution. One dimer is observed in the asymmetric unit of the crystal. The most striking feature of the structure is the presence of a zinc finger mediating the dimerization. The monomer structure consists of two domains, one entirely alpha-helical and one including the zinc finger. The dimer has a crescent shape holding a highly acidic region at both ends. We propose that this acidic region is involved in the interactions with the polyamines and/or catalytic subunits. Interestingly, conserved amino acid residues among beta subunit sequences are clustered along one linear ridge that wraps around the entire dimer. This feature suggests that protein partners may interact with the dimer through a stretch of residues in an extended conformation.     

Crystal structure of human platelet-derived growth factor BB.

The crystal structure of the homodimeric BB isoform of human recombinant platelet-derived growth factor (PDGF-BB) has been determined by X-ray analysis to 3.0 A resolution. The polypeptide chain is folded into two highly twisted antiparallel pairs of beta-strands and contains an unusual knotted arrangement of three intramolecular disulfide bonds. Dimerization leads to the clustering of three surface loops at each end of the elongated dimer, which most probably form the receptor recognition sites.     

Transient dimerization and interaction with ERGIC-53 occur in the fibroblast growth factor receptor 3 early secretory pathway

The fibroblast growth factor receptor 3 (FGFR3) secretory pathway includes N-linked glycosylation in the endoplasmic reticulum where a stringent quality control system ensures that only correctly folded receptor reaches the cell surface from where mature-functional FGFR3 signals upon ligand-mediated dimerization. We have previously shown that the increased kinase activity associated with FGFR3 bearing the thanatophoric dysplasia type II (TDII) mutation hampers its maturation, enabling the receptor to signal from the endoplasmic reticulum. Here we investigate if this biosynthetic disturbance could be explained by premature dimerization of the receptor. Our observations show that a limited fraction of the immature high-mannose, mutant receptor dimerizes in the early secretory pathway, as does the immature wild type FGFR3. In contrast, the mature fully glycosylated wild type receptor reaches the cell surface as monomer suggesting that dimerization is a transient event. The kinase activity of mutant FGFR3 is not required for dimerization to occur, although it increases dimerization efficiency. Furthermore, mutant FGFR3 trans-phosphorylates the immature wild type receptor indicating that dimerization occurs in the endoplasmic reticulum. Visualization of protein interaction inside the secretory pathway confirms receptor dimerization. In addition, it shows that both wild type and TDII FGFR3 interact with the mannose-specific lectin ERGIC-53. We conclude that transient dimerization is an obligatory step in FGFR3 biosynthesis acting as a pre-assembly quality control mechanism. Furthermore, the TDII/ERGIC-53 complex formation may function as a checkpoint for FGFR3 sorting downstream the endoplasmic reticulum. These findings have implications for understanding the pathogenesis of FGFR3-related disorders.     

Crystal structure of the factor XI zymogen reveals a pathway for transactivation

Factor XI (FXI), a coagulation protein essential to normal hemostasis, circulates as a disulfide-linked dimer. Here we report the full-length FXI zymogen crystal structure, revealing that the protease and four apple domains assemble into a unique 'cup and saucer' architecture. The structure shows that the thrombin and platelet glycoprotein Ib binding sites are remote within the monomer but lie in close proximity across the dimer, suggesting a transactivation mechanism.     

An atomic resolution structure for human fibroblast growth factor 1

A 1.10-A atomic resolution X-ray structure of human fibroblast growth factor 1 (FGF-1), a member of the beta-trefoil superfold, has been determined. The beta-trefoil is one of 10 fundamental protein superfolds and is the only superfold to exhibit 3-fold structural symmetry (comprising 3 "trefoil" units). The quality of the diffraction data permits unambiguous assignment of Asn, Gln, and His rotamers, Pro ring pucker, as well as refinement of atomic anisotropic displacement parameters (ADPs). The FGF-1 structure exhibits numerous core-packing defects, detectable using a 1.0-A probe radius. In addition to contributing to the relatively low thermal stability of FGF-1, these defects may also permit domain motions within the structure. The availability of refined ADPs allows a translation/libration/screw (TLS) analysis of putative rigid body domains. The TLS analysis shows that beta-strands 6-12 together form a rigid body, and there is a clear demarcation in TLS motions between the adjacent carboxyl- and amino-termini. Although separate from beta-strands 6-12, the individual beta-strands 1-5 do not exhibit correlated motions; thus, this region appears to be comparatively flexible. The heparin-binding contacts of FGF-1 are located within beta-strands 6-12; conversely, a significant portion of the receptor-binding contacts are located within beta-strands 1-5. Thus, the observed rigid body motion in FGF-1 appears related to the ligand-binding functionalities.     

Conversion of a paracrine fibroblast growth factor into an endocrine fibroblast growth factor

FGFs 19, 21, and 23 are hormones that regulate in a Klotho co-receptor-dependent fashion major metabolic processes such as glucose and lipid metabolism (FGF21) and phosphate and vitamin D homeostasis (FGF23). The role of heparan sulfate glycosaminoglycan in the formation of the cell surface signaling complex of endocrine FGFs has remained unclear. Here we show that heparan sulfate is not a component of the signal transduction unit of FGF19 and FGF23. In support of our model, we convert a paracrine FGF into an endocrine ligand by diminishing heparan sulfate-binding affinity of the paracrine FGF and substituting its C-terminal tail for that of an endocrine FGF containing the Klotho co-receptor-binding site to home the ligand into the target tissue. In addition to serving as a proof of concept, the ligand conversion provides a novel strategy for engineering endocrine FGF-like molecules for the treatment of metabolic disorders, including global epidemics such as type 2 diabetes and obesity.     

Cysteine-rich fibroblast growth factor receptor alters secretion and intracellular routing of fibroblast growth factor 3

Expression of the cysteine-rich fibroblast growth factor (FGF) receptor (CFR) in COS-1 cells strongly inhibits the secretion of co-expressed FGF3. By using a column retention assay and affinity chromatography, we demonstrate that at physiological salt concentrations FGF3 binds with strong affinity to CFR in vivo and in vitro. Furthermore, to show that FGF3 binds to CFR in vivo, truncation mutants of CFR with changed subcellular distributions were shown to cause a similar redistribution of FGF3. Although CFR is a 150-kDa integral membrane glycoprotein that is primarily located in the Golgi apparatus, we show here that in COS-1 cells a substantial proportion of CFR is secreted. This is due to a carboxyl-terminal proteolytic cleavage that releases the intraluminal portion of the protein for secretion. However, the apparent size of the integral membrane and secreted CFR appears similar, since the loss of protein mass is balanced by a gain of complex carbohydrates. The released CFR is associated with the extracellular matrix through its affinity for glycosaminoglycans. These findings show that CFR can modulate the secretion of FGF3 and may control its biological activity by regulating its secretion.