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• W2010476505 abstract "Article25 January 2007free access Structural basis for stem cell factor–KIT signaling and activation of class III receptor tyrosine kinases Heli Liu Heli Liu Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Feinberg School of Medicine, Chicago, IL, USA Search for more papers by this author Xiaoyan Chen Xiaoyan Chen Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Feinberg School of Medicine, Chicago, IL, USA Search for more papers by this author Pamela J Focia Pamela J Focia Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Feinberg School of Medicine, Chicago, IL, USA Search for more papers by this author Xiaolin He Corresponding Author Xiaolin He Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Feinberg School of Medicine, Chicago, IL, USA Search for more papers by this author Heli Liu Heli Liu Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Feinberg School of Medicine, Chicago, IL, USA Search for more papers by this author Xiaoyan Chen Xiaoyan Chen Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Feinberg School of Medicine, Chicago, IL, USA Search for more papers by this author Pamela J Focia Pamela J Focia Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Feinberg School of Medicine, Chicago, IL, USA Search for more papers by this author Xiaolin He Corresponding Author Xiaolin He Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Feinberg School of Medicine, Chicago, IL, USA Search for more papers by this author Author Information Heli Liu1,‡, Xiaoyan Chen1, Pamela J Focia1 and Xiaolin He 1 1Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Feinberg School of Medicine, Chicago, IL, USA ‡These authors contributed equally to this work *Corresponding author. Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Feinberg School of Medicine, Searle 8-417, 303 E Chicago Ave, Chicago, IL 60611, USA. Tel.: +1 312 503 8030; Fax: +1 312 503 5349; E-mail: [email protected] The EMBO Journal (2007)26:891-901https://doi.org/10.1038/sj.emboj.7601545 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Stem cell factor (SCF) binds to and activates the KIT receptor, a class III receptor tyrosine kinase (RTK), to stimulate diverse processes including melanogenesis, gametogenesis and hematopoeisis. Dysregulation of KIT activation is associated with many cancers. We report a 2.5 Å crystal structure of the functional core of SCF bound to the extracellular ligand-binding domains of KIT. The structure reveals a ‘wrapping’ SCF-recognition mode by KIT, in which KIT adopts a bent conformation to facilitate each of its first three immunoglobulin (Ig)-like domains to interact with SCF. Three surface epitopes on SCF, an extended loop, the B and C helices, and the N-terminal segment, contact distinct KIT domains, with two of the epitopes undergoing large conformational changes upon receptor binding. The SCF/KIT complex reveals a unique RTK dimerization assembly, and a novel recognition mode between four-helix bundle cytokines and Ig-family receptors. It serves as a framework for understanding the activation mechanisms of class III RTKs. Introduction Stem cell factor (SCF) binds to the extracellular domains of the KIT receptor tyrosine kinase (RTK) and plays a key role in diverse biological processes. KIT, the product of c-kit gene, was first identified as the cellular homologue of the transforming gene of Hardy-Zuckerman 4-feline sarcoma virus (Besmer et al, 1986; Yarden et al, 1987). Subsequently, KIT was mapped to the mouse White Spotting locus, followed by the identification of its ligand SCF (KitL) and the demonstration that it is allelic with the murine steel locus (for a review, see Besmer, 1991). These pioneer studies have brought to light the pleiotropic functions of the SCF/KIT system in melanogenesis, gametogenesis and hematopoiesis (Lennartsson et al, 2005). Recent findings of SCF and KIT's role in brain angiogenesis suggest additional function for this widely implicated system (Sun et al, 2006). Dysregulation of SCF–KIT signaling and gain-of-function KIT mutations contribute to the genesis of many cancers, with acute myeloid leukemia, gastrointestinal stromal tumors and mastocytosis being the most prevalent types (Lennartsson et al, 2005). KIT is a member of the class III subfamily of RTKs that includes KIT (SCFR), FMS (MCSFR or CSF-1R), FLT3, and PDGFR-α and -β. This class of RTKs are also called PDGFR family and are key receptors in the regulation of hematopoiesis and embryonic development (Reilly, 2003; Tallquist and Kazlauskas, 2004). They are characterized by an extracellular fragment consisting of five immunoglobulin (Ig)-like domains, a single transmembrane domain, two intracellular tyrosine kinase domains divided by a kinase insert domain and a C-terminal domain. Among class III RTKs, KIT and FLT3 mediate most of the early hematopoietic signaling (Fichelson, 1998); modulating their activities has the potential to facilitate the regeneration, isolation and expansion of stem cells in clinical applications. In addition to their physiological roles, most class III RTKs are involved in the genesis and development of cancers, and are widely pursued targets in the development of anti-cancer drugs and therapies (Krause and Van Etten, 2005). The ligands for class III RTKs are two groups of topologically unrelated growth factors. The first group, including SCF, the ligand for KIT, macrophage-colony stimulating factor (MCSF), the ligand for FMS, and FLT3L, the ligand for FLT3, are four-helix bundle type cytokines (Sprang and Bazan, 1993). They are non-covalently or covalently linked dimers existing naturally as membrane-anchored and soluble isoforms as a result of alternative RNA splicing and proteolytic processing. Their N-terminal global domains have been identified as a functional core, which includes the dimerization interface and portions that bind and activate receptors (Langley et al, 1994). The other group of class III RTK ligands, PDGF-AA, -AB, -BB, -CC and -DD, are VEGF-like cysteine-knot type growth factors. Class III RTKs therefore represent a rare receptor family that use the same scaffold to receive ligands in fundamentally different folds, prompting a fascinating structural question to be answered. The pivotal roles of class III RTKs in hematopoiesis and tumorigenesis have led to extensive functional and biochemical investigations, but it has not been structurally understood how these receptors recognize ligands and become activated. Despite a handful of ligand structures (Oefner et al, 1992; Pandit et al, 1992; Jiang et al, 2000; Savvides et al, 2000; Zhang et al, 2000) and mutagenesis mapping studies on ligands (Taylor et al, 1994; Matous et al, 1996; Graddis et al, 1998; Hsu et al, 1998), a structure of ligand–receptor complex has been elusive for this important receptor family. Here, we present the first crystal structure of a class III RTK ligand–receptor complex, the core domain of SCF bound to the extracellular ligand-binding domains of KIT. The structure shows a unique RTK/ligand assembly, in which SCF dimer is ‘wrapped’ by KIT domains to allow engagement of two receptors, providing a framework for understanding ligand–receptor interactions and activation mechanisms of class III RTKs. It also reveals a new recognition mode between four-helix bundle cytokines and Ig-family receptors. Results Overall structure of SCF/KIT complex Crystals of SCF/KIT complex were prepared using refolded mouse SCF from Escherichia coli and soluble, baculovirus-expressed mouse KIT extracellular domains 1–3. Initial trials using fully glycosylated, heterogeneous KIT yielded poorly diffracting crystals. To improve crystal quality, we reduced KIT glycosylation by mutagenesis (N146Q). This material behaved identically to the fully glycosylated proteins and yielded crystals that diffracted to 2.5 Å resolution. The SCF/KIT structure was solved by single isomorphous replacement with anomalous scattering from an iodide derivative of the binary complex (Table I). Table 1. Data collection, phasing and refinement statistics SCF/KIT complex SCF Native NaI Wavelength (Å) 0.9798 1.5498 0.9184 Resolution range (Å) (highest resolution shell) 20–2.5 (2.60–2.50) 50–3.21 (3.31–3.21) 50–2.18 (2.26–2.18) Space group P21 P21 P65 Unique reflections 82 592 40 563 13 625 Completeness (%) 96.8 (93.3) 99.8 (99.8) 98.9 (100.0) I/σ(I) 17.1 (2.6) 22.5 (5.6) 43.0 (5.0) Redundancy 2.9 9.3 13.0 Rmerge (%) 4.0 (38.9) 8.6 (44.6) 10.0 (49.0) SIRAS phasing Resolution (Å) 20–3.2 Number of heavy atom sites 27 Rano (%) 5.8 Riso (%) 15.4 Figure of merit 0.41 Refinement SCF/KIT complex SCF Resolution range (Å) 20–2.5 (2.59–2.50) 15–2.2 (2.28–2.10) Rcryst 0.237 (0.416) 0.241 (0.267) Rfree 0.270 (0.427) 0.266 (0.286) Average B factor (Å2) 97.4 54.5 R.m.s.d. bond length (Å) 0.007 0.009 R.m.s.d. bond angle, dihedral, improper (deg) 1.4, 25.3, 1.1 1.5, 22.2, 1.0 R.m.s.d. bonded B factor (Å) 3.7 2.3 Ramachandran (favored, allowed, generally allowed, disallowed) (%) 84.6, 15.3, 0.1, 0 85.3, 13.8, 0.9, 0 The overall structure of the 2:2 SCF/KIT complex roughly resembles an ‘H’ letter with its top pushed down (Figure 1A and B). The lower parts of KIT stand vertical to the long axis of a flat-lying SCF dimer, and the N-terminal parts of KIT are bent back like horns. The orientation of the complex in Figure 1 places the SCF dimer parallel to the cell surface and the KIT C-termini proximal to the membrane. The SCF structure is similar to the free human SCF structures (Jiang et al, 2000; Zhang et al, 2000), with a core of four α-helices (A, B, C and D) and two β-strands (one between helices A and B and one between helices C and D). The head-to-head dimerization mode of SCF is unchanged from free human SCF structures. Two KIT are tethered together entirely through SCF dimerization. Each KIT interacts with only one SCF, and no KIT-to-KIT interaction is observed. Each of the KIT domains, designated D1 (residues 33–114), D2 (residues 115–208) and D3 (residues 209–310), interacts with SCF through spatially adjacent, but largely separated, binding epitopes. To facilitate analysis, these epitopes are designated as sites 1, 2 and 3, respectively (Figure 1A). Overall, the binding of each KIT to an SCF monomer resembles a taco, with KIT wrapping around SCF in a half-closed fashion. This binding fashion differs substantially from the previous models of SCF/KIT complex (Jiang et al, 2000; Zhang et al, 2000) (Supplementary Figure S1). Jiang et al (2000) placed KIT-D2 at approximately correct positions, and successfully predicted the charge complementarity between SCF and KIT (discussed below), but the orientation of each modeled KIT domain (D2 and D3) is different from actual. Additionally, the modeled D2–D2 distance, mimicking the VEGF/FLT1 complex, is longer than the actual distance, and the modeled D2–D3 hinge angle, mimicking VCAM, is drastically different from that seen in this structure. Figure 1.Structure of the SCF/KIT complex. (A) Ribbon model of the binary complex, SCF molecules are in cyan and green, and KIT molecules in orange and pink. (B) Side view of the complex in surface model, rotated 90° vertically relative to (A). Download figure Download PowerPoint Structure of KIT The extracellular SCF-binding region of KIT, comprising three Ig-like domains, resembles the perpendicular stroke of the letter ‘f’ (Figure 2). It adopts an elongated, and surprisingly, bent conformation. Each D1–D2 or D2–D3 hinge angle is identical between different copies of KIT in the asymmetric unit, suggesting that this bent conformation is rigid rather than flexible. The D1–D2 hinge angle is abrupt (the β-strands directions of D1 and D2 are ∼80° relative to each other) and D1 folds back toward D2. The tight D1–D2 packing observed in KIT has also been observed among some cytokine receptors, Fc receptors and NK receptors, but has not observed in other RTKs. The D2–D3 hinge angle, in comparison, is much more linear (∼150°). D1 is a non-canonical intermediate between I-set and S-set Ig domains. Its first strand is divided into A and A′ strands and is shared by both layers of β-sheets (GFC face and BED face), characteristic of I-set configuration (Harpaz and Chothia, 1994), but its fourth strand switches from the BED face to the GFC face (D to C′), characteristic of S-type configuration (Bork et al, 1994) (Figure 2A). Unusually, D1's short CC′ loop and the C′ strand protrude from the domain core; they seem to be stabilized more by D1–D2 and D1–SCF interactions than by intra-domain interactions. D2 is a distorted I-set Ig domain. Its GFC face is twisted into two 90°-related β-sheets: the lower sheet (G, F and A′ strands) faces SCF to form D2–SCF interactions, and the upper sheet (C, G′ and F′ strands) faces outwards (Figure 2A). Domain D3 is a canonical I-set Ig domain. There are three predicted N-linked glycosylation sites in KIT, all on the opposite side of SCF-binding sites. Among these sites, Asn146 in D1 is mutated to Gln, whereas Asn296 and Asn303 in D3 appear fully glycosylated and three glycan residues were modeled for each site. Figure 2.Structure of KIT. (A) Structural details of KIT domains. In each domain, the top layer of β-strands is colored in green and the bottom layer in red, with the exceptions that the switched strand (C′ strand) in D1 is green in color, and the upper part of the D2 bottom strands, vertical to the lower part, is colored in orange. (B) The D1–D2 interface. The side chains are colored in green for D1 residues and in cyan for D2 residues. (C) Structure-based sequence alignment of N-terminal three domains between KIT and related receptors. Residues involved in hydrophobic interaction at D1–D2 interface are shaded in green. Conserved residues at D1–D2 linker are shaded in pink. Residues involved in ligand binding as proven in structures of complexes are boxed in gray. Download figure Download PowerPoint The segmental rigidity of KIT is ensured by the extensive inter-domain interactions. The D2–D3 junction is reinforced by both hydrophobic stacking and hydrogen bonds, with a total area of 630 Å2 buried in the interface. The D1–D2 interaction is even more extensive, burying a large area of the surface area (1380 Å2), which may suggest that D1 and D2 are integral and inseparable. The D1–D2 junction is exquisitely designed, encompassing primarily hydrophobic inter-domain interactions (Figure 2B). The D1–D2 interface can be divided into two areas. The first, larger area is a hydrophobic cluster, including Ile47, Ala90, Thr93 (Cγ2), Tyr109, Phe111, Asp114 (Cβ) from D1 and Lys117 (aliphatic part), Leu120, Thr140 (Cγ2), Pro142 from D2, further consolidated by two salt bridges (Arg113–Asp141 and Glu45–Lys168). The area is entirely conserved across mammalian KIT molecules (Supplementary Figure S2). The second, smaller area is formed between hydrophobic residues (Tyr71 and Phe72) extended from the D1 CC′ loop and the hydrophobic patch, Leu120, Leu123, Arg136 (aliphatic part), Pro138, from D2. These residues are also either conserved or similarly hydrophobic among mammalian species (Supplementary Figure S2). The sequence conservation of both patches suggests that the configuration between D1 and D2, and thereby the bent-back orientation of D1, is structurally conserved. The bent-back orientation of D1, together with the anchoring of the D1 CC′ loop to D2 through hydrophobic interaction, serves to position the D1 C′ strand, which is involved in SCF binding (discussed below). The D1–D2 configuration and the bent-back orientation of D1 may be a conserved feature among many Ig-containing RTKs, including other class III RTKs and closely related class V RTKs. Structure-based sequence alignment (Figure 2C) shows that the residues contributing to the hydrophobic interactions in the D1–D2 interface are highly conserved among helical ligand-binding class III RTKs (e.g., KIT and FMS), β-sheet ligand-binding class III RTKs (e.g., PDGFRα) and class V RTKs (e.g., FLT1). In particular, FLT1 shows the highest similarity to KIT in D1–D2 interface composition. In addition, the D1–D2 linker sequences are the same length and similar among these RTKs (Figure 2C). The conservation of both D1–D2 interface and D1–D2 linker suggests that many class III and class V RTKs share similar D1–D2 configuration. This configuration then dictates a bent-back D1, which can bear the potential to participate in ligand binding. Interactions between SCF and KIT Overall, about 2200 Å2 of solvent-accessible surface is buried between each pair of SCF and KIT, which can be subdivided into 610 Å2 for site 1, the D1/SCF epitope, 760 Å2 for site 2, the D2/SCF epitope, and 830 Å2 for site 3, the D3/SCF epitope. Consistent with early thermodynamic data showing that SCF/KIT binding is largely enthalpy-driven (Philo et al, 1996; Lemmon et al, 1997), the SCF/KIT-binding interface buries large areas of hydrophilic surface, with abundant salt bridges and hydrogen bonds. Only 12% (268 Å2) of the buried surface is hydrophobic. As predicted by Jiang et al (2000), charge complementarity appears to be an important feature of SCF/KIT recognition: KIT is positively charged at site 2 and slightly positively charged at site 3, whereas SCF is rich in negative charge at both these sites to meet KIT (Figure 3E). An exception is site 1, where the charge usage of receptor and ligand is reversed, that is, SCF uses positive charge and KIT uses negative charge (Figure 3E). Figure 3.Interaction between SCF and KIT. (A) Site 1, the KIT-D1/SCF interface. SCF's main chain is colored in cyan, side chain in blue; KIT's main chain in pink, side chain in purple. (B) Site 2, the KIT-D2/SCF interface. (C) Site 3, the KIT-D3/SCF interface. (D) Composite 2Fo−Fc omit map contoured at 1.0σ showing a portion of the site 2 interface. (E) Charge complementarity between SCF and KIT. Left part is KIT as tube and SCF as GRASP surface colored with electrostatic potential, red being negative and blue being positive; right part vice versa. (F) Sequence alignment between mouse and human SCF/KIT. Site 1, 2 and 3 residues are shaded in blue, green and orange, respectively. Download figure Download PowerPoint The small site 1 interface consists of the C′ strand (residues 73–77) from KIT-D1 and an extended loop (residues 95–104) from SCF (Figure 3A). It encompasses two salt bridges, three hydrogen bonds and a number of van der Waals interactions (Table II). The site 2 interface is formed by the lower part of the KIT-D2 bottom β-sheet, including the A′, F and G strands (residues 124–126, 181–182 and 200–206), crossing vertically above the B and C helices of SCF (residues 50–61 and 77–88) (Figure 3B). This interface can be divided into two side-by-side, chemically distinct patches, one hydrophobic and the other hydrophilic. The first, hydrophobic patch is formed between SCF and the A′ strand of KIT-D2, in which KIT inserts Pro124 and Phe126 into the cavity formed by SCF Ile50, Leu54, Val87 and Leu88 (Table II, Figure 3D). The second, hydrophilic patch is formed between SCF and the G and F strands of KIT-D2. This patch consists entirely of hydrophilic residues and is rich in charges (Table II). The charge complementarity at this patch is also clearly reflected in Figure 3E. The site 3 interface is formed between SCF N-terminal segment and the BC loop (residues 263–274) plus the DE loop (residues 239–243) of KIT-D3 (Figure 3C). Compared to sites 1 and 2, this interface has the largest buried area (830 Å2) and the largest number of SCF/KIT contacts (Table II). Table 2. Contacts between SCF and KIT KIT-D1 SCF Distance (Å) Hydrogen bonds and salt bridges Asn3 O Asn97 Nδ2 2.6 Glu74 Oε1 Arg104 Nη2 3.1 Met75 N Asn97 Oδ1 3.3 Met75 O Lys99 N 2.9 Glu77 Oε1 Lys99 Nζ 3.3 van der Waals contacts Thr70 Arg104 Phe72 Arg104 Asn73 Asn97 Glu74 Asn97 Met75 Ile98 KIT-D2 Hydrogen bonds and salt bridges Arg182 Nη2 Asp77 Oδ1 2.5 Lys200 Nζ Asp61 Oδ1 3.5 Lys204 Nζ Asp84 Oδ2 3.0 van der Waals contacts Pro124 Ile50 Phe126 Leu88, Val87 His181 Lys81 Thr202 Thr57 Arg206 Leu88 KIT-D3 Hydrogen bonds and salt bridges Asn263 Nδ2 Asp10 Oδ1 3.3 Ser264 N Asp10 Oδ2 3.3 Ser264 O Asp10 N 2.7 His266 N Asn11 Oδ1 2.9 Arg274 Nη1 Asn6 Oδ1 3.3 van der Waals contacts Thr241 Asn6, Asp85 Ser264 Val8, Asn6, Thr9, Asp10 Trp265 Asp10 His266 Asn11, Thr9 Asp269 Lys81 The SCF/KIT structure can now be used to rationalize previous biochemical and functional data mapping SCF/KIT interaction. Mutagenesis and antibody mapping data on SCF implicate the first few N-terminal residues before Cys4 (Langley et al, 1994), the regions flanked by residues 61–65 and 91–95 (Mendiaz et al, 1996) or the region of residues 79–97 (Matous et al, 1996), being important for KIT binding. These regions, to some extent, differ from the SCF/KIT interfaces in the complex. Overall, these studies emphasize the end, or dimerization interface-distal, region of SCF being critical in KIT binding. In comparison, the SCF/KIT structure shows that the receptor-binding surface is located in the middle of SCF helices, and slightly closer to the dimerization interface than to the end. In another study, a quadruple mutant of SCF (R121N, D124N, K127D, D128K) is deficient in KIT binding (Matous et al, 1996). These residues, part of the D helix, are on the opposite side to the KIT-binding surface. This deficiency is likely due to indirect structural effect: Arg121 forms salt bridge with Asp37 and its long aliphatic region protects Met36 and Trp44 from the core; mutation of this residue may be structurally disruptive. Human SCF/KIT complex should have the same overall configuration between SCF and KIT as in mouse SCF/KIT complex in this study, given the high sequence similarity between species for both SCF and KIT (Figure 3F and Supplementary Figure S2). Although most SCF/KIT interactions are likely conserved, there is a notable difference between mouse and human complexes. In site 2, the hydrophobic interactions at the patch between mouse SCF (Ile50, Leu54, Val87 and Leu88) and mouse KIT (Pro124 and Phe126) are significantly altered in the human complex (Asp54 and Glu88 in human SCF, Ser 123 and Tyr125 in human KIT) (Figure 3F). This difference likely explains the species specificity in SCF/KIT binding. Human SCF does not detectably recognize murine KIT, but murine SCF can activate human KIT, albeit at a reduced affinity (Lev et al, 1992). This may be because Pro124 and Phe126 of mouse KIT are highly hydrophobic and require the corresponding SCF residues to be also hydrophobic. By comparison, Ser123 and Tyr125 in human KIT are less hydrophobic and can be more promiscuous at this site. Glycosylation of SCF has been shown to affect its activity (Lu et al, 1992). Three of the four potential N-linked glycosylation sites, Asn65, Asn93 and Asn120, are occupied in the CHO-cell-derived human SCF. Asn120 glycosylation does not affect receptor binding. In the SCF/KIT complex, it is located on helix D of SCF, the back of the SCF/KIT interface. By comparison, the Asn65 and Asn93 sites, which reduce SCF's biological activity by 10-fold, are much closer to the SCF/KIT interface. The Asn93 site, being immediately before the flexible loop that binds KIT-D1, may interfere with re-ordering of this loop upon receptor binding (discussed below). The side chain of SCF Asn65 is adjacent to site 3 and points to KIT-D3, suggesting that the glycan on this site could hinder the incoming KIT-D3. KIT-induced SCF conformational change The mouse SCF structure in the complex is generally similar to the free human SCF structures reported previously (Jiang et al, 2000; Zhang et al, 2000), but differences exist in several surface segments. To facilitate comparison, we also crystallized free mouse SCF and solved its structure at 2.2 Å resolution (Table I). The free mouse SCF has a significant amount of surface regions disordered or poorly ordered, including the N-terminal polypeptide chain (up to residue 11), the 92–104 loop and the 129–136 loop (Figure 4A). The flexibility of these regions was also observed in free human SCF (Jiang et al, 2000; Zhang et al, 2000). In the SCF/KIT complex, however, this segmental flexibility is changed. One significant change is associated with the N-terminal segment. In the SCF/KIT complex, all four individual mouse SCF subunits have good electron densities at the N-terminal regions. But in free human SCF or mouse SCF, this segment is disordered in every subunit; even the disulfide bond (Cys4–Cys89) appears incapable of stabilizing its conformation. The disparity between unbound and bound states suggests that the structure of N-terminal segment in bound SCF is imposed by the receptor. Of particular importance are the hydrogen bonds between KIT main-chain atoms and SCF residues 10–11, as well as the hydrogen bond between KIT Arg274 and SCF Asn6, in orientating the exterior, hydrophilic side of the SCF N-terminal segment. Upon determination of the exterior side, SCF's Pro7, Val8 and Val12 side chains, the interior side, form hydrophobic interactions with SCF's core (Leu79, Ile82, Leu86, Phe119, Ile123 and Phe126), further stabilizing the local structure (Figure 4C). Figure 4.Conformational change of SCF. (A) Superimposition of the Cα atoms of free human SCF, free mouse SCF and bound SCF as in the SCF/KIT complex, highlighting the differences at the 92–104 loop and the N-terminal segment. (B) Comparison of the 92–104 loops of free human SCF and bound mouse SCF. (C) Comparison of the N-terminal segments of free and bound mouse SCF. Download figure Download PowerPoint Another significant change is associated with the 92–104 loop of SCF. This loop is clearly visible and well defined in the SCF/KIT complex. It is, however, invisible in all but one of the four human SCF subunits (Jiang et al, 2000). In a different crystal form of human SCF, this loop is modeled, albeit with high temperature factors (Zhang et al, 2000). Comparison of the human SCF subunits containing visible 92–104 loops, solved independently in different crystal forms, shows that their 92–104 loops are roughly superimposable. This suggests that the 92–104 loop is only partially flexible and has a favorable conformation. As this loop in our free mouse SCF is disordered and does not support comparison, we compared one of the defined 92–104 loops in human SCF (Jiang et al, 2000) with that in our SCF/KIT complex (Figure 4B). The 92–104 loop appears to undergo a large conformational change upon receptor binding. In free SCF, the hydrophobic Leu98 side chain, being in the middle of this long loop, is anchored deeply to the SCF hydrophobic core (Figure 4B). This restraint limits the mobility of the 92–104 loop and brings it close to the end of the SCF B helix, adding numerous contacts between this loop and the SCF core structure. In bound SCF, however, the loop is deviated away from the core structure to form interactions with KIT-D1. The Cα atoms of residues 98–99 in bound SCF are 11–12 Å away from these atoms in free SCF. None of the residues 95–104 has contact with the rest of SCF; the loop is entirely stabilized by receptor–ligand interactions. Collectively, the conformational changes at both the N-terminal segment and the 92–104 loop of SCF are induced by KIT binding. The necessity of these changes for receptor binding adds another dimension to the specificity requirement in KIT activation. Discussion A unique RTK dimerization assembly Ligand-induced receptor dimerization is believed to be the triggering step for RTK activation (Schlessinger, 2000). Indeed, clustering of two receptors is a common feature of all known structures of RTK/ligand complexes. However, among different RTK subfamilies, the scheme used to achieve receptor clustering is widely diverse: each subfamily appears to have its own configuration of assembling receptor/ligand complex. In general, the dimerized RTK complexes can be divided into two groups: dimeric ligand-driven and monomeric ligand-driven. The SCF/KIT complex, together with VEGF/FLT1 (Wiesmann et al, 1997) and NGF/TrkA (Wiesmann et al, 1999), defines a group of RTK complexes that are dimerized primarily by the dimeric nature of their ligands, although receptor-to-receptor contacts can add to dimerization in some cases (Blechman et al, 1995; Barleon et al, 1997). This group may also include Ret, Tie and Met subfamilies, for which structures of fully associated, dimerized complexes remain to be determined. The other group of RTK complexes induced by monomeric ligands are dimerized by either ligand–receptor crosslinking, for example, Ephrin/Eph (Himanen et al, 2001), gas6/Axl (Sasaki et al, 2006) and FGF/FGFR (Mohammadi et al, 2005), or by ligand-induced and conformation-regulated receptor–receptor contact, for example, EGF/EGFR (Schlessinger, 2002). A third possible assembly, in which two receptors bind to different sites on a single ligand, has not been found for RTKs, although has been reported redundantly fo" @default.
• W2010476505 created "2016-06-24" @default.
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• W2010476505 creator A5081557873 @default.
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• W2010476505 date "2007-01-25" @default.
• W2010476505 modified "2023-10-16" @default.
• W2010476505 title "Structural basis for stem cell factor–KIT signaling and activation of class III receptor tyrosine kinases" @default.
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