Growth factors
3D structure of the kinase domain of Activin receptor1 (Acvr1) complex with inhibitor shows the inhibitor forming various interactions with the protein including a hydrogen bonds to His residues and a water bridged hydrogen bond to the catalytic lysine[1]. Water molecule is shown as red sphere. Rabbit Phosphoglucose isomerase (autocrine motility factor) active site containing the substrate D-fructose 6-phosphate shows the substrate interacting mainly with residues of one subunit[2]. Water molecules are shown as red spheres.
The structure of the complex between M-CSF and its receptor shows that a dimer of M-CSF binds to 2 molecules of the receptor. There are two binding sites between the molecules designated site I and site II[3]. All interactions. The kinase domain of M-CSF receptor interacts with a drug-designed inhibitor via the conserved kinase DFG motif (colored in salmon) and its gatekeeper threonine residue (colored in magenta)[4].
Lapatinib is a EGFR inhibitor used in breast cancer treatment. EGFRs are overexpressed in many types of human carcinomas including lung, pancreatic, and breast cancer, and are often mutated. This overexpression leads to excessive activation of the anti-apoptotic Ras signaling cascade, resulting in uncontrolled DNA synthesis and cell proliferation. The EGFR tyrosine kinase domain is responsible for activating this Ras signaling cascade. Upon binding ligands like Epidermal Growth Factor, EGFR dimerizes and autophosphorylates several tyrosine residues at its C-terminal domain. Upon phosphorylation, EGFR undergoes a significant conformational shift, revealing an additional binding site capable of binding and activating downstream signaling proteins. Gefitinib inhibits the EGFR by binding to the ATP-binding site located within the kinase domain. Residues Lys745, Leu788, Ala743, Thr790, Gln791, Met193, Pro794, Gly796, Asp800, Ser719, Glu762, & Met766 tightly bind the inhibitor. Unable to bind ATP, EGFR is incapable of autophosphorylating its C-terminal tyrosines, and the uncontrolled cell-proliferation signal is terminated. Erlotinib inhibits the EGFR by binding to the ATP-binding site located within the kinase domain. EGFR uses residues Asp831, Lys721, Thr766, Leu820, Gly772, Phe771, Leu694, Pro770, Met769, Leu768, Gln767 & Ala719 to tightly bind the inhibitor. Unable to bind ATP, EGFR is incapable of autophosphorylating its C-terminal tyrosines, and the uncontrolled cell-proliferation signal is terminated. See also Herceptin - Mechanism of Action
Ephrins (Eph) are the membrane-bound ligands of ephrin receptors. The binding of Eph and ephrin receptors is achieved via cell-cell interaction. Eph/Eph receptor signaling regulates embryonic development, guidance of axon growth, long-term potentiation, angiogenesis and stem-cell differentiation [5]. Eph-A5 is implicated in spinal cord injury. Eph-A1 is implicated in myocardial injury and renal reperfusion injury. Ephrin-A5 receptor-binding domain complex with ephrin type A receptor 2 (PDB code 3mx0).[6] Ephrin A3 receptor with peptide substrate, nucleotide derivative and Mg+2 ion. Water molecules are shown as red spheres. Nucleotide derivative and Mg+2 ion binding site. Peptide substrate binding site (3fxx).[7] The extracellular part of Eph receptors includes the N-terminal ephrin (Ligand)-binding domain (LBD), a cysteine-rich domain (CRD), and 2 fibronectin Type-III Repeats (FN3). EphA binds ephrins with its LBD. Most ephrins have a similar rigid structure which includes four loops, AB, CD, FG, & GH. The LBD of EphA4 is said to be a “structural chameleon” able bind both A and B class ephrins. This explains why Ephrin Type-A receptors exhibit cross-class reactivity. The overall structure of the EphA4 LBD includes four important loops, the BC, DE, GH, & JK loops. EphA4 binds the GH loop of the ephrin ligand within a deep pocket created by the EphA4 DE and JK loops. It is these loops, DE and JK, which undergo the greatest conformational shifts when binding either EphrinA2 or EphrinB2. When binding EphrinA2, EphA4-Arg 162 forms a hydrogen bond with EphrinA2-Leu 138, while EphA4-Met 164 and EphA4-Leu 166 participate in hydrophobic interactions with EphrinA2-Leu 138 and EphrinA2-PHe 136. Although EphA4 binds EphrinB2 in the same binding pocket, the local interactions are significantly different. Most notably, the α-helix present in the EphA4-EphrinA2 JK loop is disrupted in the EphA4-EphrinB2 structure. This is due to the steric clash that would occur between EphrinB2-Trp 122 and EphA4 Met 164. Instead, EphA4-Arg 162 and EphrinB2-Trp 122 form hydrophobic stacking interactions among other interactions which stabilize the receptor-ligand complex. A morph of the movements EphA4 undergoes to bind EphrinA2 and EphrinB2 can be seen here. Eph-Ephrin complexes form two unique heterotetrameric assemblies consisting of distinct EphA2-EphA2 interfaces. The 1st tetrameric form is generated by Eph-Eph interactions only within the LBD. The 2nd tetrameric form involves complex interactions in the LBD and in the region near the CRD.[8] These two heterotetramers generate a continuous Eph-ephrin assembly when combined (Alternative Coloring). The proximity of kinase domains in an eph-ephrin tetramer, favors transphosphorylation of tyrosines in the cytoplasmic domains. Phosphorylation promotes kinase activity by orienting the activation segment of the kinase domain in a way that favors subsrate binding and subsequent signaling. Erythropoietin (EPO) is a glycoprotein composed of only Alpha Helices. The sulfur of the cysteine residues links to form disulfide bonds. These disulfide bonds help keep EPO's structure. Helix A is connected to Helix D by Cys7 and Cys161, while Helix A and Helix B are connected by Cys29 and Cys33 . EPO’s structure was determined in 1993. It is made up of four alpha helixes. EPO is produced mainly in the kidney, but further research has shown the brain and liver still produce small amounts. The EPO receptor of the blood marrow is part of the hematipoietic cytokine family. This receptor has a single transmembrane domain, that forms a homodimer complex until it is activated by the binding of EPO. This receptor is 484 amino acids long and weigh 52.6 kDa. Once the homodimer is formed after the binding, autophosphorlation of the Jak2 kinases, which activates other cellular processes. This transmembrane receptor has two extracellular domains. This receptor has two disulfide bonds that are formed from 4 cystine residues, Cys67 and Cys83 and Cys28 and Cys38. The intracellular domain of this receptor does not possess any enzymatic activity like other receptors. When EPO comes in contact with the extracellular domains form a ligand bond. The extracellular sinding site 1 and Binding site 2 are composed of D1 and D2. When EPO binds, all loops on D1 and D2 of binding site one form a bind with EPO. However loop 4 of D1 on binding site 2 does not participate in the binding of EPO [9]. After the biniding of EPO, 8 tyrosine residues are phosphoralated which activates the Jak2 kinase. This kinase helps regulate the transcription of different genes and expression of other proteins. Human erythropoietin receptor with erythropoietin (PDB code 1cn4).[10]
The heparin binds in the dimer interface of the rat fibroblast growth factor 1[11]. Water molecules are shown as red spheres. FGFR consist of an extracellular ligand-binding domain (LBD), transmembrane helix domain and cytoplasmic tyrosine kinase activity domain (TKD) with phosphorylated tyrosine designated PTR. FGFR LBD contains 3 immunoglobulin-like domains D1, D2 and D3. Human fibroblast growth factor receptor 1 ligand-binding domain modules D2 and D3 with 2 molecules of fibroblast growth factor 1 (PDB code 1evt). For more details see Group:MUZIC:Myostatin. See also Bone morphogenetic protein.
The A loop of the wt receptor contains 2 tyrosines at position 1234 and 1235. When these 2 residues become phosphorylated, the kinase can become active. A unique part of the c-met structure is the pair of tyrosine residues (1349 and 1356). These tyrosines are necessary for normal c-met signaling. When these 2 tyrosines were substituted with with phenylalanine in mice, the mice had an embryonically lethal phenotype and defects were found in placenta, liver, muscles and nerves. In a wt c-met, these sites will become phosphorylated and act as docking sites for many different transducers and adapters. Upon phosphorylation, these tyrosines can bind with Src homology 2 (SH2) domains and phophotyrosine-binding (PTB), and therefore bind many effectors that will cause downstream effects such as cell proliferation, scattering and inhibition of apoptosis. This receptor follows the typical structure of a protein kinase, with a bilobal structure. The N-terminal contains β-sheets and is linked through a hinge to the C lobe, which is full of α helices. This particular kinase domain is very similar to the domains of the insulin receptor kinase and fibroblast growth factor receptor kinase.[12] This structure is made up of many α-helices that move in the transformation from inactive to active kinase. Some of these helices are conserved in many different tyrosine kinases. C-met does show a divergence from other tyrosine kinases (such as IRK and FGFRK) in the helix formed at the N-terminus, before the core kinase domain, in residues 1060-1069. The αA is in contact with αC and so causes αC to be in a slightly different orientation than in FGFRK and IRK. Residues Leu-1062, Val-1066, and Val-1069 of αA intercalate with with residues Leu-1125 and Ile-1129 of αC. There is another interaction between the residues Ile-1053, Leu-1055 and Leu-1058 of αA and Ile-1118 and Val-1121 of αC. Because of the movement of αC during activation of the kinase, it is an assumption that αA is also part of the kinase activation upon ligand binding. The particular structure of the hepatocyte growth factor tyrosine kinase domain is one harboring a human cancer mutation. The 2 tyr1234 and tyr1235 are replaced by a phenylalanine and aspartate, respectively. This mutation normally causes the receptor to be constitutively active, and is found in HNSC (Head, Neck squamous cell) carcinoma. Although there is no longer phosphorylation at these sites, it is believed that the negative charge of the aspartate resembles the negative phosphate that would normally cause activation, and therefore keeps the protein in its active form. [13] There is a 3rd mutation at Tyr-1194 which is substituted for a phenylalanine. This is shown to point into the pocket formed by Lys-1198 and Leu-1195 from αE. [14] This structure is conserved in the wild type protein, suggesting that the mutation at residue 1149 is not changing the structure at this position. K-252a is a staurosporine analog. Staurosporine is an inhibitor of many Ser/Thr Kinases, and has been shown to also inhibit c-Met activation by inhibiting its autophosphorylation. The structures of K-252a and staurosporine are very similar, with the main difference being that K-252a has a furanose instead of a pyranose structure. The binding of K-252a causes the c-Met to adopt an inhibitory conformation of the A-loop, specifically with residues 1231-1244. This segment blocks the place where the substrate tyrosine side chain would bind, if the protein were in an active conformation. Residues 1223-1230 also enhance this inhibitory conformation, as they constrain αC into a conformation that does not allow the catalytic placement of Glu-1127 keeping αC in an inactive conformation. In an active kinase, Glu-1127 would form a salt bridge with Lys-1110. Residues 1229-1230 pass through the triphosphate subsite of bound ATP blocking ATP binding. The K-252a itself binds in the adenosine pocket, therefore also inhibiting the binding of ATP. The binding of K-252a is very favorable. This is probably due to polar interactions as well as a change in conformation upon binding. There is a concerted conformational change in the complex upon K-252a binding. One of these changes involves the A-loop, specifically residues 1228-1230. In the Apo-Met structure, the side chain of Met-1229 would pass through the ring of the inhibitor, and so, in order to make room for K-252a, the segment must move, with residues 1229 and 1230 moving by 3-4 Å. In order to make room for the side chain of Tyr-1230, Arg-1208 moves by 8 Å toward Asp-1204. Arg-1208, which in the uninhibited complex would stack with tyr-1230, now stacks with Phe-1234. K-252a binds in the adenosine pocket. It has 4 hydrogen bonds to the enzyme, with two of these mimicking hydrogen bonds of an adenine base. There is a hydrogen bond between the K-252a nitrogen and the carbonyl oxygen of Pro-1158, and another between the K252-a carbonyl oxygen and the hydrogen of the amide of Met-1160. There are 2 more hydrogen bonds between the 3' hydroxyl and carbonyl oxygen and the tyr-1230 of the A loop. There are also many hydrophobic interactions between the interface of the enzyme and K-252a. The residues involved in this are Ile-1084, Gly-1085, Phe-1089, Val-1092, Ala-1108, Lys-1110, and Leu-1140 (N lobe); Leu-1157, Pro-1158, Tyr-1159, and Met-1160 (hinge region); and Met-1211, Ala-1226, Asp-1228, Met-1229, and Tyr-1230 (C lobe). Met-1229, Met-1211 and Met-1160 all make up the platform for the indolocarbazole plane as they are all within van der Waals distance of it. In c-Met, there are 2 tyrosines located in the C-terminal tail sequence, which, upon phosphorylation, act as the docking sites for many signal transducers. These tyrosines correspond to residues 1349 and 1356. Both of these sites interact with SH2, MBD and PTD domains of signal transducers. The residues 1349-1352 form an extended conformation, which is seen in other phosphopeptides that bind to SH2 domains. Residues 1353-1356 form a type I β turn, which is similar to sequences that bind to Shc-PTB domians. Whether binding to SH2 domains or PTB domains, upon binding, these motifs would move to avoid clashes with the C lobe. The 3rd binding motif is found in residues 1356-1359, which form a type II β turn, and is similar to pohsphopeptides that bind Grb2. When comparing the unphosphorylated conformation of the motif to one that is phosphorylated, and bound to the Grb2 complex, there is a peptide flip between the bind of Val-1357 and Asn-1358. This suggests that when Grb2 docks onto c-Met, there is a change in orientation of this motif. These 3 binding motifs of the mutated structure are very similar to binding motifs that would be recognized by their binding partners, implying that the C-terminal supersite of this structure is very similar to that of an active c-met.
Memory-Enhancement by Traditional Chinese Medicine? [15] IGF receptor (IGF-IR, in darkmagenta) activation is critical for IGF-I to elicit desirable cognitive functions. Molecular dynamics simulation revealed that the Traditional Chinese medicine (TCM) ligands were secured at the opening of the IGF-IR binding site for the duration of the MD. 3-(2-carboxyphenyl)-4(3H)-quinazolinone was stabilized by Asp1056, (+)-N-methyllaurotetanine was stabilized by Leu975 and Asp1056, and (+)-1(R)-Coclaurine was stabilized by Leu975 and Gly1055 (key residues are colored in yellow). Interleukin (IL) is a cytokine which functions in the immune system. IL families are denoted by numbers[16]. The complex between NT3 and p75 neurotrophin receptor (p75NTR) shows a homodimer of NT3 with two symmetrically arranged p75NTR molecules. There are 3 sites of interactions between NT3 and p75NTR - site 1, site 2 and site 3. TrkA. Trk stands for Topomyosin-Related Kinase. TrkA ligand - nerve growth factor activates the receptor by stabilizing homodimer formation which initiates transautophosphorylation. Structure of Nerve Growth Factor Complexed with the Extracellular Domain of TrkA. An Arg residue, conserved in all neutrophins, forms the most important binding determinant between TrkA and its ligand - nerve growth factor - which forms the active homodimer of the receptor. All interactions between TrkA chain A and NGF. TRK-A contains an extracellular ligand binding domain (LBD), a transmembrane helix and an intracellular region which contains the kinase domain. The kinase domain (4yne) contains the tripeptide DFG which flips out in TRK-A inactivated form. Inhibitor binding site (4yne). The structure of the complex of TRK-A with the phenylpyrrolidine derivative shows the inhibitor forming hydrogen bonds to Met620 and Lys572 residues and π-π interactions of it with Phe617 and Phe 698. The complex between TRK-A and the nerve growth factor (2ifg) is a 2:2 dimer. The C-terminal immunoglobulin-like domain interacts with the NGF. The extracellular domain of TRK-A contains 3 Leu-rich regions flanked by Cys-rich regions (in yellow), 2 immunoglobulin-like domains and the nerve growth factor (NGF) binding domain. Structure of the TrkB-d5:NT-4/5 Complex, comprising one homodimer of NT-4/5 bound to two monomers of TrkB-d5. TrkB and neutrotrophin-4/5 interact via a specificity interaction site and via a conserved interaction site.
The biological assembly of human tumor necrosis factor is homotetramer (PDB entry 2az5). Inhibitor binding site. TRAIL-R2 is called DR5. TRAIL trimer residues complex with death receptor-5 extracellular domain (1d0g). The extracellular domain of TNFR contains 2 to 6 cysteine-rich domains (CRD). The CRD domains are ca. 40 amino-acid long and contain 4-6 cysteine residues. The CRDs are involved in binding of TNF[17]. Mg coordination site. Water molecules are shown as red spheres.
VEGF-A is a homodimer composed of two 23 kDa subunits. VEGF-A exists in a number of different isoforms following alternative splicing of its precursor mRNA [18]. In humans, 6 variants have been found: VEGF-A-121, VEGF-A-145, VEGF-A-165, VEGF-A-183, VEGF-A-189, and VEGF-A-206, with VEGF-A-165 the most abundantly expressed. All VEGF-A isoforms bind to VEGFR-1 and -2. The amino acids determined to be critical to binding to VEGFR-1 are D63, L66, and E67. VEGF-A binding by VEGFR-1 leads to cellular proliferation, migration, and increased cellular permeability resulting in vasculogenesis and angiogenesis. Those residues critical to binding to VEGFR-2 are I43, I46, Q79, I83, K84 and P85.[19] Binding of VEGF-A to VEGFR-2 results in similar Vasculogenesis and angiogenesis, but also lymphangiogenesis in embryos. The remainder of the binding pocket is formed by D34, S50, E64, and F36. It is upon binding of VEGFR by VEGF that the subsequent signal cascade is initiated leading to angiogenesis, etc.[20] VEGF-E consists of a homodimer that is covalently linked by two intermolecular disulfide bonds between Cys51 and Cys 60. Each monomer contains a central antiparallel beta sheet, with the canonical cysteine knot found in other VEGFs. [21] The knot consists of an eight residue ring formed by the backbone of residues 57-61 and 102-104 and intramolecular disulfide bridges Cys57-Cys102 and Cys61-Cys104, and a third bridge, Cys26-Cys68, that passes perpendicularly through the ring. Each VEGF-E monomer contains an amino terminal alpha helix and three solvent accessible loop regions, L2, L1 and L3 . are able to form a complex hydrogen bond network as well as extensive hydrophobic contacts with VEGFR making these loops ideal receptor specificity determinants. Residues: P34, S36, T43, P50, R46, D63, E64, and E67 make up the VEGF-E binding pocket and are critical for binding to VEGFR-2 as determined by alanine mutagenesis.[22] Further, the salt bridge between R46 and E64 is believed to be the source of VEGF-E’s VEGFR-2 specificity by preventing binding to VEGFR-1. [23] Vascular Endothelial Growth Factor Receptors (VEGFRs) are tyrosine kinase receptors responsible for binding with VEGF to initiate signal cascades that stimulate angiogenesis among other effects. The tyrosine kinase domain of VEGFR-2 is separated into 2 segments with a 70 amino acid long kinase insert region. Upon binding VEGFA and subsequent dimerization, VEGFR-2 is autophosphoryalted at the carboxy terminal tail and kinase insert region, 6 tyrosine residues of VEGFR2 are autophosphorylated. Auto-phosphorylation of residues 1054 and 1059 within the activation loop of VEGFR2 leads to increased kinase activity. Anti-tumor inhibitor binding site (3c7q). See also Bevacizumab. See also:
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