Receptor tyrosine kinases (RTKs) are part of the larger family of protein tyrosine kinases. They are the high-affinity cell surface receptors for many polypeptide growth factors, cytokines, and hormones. Approximately 20 different RTK classes have been identified.[1]
Lapatinib is a EGFR inhibitor used in breast cancer treatment. ERBB2 is necessary for heart cells proliferation and regeneration[2].
Epidermal Growth Factor Receptors 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 signalling cascade, resulting in uncontrolled DNA synthesis and cell proliferation. Studies have revealed that the 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.[3] Erlotinib inhibits the EGFR tyrosine kinase by located within the kinase domain. Residues Met 774, Leu 825, Val 707, Thr 835, Asp 836, Phe 837, Thr 771, Lys 726, Ala 724, & Leu 769 tightly bind the inhibitor in place. Unable to bind ATP, EGFR is incapable of autophosphorylating its C-terminal tyrosines, and the uncontrolled cell-proliferation signal is terminated.[4]
Gefitinib inhibits the EGFR by located within the kinase domain. Residues Lys 745, Leu 788, Ala 743, Thr 790, Gln 791, Met 193, Pro 794, Gly 796, Asp 800, Ser 719, Glu 762, & Met 766 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.[5]
Erlotinib inhibits the EGFR by located within the kinase domain. EGFR uses residues Asp 831, Lys 721, Thr 766, Leu 820, Gly 772, Phe 771, Leu 694, Pro 770, Met 769, Leu 768, Gln 767 & Ala 719 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.[6]
A Possible Strategy against Head and Neck Cancer: In Silico. Investigation of Three-in-One inhibitors[7]
(in darkmagenta), which is an enzyme with decarboxylation reaction of uroporphyrinogen III to (in salmon), is overexpressed in tumor tissues and has potential to sensitize cancer patients to radiotherapy. Moreover, (in magenta) and (in deeppink), which are tyrosine kinase receptors in the erbB family, are also overexpressed in tumor tissues and have been indicated as the important targets of therapy for cancer. In this research, we discuss the possible conformation for an inhibitor against three target proteins, UROD, EGFR, and Her2.
Virtual screening of the UROD (PDB ID: 1r3y), EGFR (PDB ID: 3poz), and Her2 (PDB ID: 3pp0) was conducted using the binding site defined by the volume and location of the co-crystallized compounds in each crystal structure. In silico results indicate the traditional Chinese medicine (TCM) compounds had high binding affinity with all 3 target proteins. For (in magenta), the top 3 compounds, (in yellow), (in cyan), and (in orange), formed hydrogen bonds with the residues, Arg803, Lys913 and some other residues in the binding domain. The docking poses of (in deeppink) with (in yellow), (in cyan<), and (in orange), exhibited hydrogen bonds between ligands and the residues in the binding site. For protein (in darkmagenta), (in yellow), (in cyan), and (in orange), have hydrogen bonds with the 3 important binding and catalytic residues Arg37, Arg41, Tyr164, and the residue His220. The 3 TCM compounds hint towards a probable molecule backbone which might be used to evolve drug-like compounds against EGFR, Her2, and UROD, and have potential application against head and neck cancer.
See also Herceptin - Mechanism of Action
Insulin receptor
The insulin receptor (IR) is a dimer of made of 2 and 2 . Within the extracellular ectodomain, there are 4 potential that can interact with insulin on the extracellular side of the membrane. The full extracellular and intracellular components of the IR have only been imaged in separate sections but a larger picture of how these sections combine to initiate downstream tyrosine autophosphorylation is emerging.
The make up the extracellular domain of the IR and are the sites of insulin binding. The α-subunit is comprised of 2 Leucine rich domains (L1 & L2), a Cysteine rich domain (CR), and a . α-CT has a unique position that allows it to reach across the receptor and interact with the insulin at the binding site on the opposing side of the receptor. The α-subunits are held together by a disulfide bond between on each α-subunit. The disulfide bonds are important to the overall stabilization of the molecule as it binds to insulin. Two types of insulin binding sites are present in the α-subunits, and . The sites are in pairs because of the heterodimeric nature of the receptor. Due to structural differences, as well as greater surface area and accessibility, binding sites 1 and 1' have much higher affinity for insulin binding than sites 2 and 2'. Insulin can also bind at sites 2 and 2', but the location on the back of the beta sheet of the FnIII-1 domain and lack of surface area decreases the likelihood of their binding site becoming occupied as quickly.
The spans from the extracellular domain across the TM region and into the intracellular portion of the IR. The β-subunit is composed of part of fibronectin domain III-2 and all of Fibronectin domain III-3. The β-subunit's FnIII-3 domain has links through the TM region into the intracellular part of the membrane. Cryo-EM provided clear representations of the FnIII-2 and FnIII-3 domains, but are missing the TM and intracellular regions. Although the FnIII-3 domain is connected to the TM and intracellular regions, the active conformation likely extends all the way to the tyrosine kinase domain region (4xlv).
The α and β subunits of the extracellular domains fold over one another and form a when the IR is inactivated. Upon activation, the extracellular domain undergoes a conformational change and forms a . An additional component to the ectodomain is . Each of the dimers has an α-CT helix. The α-CT helix is a single α-helix that plays an important role in insulin binding and stabilization of the "T" shape activated conformation. α-CT interacts with a leucine-rich region of the α subunit and a fibronectin type III region of the β subunit to form the insulin binding sites known as .
The structure of the extracellular domain is stabilized through multiple disulfide bonds. The α-subunits are linked through 2 disulfide bonds, with the main one being between of 2 adjacent α-subunits. of both α-subunits are also held together with a disulfide bond. The α-subunit is also attached to the β-subunit by a disulfide bond between the . The IR unit has 4 separate sites for the insulin binding. There are 2 pairs of 2 identical binding sites referred to as and .
The insulin molecules bind to these sites mostly through hydrophobic interactions, with some of the most crucial residues at sites 1 and 1' being between of the IR FnIII-1 domain. Despite some of the residues included being charged, the main interactions are still hydrophobic in this binding site. For example, due to arginine carrying its positive charge at the end of the side chain, to allow the hydrophobic part of the side chain to interact with the other hydrophobic residues. The α-subunits also have significant that help maintain a compact binging site. At sites 2 and 2', the major residues contributing to these hydrophobic interactions are the .
Sites 1 and 1' have a higher binding affinity than sites 2 and 2' due to site 1 having a larger surface area (706 Å2) exposed for insulin to bind to compared to site 2 (394 Å2). The binding interactions of the insulin molecules in sites 1 and 1' are facilitated by hydrophobic residues of an of the IR. The insulin molecules in sites 2 and 2' primarily interact with the residues that comprise some of the of the IR.
At , a occurs between 3 critical parts of the α subunits of the IR. The entire interface of the tripartite interaction involves many residues that are involved with intra-protomer ionic and hydrogen bonding at the binding site. The α-CT chain and the FnIII-1 domain region come into close proximity during the conformational change of the IR and their interaction involves the following residues: and the . This duo then interacts with the L1 region, specifically ARG14, creating an ideal for the insulin ligand. The FnIII-1 and α-CT are interacting from the 2 different α-subunits, which displays a "cross linking" scenario where the domains of the heterodimer can intertwine with each other. The tripartite interaction between α-CT, the FnIII-1 domain, and the L1 region is important because it allows for a strong interaction between 2 subunits of the IR that maintains and stabilizes the T-shape activation state for the rest of the downstream signaling to occur.
It has been hypothesized that activation of the IR can change based on the concentration of insulin. These structures of the IR have demonstrated that at least 3 insulin molecules have to bind to the IR to induce the active conformation, as binding of 2 insulin molecules is insufficient to induce a full conformational change. However, this conclusion has not yet been widely confirmed. In low concentrations of insulin, the IR may not require binding of 3 insulin molecules in order to exhibit activation. Rather, the level of activity will change in accordance to the availability of insulin. When higher concentrations of insulin are present, the conformational difference between the 2-insulin-bound state and the 3-insulin-bound state is drastic as the IR transitions from the inactive to the active . However, in conditions of low insulin availability, the 2-insulin-bound state may be enough to induce partial activation of the receptor.
The conformational change between the inverted, inactive and the active of the IR is induced by insulin binding. The T shape conformation is well observed in the α subunit. It is horizontally composed of L1, CR (including the ), and L2 domains and vertically composed of the FnIII-1, 2, and 3 domains. The proper conformational change of the ectodomain of the IR is crucial for transmitting the signal into the cell. The movements extracellularly cause the 2 receptor tyrosine kinase domains intracellularly to become close enough to each other to autophosphorylate. This autophosphorylation activates the tyrosine kinase domain, initiating intracellular insulin signaling cascades.
When an insulin molecule binds to site 1 of the α-subunit, the respective protomer is recruited and a slight inward movement of the of the β-subunit is initiated. This is accomplished by the formation of several salt bridges, specifically between . Binding of insulin to both protomers establishes a full activation of the IR. This activation is demonstrated through the inward movement of both protomers. This motion has been referred to as a "hinge" motion as both protomers "swing" in towards one another. The conformational change and "hinge motion" between the inactive and active forms of an IR protomer. Upon insulin binding, the β subunits of the inactive form, shown in blue, are "swung" inward to the active form, shown in orange. When the receptor is in an , the FnIII-3 domains are separated by about 120Å. This distance prevents the initiation of autophosphorylation and downstream signaling by the tyrosine kinase domains on the intracellular side of the receptor. Upon the binding of insulin to multiple binding sites, this conformation change brings the FnIII-3 domains within 40Å of each other to induce the conformation. As the fibronectin type III domains of the β subunit swing inward, the α subunits also undergo a conformational change upon insulin binding. As insulin binds to site 1, the leucine-rich region of one protomer interacts with α-CT and the FNIII-1 domains of the other protomer to form the binding site. For the tripartite interface to form, the α subunits of each protomer must undergo a "folding" motion.
(3bu5).
TK domain of IR contains an activation loop and a catalytic loop and . The bound IR substrate 2 peptide tyrosine is the phosphorylated residue[8].
In type 2 diabetes, the TK domain is thought to be down-regulated through phosphorylation of by protein kinase C[9].
. Water molecules shown as red spheres.
Student Projects for UMass Chemistry 423 Spring 2012-1
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. within the activation loop of VEGFR2 leads to increased kinase activity. (3c7q).
See also Bevacizumab.
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.
(PDB code 1evt).
The A loop of the wild type receptor contains two tyrosines at position 1234 and 1235. When these two residues become phosphorylated, the kinase can become active. A unique part of the c-met structure is the pair of . Studies have shown that these tyrosines are necessary for normal c-met signaling. When these two 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. [10] In a wild type c-met, these sites will become phosphorylated and act as docking sites for many different transducers and adapters. [11] 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. [12]
This receptor follows the typical structure of a protein kinase, with a bilobal structure. The N-terminal contains 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.[13]
Helices
This structure is made up of many α helical structures 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 . [14] 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 with with residues Leu-1125 and Ile-1129 of αC. There is another 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. [15]
Mutation
This particular structure of the hepatocyte growth factor tyrosine kinase domain is one harboring a human cancer mutation. The two
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. [16] There is a third mutation at Tyr-1194 which is substituted for a . This is shown to point into the formed by Lys-1198 and Leu-1195 from αE. [17] 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 . This segment blocks the place where the substrate tyrosine side chain would bind, if the protein were in an active conformation. Residues
also enhance this inhibitory conformation, as they constrain αC into a conformation that does not allow the catalytic placement of 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 (enthalpy change of -17.9 kcal/mol). This is probably due to polar interactions as well as a change in conformation upon binding. [18]
There is a concerted conformational change in the complex upon K-252a binding. One of these changes involves the A-loop, specifically residues . 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, moves by 8 Å toward . Arg-1208, which in the uninhibited complex would stack with tyr-1230, now stacks with
[19]
K-252a binds in the adenosine pocket. It has four hydrogen bonds to the enzyme, with 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 two more hydrogen bonds between the 3' hydroxyl and carbonyl oxygen and the of the A loop. [20]
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 (); Leu-1157, Pro-1158, Tyr-1159, and Met-1160 (); and Met-1211, Ala-1226, Asp-1228, Met-1229, and Tyr-1230 (). [21]
Met-1229, Met-1211 and Met-1160 all make up the for the indolocarbazole plane as they are all within van der waals distance of it. [22]
C-Terminal Docking Site
In c-Met, there are two 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 . Both of these sites interact with SH2, MBD and PTD domains of signal transducers. The residues form an extended conformation, which is seen in other phosphopeptides that bind to SH2 domains. Residues
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 third binding motif is found in residues , 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 . This suggests that when Grb2 docks onto c-Met, there is a change in orientation of this motif. These three 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. [23]
See also Receptor
