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 consitutively active, and is found in metastatic HNSC carcinoma. Although there is no longer phosphorylation at these sites, it is believed that the aspartate negative charge resembles the negative phosphate that would normally cause activation, and therefore keeps the protein in its active form. ^[6] There is a third mutation at Tyr-1194 which is substituted for a phenylalanine. This is shown to point in a the pocket formed by Lys-1198 and Leu-1195 from αE. ^[7] This structure is conserved in the wild

Helices

This structure is made up of many α helial structures that move in the transformation from inactive to active kinase. Residues Leu-1062, Val-1066, and Val-1069 of αA intercilate 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-118 and Val-1121 of αC. Because of the movement of αC during activation of the kinase, αA also probably moves during activation. ^[8]

K-252a

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 Moiety. The binding of K-252a causes the c-Met to adopt an inhibitory conformation of the A-loop. 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. ^[9]

K-252a binds in the adensine pocket. It has four hydrogen bonds to the enzyme, with two of these mimicking hydrogen bonds of an adenine base. There is a hydrogen bond between the lactam nitrogen and the carbonyl oxygen of Pro-1158, and another between the lactam carbonyl oxygen and the hydrogen of the amide of Met-1160. there is another hydrogen bond between the 3' hydroxyl and carbonyl oxygen and the tyr-1230 of the A loop. ^[10]

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). ^[11]

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. ^[12]

In this conformation, the peptide chain blocks the area where peptide binding would occur. Residues 1223-1226 of the A loop bulge toward the N loop in an type II' β turn formation, with Leu-1225 at the apex of this turn. This Leucine forms a van der Waals interaction with Gly-1128. The K-252a shows a divergence from Apo-Met in residues 1228-1230. In the active confromation, Glu-1127 would form a salt bridge with Lys-1110, but in the K-252a complex, the A loop does not allow proper positioning of this residue, and so blocks nucletide binding. Asp-1235 of the chain is interacting with the amide nitrogens of Ala-1243 and Ala-1244. There is also an interaction between Phe-1234 and Arg-1208 and Trp-1249. This confomation seems to be conserved in wild type c-met inhibited structures, and so is not causes by the mutations. [13]

Some of the main conformational changes involve the A-loop, specifically residues 1228-1230. In the Apo-Met structure, the side chain of Met-1229 would pass through the six-membered ring of the indolocarbzole moiety. But, in this structure, because of the binding of K-252a, Met-1229 and Tyr-1230 move by 3.8 and 3.1 A. In order to make room for the side chain of Tyr-1230, rg-1208 moves by 8 A toward Asp-1204.^[14]

C-Terminal Docking Site

The c-terminal tail consists of two important tyrosines (1349 and 1356). Both of these become phosphorylated, and act as docking sites for many signal transducers through interaction with their SH2, MBD and PTB domains. In this structure, neither tyrosines are phosphorylated, and the protein chain for residues 1249-1352 is in an extended conformation while those of1353-1356 and 1356-1359 are β turns.^[15]