Nuclear magnetic resonance analysis of the conformational state of cancer mutant of fibroblast growth factor receptor 1 tyrosine kinase domain

Abstract

Tyrosine kinases are key enzymes that play critical roles in growth signaling, the abnormal activation of which is associated with various human cancers. Activation of tyrosine kinases is mediated by tyrosine phosphorylation in the activation‐loop, which transforms the catalytic domain to the active state conformation. Cancer mutations are supposed to transform the conformation of the catalytic domain into the active‐form independent of the phosphorylation state of the activation‐loop. Here, we report structural and biophysical analyses of cancer mutations of the tyrosine kinase domain of fibroblast growth factor receptor 1 (FGFR1). Based on the nuclear magnetic resonance analyses, phosphorylation of the activation‐loop exhibited cooperative structural transition in the activation‐loop, C‐helix and P‐loop regions, whereas cancer mutations induced structural transformation at either one or two of these regions.

Biophysical characterization of drug‐resistant mutants of fibroblast growth factor receptor 1

“…Nuclear magnetic resonance analysis of the effect of the gatekeeper mutation on the conformational state of the P-loop region of FGFR1 Upon activation by phosphorylation, FGFR1 shows substantial structural change around the nucleotidebinding loop (P-loop), the activation loop and the C- helix (Chen et al 2007). Our NMR analysis showed that the gatekeeper mutation of FGFR1, V561M, showed a locally activated structure around the P-loop (Kobashigawa et al 2016). The crystal structure of the complex formed between FGFR1 and PD173074 showed that the tert-butyl group interacted with the P-loop (Mohammadi et al 1998).…”

“…We introduced an R627E mutation, which greatly improved the solubility and stability without substantially perturbing the structure of the FGFR1 kinase domain (Kobashigawa et al . , ). Figure shows the overlay of the NMR spectra of the Met 13 CH 3 ‐labeled FGFR1 gatekeeper mutants.…”

“…Our NMR analysis showed that the V561M mutant showed a locally activated structure around the P‐loop (Kobashigawa et al . ). The crystal structure of the complex formed between FGFR1 and PD173074 showed that the tert ‐butyl group interacted with the P‐loop, and the clearance around this loop was markedly limited.…”

“…In previous studies (Kobashigawa et al . , ), we carried out NMR analysis of cancer mutants of FGFR1 and showed that the conformational state of the N546K mutant was different from that of the gatekeeper mutant, although the relevance of this difference to the mechanism of drug resistance remains unclear. Moreover, the molecular brake residues are conserved among FGFR1, FGFR2, FGFR3, FGFR4, PDGFR, CSF1R, FLK1, FLT1, FLT4, TEK and TIE (Chen et al .…”

“…In the FGFR family, the hinge residue N546 in FGFR1 has been reported to form a molecular brake and is one of the consensus mutation sites in cancers that show drug resistance (Byron et al 2013;Chell et al 2013;Sohl et al 2015). In previous studies (Kobashigawa et al 2015(Kobashigawa et al , 2016, we carried out NMR analysis of cancer mutants of FGFR1 and showed that the conformational state of the N546K mutant was different from that of the gatekeeper mutant, although the relevance of this difference to the mechanism of drug resistance remains unclear. Moreover, the molecular brake residues are conserved among FGFR1, FGFR2, FGFR3, FGFR4, PDGFR, CSF1R, FLK1, FLT1, FLT4, TEK and TIE (Chen et al 2007), and thus, mutations to this site might also include conserved drug-resistant mutants.…”