利用X-ray單晶繞射法分析Focal adhesion kinase之N端FERM domain的結構

Abstract

Focal adhesion kinase (FAK) is a protein tyrosine kinase that can influence the structure of cytoskeleton, cell adhesion sites, and membrane protrusions to regulate cell movement. The N-terminal domain of FAK (FAKN), which is homologous to the so-called FERM domain, has been shown to mediate both protein-protein and protein-membrane interactions. The FAKN structure reveals a compact clovercleaf-like three-lobed architecture with the three subdomains (termed F1, F2, and F3) intimately associate with one another. The F1 subdomain resembles ubiquitin-like fold, the F2 subdomain has a protein fold similar to that of acyl coenzyme A (acyl-CoA) binding protein, and structural similarity has been detected between the F3 subdomain and the PH/PTB/EVH1 domains. The hepatocyte growth factor (HGF) receptor is encoded by the proto-oncogene c-met, which can be uncongenially activated through a chormosomal rearrangement that creates a hybrid oncogene tpr-met. The resulting fusion protein Tpr-Met has been found in several human cancers. Recent studies autophosphorylated Tpr-Met interacts with F2 subdomain of FAKN domain and leads to the phosphorylation of its Tyr-5 and continuous activation of FAK, in turn leading to formation of tumors and increased metastasis. To further examine how the phosphorylation of Tpr-Met on its Tyr-482 and Tyr-489 enables its interaction with F2 subdomain which contains a patch of basic residues (KAKTLRK) , we designed a synthetic phosphopeptide based on the sequence flanking Tyr-482 and Tyr-489 of Tpr-Met for a co-crystallization study with FAKN. Moreover, although the structure of the FAKN domain (31-399) have been published, attempts to understand the mechanism of tumors formation by activated FAK have been hampered by the lack of a more complete FAKN structure. To provide structural information for the N-terminal residues of FAKN, we have crystallize the full-length FAKN (1-363) in a crystal form different from the ones published previously. And the crystal structure has been determined at 2.7 A. However, we did not observe any electron density for the N-terminal residues, likely due to pronounced structural flexibility of this region. Minor structural differences between the F3 subdomain of the FAKN1-363 and the published structures were recorded, which may result from differences in the crystal packing of FAKNs in different crystal forms. We observed a non-crystallographic 2-fold FERM-FERM interaction in the crystal lattice, the functional significance of this interface remains to be explored. No electron density can be recognized for phosphopeptide in the co-crystal structure, which may indicate a heterogenous mode of binding.