New Insights into FAK Signaling and Localization Based on Detection of a FAT Domain Folding Intermediate

Abstract

Mounting evidence suggests that the focal adhesion targeting (FAT) domain, an antiparallel four-helix bundle, exists in alternative conformations that may modulate phosphorylation, ligand binding, and the subcellular localization of focal adhesion kinase (FAK). In order to characterize the conformational dynamics of the FAT domain, we have developed a novel method for reconstructing the folding pathway of the FAT domain by using discrete molecular dynamics (DMD) simulations, with free energy constraints derived from NMR hydrogen exchange data. The DMD simulations detect a folding intermediate, in which a cooperative unfolding event causes helix 1 to lose helical character while separating from the helix bundle. The conformational dynamic features of helix 1 in the intermediate state of the FAT domain are likely to facilitate Y926 phosphorylation, yet interfere with paxillin binding. The presence of this intermediate state in vivo may promote FAK signaling via the ERK/MAPK pathway and by release of FAK from focal adhesions.

The Folding Energy Landscape of the Dimerization Domain of Escherichia coli Trp Repressor: A Joint Experimental and Theoretical Investigation

“…An example of the change in HX behavior as a function of protein concentration is shown in Figure 5B. As the protein concentration was increased, the apparent number of amide hydrogens exchanging through the fast, k f , process decreased and the number exchanging through the slow, k s , process increased for peptide 26-35, segment 31-47, and peptide [42][43][44][45][46][47][48][49][50][51][52][53][54][55][56][57][58][59][60][61]. No reliable trends were observed for the k i1 and k i2 processes for these peptides or segments.…”

Protein folding: Then and now

“…In recent studies, Gsponer et al [171] and Dixon et al [172] [165]. Besides HX methods, the site-specific structural information obtained from other NMR-based techniques such as relaxation dispersion spectroscopy [173], has also been incorporated into MD simulations to study weakly-populated folding intermediates [174].…”

Simple but predictive protein models

“…Several protein models that have striking predictive power have been developed in the last six years [15,17,18,22,25]. In most cases, predictions made using these models were not validated by experiments at the time of publication.…”

“…The phosphorylation-competent domain states of FAT are weakly populated and, therefore, are not amenable to structural determination by NMR. Using the developed method, we have identified these weakly populated intermediate states of the FAT domain and found that Y926, which belongs to helix 1 of the four-helix bundle that comprises the FAT domain, is phosphorylated in these states [22]. A similar approach has also been proposed by Vendruscolo et al [42], who has related the number of native contacts per residue to its equilibrium hydrogenexchange-protection factor and reconstructed the ensemble of protein conformations that are consistent with the experimental condition.…”

“…We describe the use of simplified protein models in conjunction with the rapid simulations methodology, discrete molecular dynamics (DMD). Despite the use of DMD in simulating polymer fluids [6,7], single homopolymers [7,8], proteins [9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25], protein aggregates [13,17,23,24,26], and gases and liquids [27,28], we believe it is significantly underutilized in the molecular-modeling field.…”