Fig 1.
Minimum free energy path (MFEP) determined by the string method in collective variables.
The left column shows four changes to the backbone that occur in the pathway: (1) motion of residues 660–663 and αC helix toward one another; (2) sliding of αEF-αF loop along activation loop; (3) rotation of the backbone of the activation loop to form a β-strand; (4) outward motion of residues 660 through 663. The right column shows the sidechain motions that accompany these backbone motions, including motions of Arg664 toward ATP, as described in the text.
Fig 2.
Key distances and potential of mean force along minimum free energy path.
(A, B, C) Distances between key atoms over the course of the string method pathway. (D) Potential of mean force as a function of collective variables used in the string method study.
Fig 3.
Results of metadynamics simulations.
(A) Free energy landscape of metadynamics simulation. The brown dotted outline corresponds to the outermost blue contour in (C). (B) Network diagram of clusters from metadynamics simulation. Each cluster represents conformations whose backbone is within a 3.0 Å RMSD, and connected clusters are whose backbones are within 3.8 Å RMSD. Purple clusters represent those in which pTyr657 faces inward toward Arg649 and Arg625. In all these clusters, Arg664 faces into the active site (more than 50% of the conformations in that cluster feature Arg664:Cζ within 8 Å of ATP:Pγ), while Leu665 and Pro666 face outward without blocking the active site (the average distance between the center of mass of the side chain carbon atoms and the Cα atom of the catalytic base Asp626 is greater than 8 Å). Blue clusters represent those in which Arg664 faces inward and Leu665 and Pro666 face outward, even though pTyr657 does not face inward. All other clusters are black. (C) Contour map indicating the density of conformations in the black, blue and purple clusters, projected onto the same axes as in (A). Each contour represents an increase in density of 0.005. (D) Representative conformation in cluster 15, the most highly populated cluster in which pTyr657 faces inward.
Fig 4.
Region of 15N HSQC of FGFR2 kinase.
Curved arrows indicate chemical shift perturbations that occur to the Arg664 shift as ATP analog is added. Black/blue spectra are of the unphosphorylated kinase, while red/purple spectra are of the phosphorylated kinase.
Fig 5.
Kinetic assays of FGFR2 kinase.
Kinetic assays were performed for wild-type FGFR2 kinase as well as R664A and R664W mutants. The control assay was performed without the presence of protein.
Fig 6.
The effect of Arg664 on the precatalytic structure.
Root-mean-square fluctuations of ATP and substrate tyrosine in simulations in which Arg664 was kept in the active site, away from the active site and pointing outward, or interacting with the αC helix.
Fig 7.
QM/MM studies of the FGFR2 kinase phosphotransfer reaction.
(A) Three structures along the phosphotransfer reaction, with their corresponding reaction coordinate values. These structures correspond roughly to a reactant, transition state, and product state. (B) Electrostatic potential-derived partial charges of the phosphate group, two Mg2+ ions, and Arg664 guanidinium protons facing the phosphate group, as a function of the reaction coordinate.