Changes in the free-energy landscape of p38α MAP kinase through its canonical activation and binding events as studied by enhanced molecular dynamics simulations

Abstract

p38α is a Ser/Thr protein kinase involved in a variety of cellular processes and pathological conditions, which makes it a promising pharmacological target. Although the activity of the enzyme is highly regulated, its molecular mechanism of activation remains largely unexplained, even after decades of research. By using state-of-the-art molecular dynamics simulations, we decipher the key elements of the complex molecular mechanism refined by evolution to allow for a fine tuning of p38α kinase activity. Our study describes for the first time the molecular effects of different regulators of the enzymatic activity, and provides an integrative picture of the activation mechanism that explains the seemingly contradictory X-ray and NMR data.

Keywords: allostery; biophysics; human; kinases; metadynamics; molecular dynamics; p38 kinase; phosphorylation; structural biology.

Figure 1.. Structural models of (a) the inactive (PDB ID: 3S3I), and (b) the active, dually phosphorylated, p38α (PDB ID: 3PY3).

Main structural elements are colored as follows: A-loop in purple (inactive) and green (active), α_C helix in red, L16 loop in slate, P-loop in cyan, hinge in yellow, and MKI in orange. Key residues are shown as sticks and labeled appropriately. The ATP-binding site is indicated by the grey filled ellipse, while the α_E helix, and CD and ED sites by the green dashed ellipse. DOI: http://dx.doi.org/10.7554/eLife.22175.002

Figure 2.. Free-energy surface for p38α (apo unphosphorylated, apo dually phosphorylated, dually phosphorylated with bound ATP-Mg²⁺, and dually phosphorylated with bound ATP-Mg²⁺ and MK2 peptide) as a function of CV₁ and CV₂ which indicate the distance from the reference inactive and active structures, respectively.

The contour lines are drawn every two kcal mol⁻¹. Representative structures for the selected minima are shown and colored as follows: A-loop in purple, α_C helix in red, L16 loop in slate, P-loop in cyan, and hinge in yellow. Key residues are shown as sticks and labeled appropriately. Part of the FES of the phosphorylated p38α that is populated by structures which are likely to bind ATP are highlighted with a grey rectangle (upper right panel). DOI: http://dx.doi.org/10.7554/eLife.22175.004

Figure 2—figure supplement 1.. Free-energy surface for p38α (apo unphosphorylated, apo dually phosphorylated, and dually phosphorylated with bound ATP-Mg²⁺, and dually phosphorylated with bound ATP-Mg²⁺ and MK2 peptide) as a function of CV₁ and CV₂ which indicate the distance from the reference inactive and active structures, respectively.

The contour lines are drawn every two kcal mol⁻¹. Representative structures for the selected minima are shown and colored as follows: activation loop in purple, α_C helix in red, L16 loop in slate, P-loop in cyan, and hinge in yellow. Key residues are shown as sticks and labeled appropriately. Part of the FES of the phosphorylated p38α that is populated by structures which are likely to bind ATP are highlighted with a grey rectangle (upper right panel). DOI: http://dx.doi.org/10.7554/eLife.22175.005

Figure 2—figure supplement 2.. Representative structures of selected free-energy minima calculated for p38α in the apo unphosphorylated state located at: (a) (CV₁, CV₂) = (0.2, 0.7), (b) (CV₁, CV₂) = (0.2, 0.6), (c) (CV₁, CV₂) = (0.1, 0.8), and d) (CV₁, CV₂) = (0.3, 0.35).

The elements are colored as follows: activation loop in purple, α_C helix in red, L16 loop in slate, P-loop in cyan, hinge in yellow, and MKI in orange. Key residues are shown as sticks and labeled appropriately. DOI: http://dx.doi.org/10.7554/eLife.22175.006

Figure 2—figure supplement 3.. Representative structures of selected free-energy minima calculated for p38α in the apo phosphorylated state located at: (a) (CV₁, CV₂) = (0.3, 0.55), (b) (CV₁, CV₂) = (0.45, 0.4), (c) (CV₁, CV₂) = (0.15, 0.75), and d) (CV₁, CV₂) = (0.6, 0.2).

The elements are colored as follows: activation loop in purple, α_C helix in red, L16 loop in slate, P-loop in cyan, hinge in yellow, and MKI in orange. Key residues are shown as sticks and labeled appropriately. DOI: http://dx.doi.org/10.7554/eLife.22175.007

Figure 2—figure supplement 4.. Histogram of the minimal distances between residues R49 and D112 calculated for the structures of the most populated cluster in the global minimum of the dually phosphorylated p38α.

The average value is shown as a red dashed line, while the bin size is 0.2 Å. DOI: http://dx.doi.org/10.7554/eLife.22175.008

Figure 2—figure supplement 5.. Representative structures of selected free-energy minima calculated for p38α in the phosphorylated state with bound ATP-Mg²⁺ located at: (a) (CV₁, CV₂) = (0.5, 0.4), (b) (CV₁, CV₂) = (0.6, 0.1), and (c) (CV₁, CV₂) = (0.25, 0.5).

The elements are colored as follows: activation loop in purple, α_C helix in red, L16 loop in slate, P-loop in cyan, hinge in yellow, and MKI in orange. Key residues are shown as sticks and labeled appropriately. DOI: http://dx.doi.org/10.7554/eLife.22175.009

Figure 2—figure supplement 6.. Representative structures of selected free-energy minima calculated for p38α in the phosphorylated state with bound ATP-Mg²⁺ and MK2 peptide located at: (a) (CV₁, CV₂) = (0.7, 0.1), and (b) (CV₁, CV₂) = (0.4, 0.4).

The elements are colored as follows: activation loop in purple, α_C helix in red, L16 loop in slate, P-loop in cyan, hinge in yellow, and MKI in orange. Key residues are shown as sticks and labeled appropriately. DOI: http://dx.doi.org/10.7554/eLife.22175.010

Figure 3.. (a) Free-energy surface for the unphosphorylated p38α, obtained from metadynamics simulations, as a function of CV₁ and CV₂ which indicate the distance from the reference inactive and active structures, respectively.

The contour lines are drawn every two kcal mol⁻¹. The CV values for the unbiased MD simulation of the same system are drawn every 0.5 ns and shown in a grey gradient as a function of time. (b) The conformation assumed by the protein at the end of the unbiased MD simulation. The backbone is colored according to the RMSF values, with purple indicating higher flexibility regions, while the key residues are shown as sticks and labeled appropriately. DOI: http://dx.doi.org/10.7554/eLife.22175.012

Figure 4.. (a) Free-energy surface for the dually phosphorylated p38α, obtained from metadynamics simulations, as a function of CV₁ and CV₂ which indicate the distance from the reference inactive and active structures, respectively.

The contour lines are drawn every two kcal mol⁻¹. The CV values for the unbiased MD simulation of the same system are drawn every 0.5 ns and shown in a grey gradient as a function of time. (b) The conformation assumed by the protein at the end of the unbiased MD simulation. The backbone is colored according to the RMSF values, with purple indicating higher flexibility regions, while the key residues are shown as sticks and labeled appropriately. DOI: http://dx.doi.org/10.7554/eLife.22175.013

Figure 5.. (a) Free-energy surface for the dually phosphorylated p38α with bound ATP-Mg²⁺, obtained from metadynamics simulations, as a function of CV₁ and CV₂ which indicate the distance from the reference inactive and active structures, respectively.

The contour lines are drawn every two kcal mol⁻¹. The CV values for the unbiased MD simulation of the same system are drawn every 0.5 ns and shown in a grey gradient as a function of time. (b) The conformation assumed by the protein at the end of the unbiased MD simulation. The backbone is colored according to the RMSF values, with purple indicating higher flexibility regions, while the key residues are shown as sticks and labeled appropriately. DOI: http://dx.doi.org/10.7554/eLife.22175.014

Figure 6.. (a) Free-energy surface for the dually phosphorylated p38α with bound ATP-Mg²⁺ and MK2 docking peptide, obtained from metadynamics simulations, as a function of CV₁ and CV₂ which indicate the distance from the reference inactive and active structures, respectively.

The contour lines are drawn every two kcal mol⁻¹. The CV values (calculated every 0.5 ns) for the unbiased MD simulations of the same system are shown as orange contours based on their density. (b) Histograms of the minimal distances between ATP and D112 calculated from the unbiased simulations of the dually phosphorylated p38α with bound ATP-Mg²⁺ with or without the MK2 docking peptide (colored grey and red, respectively). The average values are shown as dashed lines, while the bin size is 0.5 Å. DOI: http://dx.doi.org/10.7554/eLife.22175.015

Figure 7.. Contact map for interactions formed between the residues of the reported X-ray structure (PDB ID: 3PY3, p38α-pTpY) (x-axis) and its symmetry related neighbors (y-axis).

The residues are considered to be in contact if the minimal distance between any of their atoms is under 4 Å. The contacts formed by the docked elements are indicated by the solid ellipses, while the ones formed between functionally important elements are indicated by the dashed ellipses. Secondary structure elements are also shown with α-helices in purple, β-sheets in green, and loops in grey. Missing residues are shown as light grey rectangles. DOI: http://dx.doi.org/10.7554/eLife.22175.016

Figure 8.. (a) Free-energy surface for the dually phosphorylated p38α, obtained from metadynamics simulations, as a function of CV₁ and CV₂ which indicate the distance from the reference inactive and active structures, respectively.

The contour lines are drawn every two kcal mol⁻¹. The CV values (calculated every 0.5 ns) for the unbiased MD simulations of the 3PY3 crystal are shown as orange contours based on their density. (b) The average conformation of the protein calculated from the unbiased MD simulations. The protein is colored according to the calculated RMSF values after the backbone alignment with respect to the 3PY3 X-ray structure. (c) Backbone RMSD values calculated with respect to the 3PY3 X-ray structure either with or without N- and C-terminal tails. The average values over 16 molecules are shown by the black lines, the extreme values by the red lines, while the error bars depicting one standard deviation are shown every 25 ns. (d) X-ray structure of the dually phosphorylated p38α (PDB ID: 3PY3) colored by refined B-factors. DOI: http://dx.doi.org/10.7554/eLife.22175.017

Figure 9.. Histograms of the minimal distances between (a) K53-E71, (b) R49-D112, and c) R49-E160 calculated from p38α X-ray structures currently deposited in PDB (March, 2017).

K53-E71 distances were calculated for structures without any mutations, while R49-D112 and R49-E160 for structures without missing R49, D112, and E160 residues. The average values are shown as red dashed lines, while the bin size is 0.5 Å. DOI: http://dx.doi.org/10.7554/eLife.22175.018

Figure 10.. Multiple alignments of the α_C helix and the activation loop (from the DFG motif to the P+1 loop) sequences of several human kinases.

Selected positively charged residues are colored green, negatively charged ones magenta, while the catalytically important residues in the α_C helix are bolded, as well as the phosphorylated residues in the activation loop. DOI: http://dx.doi.org/10.7554/eLife.22175.019

Author response image 1.

DOI: http://dx.doi.org/10.7554/eLife.22175.021

Author response image 2.. Contact maps for interactions formed between the residues of the reported X-ray structure (PDB ID: 1CM8, p38γ-pTpY) (x-axis) and its symmetry related neighbors (y-axis).

The residues are considered to be in contact if the minimal distance between any of their atoms is under 4 Å. The contacts formed by the A-loop are indicated by the solid ellipse. Secondary structure elements are also shown with α-helices in purple, β-sheets in green, and loops in grey. Missing residues are shown as light grey rectangles. DOI: http://dx.doi.org/10.7554/eLife.22175.022