NMR Characterization of Information Flow and Allosteric Communities in the MAP Kinase p38γ

Abstract

The intramolecular network structure of a protein provides valuable insights into allosteric sites and communication pathways. However, a straightforward method to comprehensively map and characterize these pathways is not currently available. Here we present an approach to characterize intramolecular network structure using NMR chemical shift perturbations. We apply the method to the mitogen activated protein kinase (MAPK) p38γ. p38γ contains allosteric sites that are conserved among eukaryotic kinases as well as unique to the MAPK family. How these regulatory sites communicate with catalytic residues is not well understood. Using our method, we observe and characterize for the first time information flux between regulatory sites through a conserved kinase infrastructure. This network is accessed, reinforced, and broken in various states of p38γ, reflecting the functional state of the protein. We demonstrate that the approach detects critical junctions in the network corresponding to biologically significant allosteric sites and pathways.

Figure 1. Long-range methyl chemical shift perturbations.

(a) Heat-map of ¹H-¹³C chemical shift perturbations (in Hz) for methyl resonances in p38γ (mutation-wildtype) caused by minimally disruptive mutations. Green represents mutation site. (b) ¹H-¹³C chemical shift perturbations due to the substitution L268V in the MAPK insert mapped onto a homology model of the inactive apo p38γ structure. Colors correspond to shift differences in Hz, using the same scale in (a). (c) The ranks of each mutation induced chemical shift perturbation for V53 and M112. Each point corresponds to a mutant as ranked among the set of mutations for the specified residue.

Figure 2. Mediation analysis.

Effects of residue A on C are mediated by B if 1) A affects mediator or residue B (2 point correlation R_AB); 2) B affects C (multiple regression coefficient ρ_bc); 3) Positive correlations; 4) Indirect effect (ρ_ac < R_AC). If these criteria are met then flow travels from A to B to C with the mediated effect of R_AB*ρ_bc.

Figure 3. Network structure and flow of inactive apo p38γ.

The method identified 15 communities with membership greater than 5 residues in inactive p38γ, but only the largest 8 communities are shown for clarity. Identified communities from chemical shift perturbation networks are colored based on tertiary structure and regulatory element: N-lobe, yellow; C-lobe, purple; active-site, green; C-spine, blue; R-spine, red; MAPK-insert, lavender. Modules depicted with similar shades of color overlap the same structural element. (a) Network map of inactive p38γ. The size of the modules represents the amount of flux and connections between residues within the module. Thickness of arrows between communities represents the amount of flux between communities. The C-spine (b) and R-spine (c) and connected communities are mapped on the homology model of inactive apo p38γ. Residues in multiple communities are depicted as multicolor spheres. The boxed region of the network map (panel a) is mapped on the structure in (d), illustrating the communities and flow linking C-lobe and N-lobe.

Figure 4. Response of p38γ network to ATP binding.

(a) Network structure of ATP + p38γ, communities are colored based on tertiary structure and regulatory element: N-lobe/C-spine, yellow; C-lobe, purple; docking site, blue; R-spine, red; MAPK-insert, lavender. (b) Comparison of N-lobe communities in inactive apo and ATP bound p38γ. Completion of the C-spine by ATP extends the N-lobe community further into the C-lobe. (c) Communities representing a pathway (left black arrow in (a)) between C-lobe (purple) and N-lobe (yellow) through the docking site (blue) and C-spine (yellow). The pathway through the C-spine is not present in apo inactive p38γ. (d) Communities involved in the R-spine (red) pathway (right black arrow in a) between N (yellow) and C (purple) lobes.

Figure 5. Network response to activation.

Network analysis identified 16 communities with membership greater than 5 residues in active p38γ. Communities are represented in shades of color based on tertiary structure and regulatory element: N-lobe/active-site, yellow; C-lobe/MAPK-insert/GHI subdomain, purple; active-site/GHI subdomain, orange; C-spine/hinge, blue; R-spine, red. The network map is depicted in (a) with flow and membership represented as in Fig. 3. Flux through communities connecting N and C-lobes are depicted: through the C-spine hinge (b), through the R-spine (c), and through the active site residues (d).

Figure 6. Network disruption by BIRB-796.

(a) The method identified 17 communities with membership greater than 5 residues in DFG-out p38γ, of which only 10 are shown for clarity. Communities are represented in shades of color based on tertiary structure and regulatory element: N-lobe, yellow; C-lobe, purple; docking site, blue. Communities in the C-lobe (purple) are heavily overlapped and include the MAPK insert, portions of the GHI subdomain, and F-helix. Darker shades of purple indicate communities that include the F-helix, lighter shades of purple indicate communities that include the H-helix. (b) Communities are mapped on the DFG-out p38γ homology model, illustrating the disruption in communication between N and C-lobes. The docking site has become insular, represented by weak flow to other communities and membership only in the C-lobe.

Figure 7. Critical network nodes reveal regulatory sites and pathways.

(a) A series of highly ranked memory nodes in inactive p38γ reveal multiple pathways that extend greater than 20 Å from the docking site to the activation loop. Large circles represent residues with a direct connection to the memory node of interest and small circles represent connected memory nodes. Semi-circles depict residues with an indirect connection to the memory node. The relative amount of flow between residues is indicated by the thickness of solid arrows. Dashed arrows indicate the common residue belonging to the next memory node in the path. Colors correspond to regulatory elements, as labeled. Memory nodes (left to right) include M109-M112, L89-L78, and L77-L174. (b) A significant memory node in the αC-helix of apo p38γ suggests a route for auto-activation by Y326 phosphorylation. (c) A highly ranked memory node in the MAPK insert of activated p38γ reveals a communication pathway to catalytic residues.