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.
Highlights
NMR chemical shifts report on the time-averaged local chemical environment and reflect conformation and dynamic processes on a broad timescale
By network analysis of chemical shift perturbations caused by methyl mutagenesis of p38γwe observe the pathways of communication between the various regulatory elements of p38γand how they respond to changes in catalytic state
Long range chemical shift perturbations caused by binding of substrate, inhibitor, and nucleotide to kinases have been observed previously, but have been described only in qualitative terms[23,24,29,30]
Summary
NMR chemical shifts report on the time-averaged local chemical environment and reflect conformation and dynamic processes on a broad timescale (ps-ms). By network analysis of chemical shift perturbations caused by methyl mutagenesis of p38γwe observe the pathways of communication between the various regulatory elements of p38γand how they respond to changes in catalytic state. ATP bound, inhibitor bound (DFG-out), and phosphorylated forms of p38γwe find that the structure of the allosteric network reflects the conserved kinase infrastructure, including distinction between N and C lobes, hydrophobic spines, as well as MAPK specific regulatory elements. The flow of information revealed by the analysis identifies novel mechanisms for allosteric effects in docking site interactions, auto-activation, and DFG-out inhibition Critical nodes in these networks - those that have the highest number of connections and greatest amount of communication flowing through them - are found to correspond very well to known allosteric sites. The results presented demonstrate the method’s effectiveness to characterize network structure and identify allosteric sites by improving our understanding of the allosteric pathways within p38γ
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