Strategies for identifying dynamic regions in protein complexes: flexibility changes accompany methylation in chemotaxis receptor signaling states

Abstract

Bacterial chemoreceptors are organized in arrays composed of helical receptors arranged as trimers of dimers, coupled to a histidine kinase CheA and a coupling protein CheW. Ligand binding to the external domain inhibits the kinase activity, leading to a change in the swimming behavior. Adaptation to an ongoing stimulus involves reversible methylation and demethylation of specific glutamate residues. However, the exact mechanism of signal propagation through the helical receptor to the histidine kinase remains elusive. Dynamics of the receptor cytoplasmic domain is thought to play an important role in the signal transduction, and current models propose inverse dynamic changes in different regions of the receptor. We hypothesize that the adaptational modification (methylation) controls the dynamics by stabilizing a partially ordered domain, which in turn modulates the binding of the kinase, CheA. We investigated the difference in dynamics between the methylated and unmethylated states of the chemoreceptor using solid-state NMR. The unmethylated receptor (CF4E) shows increased flexibility relative to the methylation mimic (CF4Q). Methylation helix 1 (MH1) has been shown to be flexible in the methylated receptor. Our analysis indicates that in addition to MH1, methylation helix 2 also becomes flexible in the unmethylated receptor. In addition, we have demonstrated that both states of the receptor have a rigid region and segments with intermediate dynamics. The strategies used in the study for identifying dynamic regions are applicable to a broad class of proteins and protein complexes with intrinsic disorder and dynamics spanning multiple timescales. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/974428v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@8a8b0forg.highwire.dtl.DTLVardef@1afb646org.highwire.dtl.DTLVardef@e7dbaaorg.highwire.dtl.DTLVardef@685c0_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIReceptors exhibit greater ns timescale dynamics in unmethylated vs methylated state C_LIO_LIMethylation helix 2 likely involved in increased flexibility of unmethylated state C_LIO_LIDynamics occur on multiple timescales in both states of the receptor C_LI