Engineered Socket Study of Signaling through a Four-Helix Bundle: Evidence for a Yin−Yang Mechanism in the Kinase Control Module of the Aspartate Receptor

摘要

The chemoreceptors of Escherichia coli and Salmonella typhimurium form stable oligomers thatnassociate with the coupling protein CheWand the histidine kinase CheA to form an ultrasensitive, ultrastablensignaling lattice. Attractant binding to the periplasmic domain of a given receptor dimer triggers antransmembrane conformational change transmitted through the receptor to its cytoplasmic kinase controlnmodule, a long four-helix bundle that binds and regulates CheA kinase. The kinase control module comprisesnthree functional regions: the adaptation region possessing the receptor adaptation sites, a coupling region thatntransmits signals between other regions, and the protein interaction region possessing contact sites fornreceptor oligomerization and for CheA CheWbinding. On the basis of the spatial clustering of known signalnlocking Cys substitutions and engineered disulfide bonds, this study develops the yin yang hypothesis fornsignal transmission through the kinase control module. This hypothesis proposes that signals are transmittednthrough the four-helix bundle via changes in helix helix packing and that the helix packing changes in thenadaptation and protein interaction regions are tightly and antisymmetrically coupled. Specifically, strongnhelix packing in the adaptation region stabilizes the receptor on state, while strong helix packing in the proteinninteraction region stabilizes the off state. To test the yin yang hypothesis, conserved sockets likely tonstrengthen specific helix helix contacts via knob-in-hole packing interactions were identified in thenadaptation, coupling, and protein interaction regions. For 32 sockets, the knob side chain was truncatednto Ala to weaken the knob-in-hole packing and thereby destabilize the local helix helix interaction providednby that socket.We termthis approach a “knob truncation scan”.Of the 32 knob truncations, 28 yielded stablenreceptors. Functional analysis of the signaling state of these receptors revealed seven lock-off knobntruncations, all located in the adaptation region, that trap the receptor in its “off” signaling state (low kinasenactivity, high methylation activity). Also revealed were five lock-on knob truncations, all located in thenprotein interaction region, that trap the “on” state (high kinase activity, low methylation activity). Thesenfindings provide strong evidence that a yin yang coupling mechanism generates concerted, antisymmetricnhelix helix packing changes within the adaptation and protein interaction regions during receptor on offnswitching. Conserved sockets that stabilize local helix helix interactions play a central role in thismechanism:nin the on state, sockets are formed in the adaptation region and disrupted in the protein interaction region,nwhile the opposite is true in the off state.