Conformational Transitions that Enable Histidine Kinase Autophosphorylation and Receptor Array Integration

Abstract

During bacterial chemotaxis, transmembrane chemoreceptor arrays regulate autophosphorylation of the dimeric, histidine-kinase CheA. The five domains of CheA (P1-P5) each play a specific role in coupling receptor stimulation to CheA activity. Biochemical and x-ray scattering studies of thermostable CheA from Thermotoga maritima find that the His-containing substrate domain (P1) is sequestered by interactions that depend upon P1 of the adjacent subunit. Non-hydrolyzable ATP analogs (but not ATP nor ADP) release P1 from the protein core (domains P3P4P5) and increase its mobility. Detachment of both P1 domains, or removal of one within a dimer, increases net autophosphorylation substantially at physiological temperature (55°C). However, nearly all activity is lost without the dimerization domain (P3). The linker length between P1 and P3 dictates inter-subunit (trans) versus intra-subunit (cis) autophosphorylation; with the trans reaction requiring a minimum length of 47 residues. A new crystal structure of the most active dimerization-plus-kinase unit (P3P4) reveals trans-directing interactions between the tether connecting P3 to P2-P1 and the adjacent ATP-binding (P4) domain. The orientation of P4 relative to P3 in the P3P4 structure supports a planar CheA conformation that is required by membrane array models, and suggests that the ATP-lid of CheA may be poised to interact with receptors and coupling proteins. Collectively, these data suggest that the P1 domains are restrained in the off-state as a result of cross-subunit interactions. Perturbations at the nucleotide-binding pocket increase P1 mobility and access of the substrate His to P4-bound ATP.

Atypical chemoreceptor arrays accommodate high membrane curvature

“…S11D) 21 . Additionally, the Td P3 domain possesses a different handedness than Tm P3 observed in previous crystal structures 22,23 .…”

“…S14D) 12 . Additionally, the handedness of the helix connection in the Td P3 domain differs from that of Thermotoga maritima CheA and rather matches helix connectivity of sensor kinase DHP domains 24,25 .…”

“…The diffraction data was scaled and integrated using XDS 17 , and phased by molecular replacement with Phaser MR using ab initio search models generated through the QUARK server and then ran through the AMPLE pipeline on the CCP4 web server 18–20 . Model improvement was done by several rounds of manual model improvement in COOT followed by automated refinement using Phenix Refine software 21,22 .…”

Complete structure of the chemosensory array core signalling unit in an E. coli minicell strain

“…Decades of work have given rise to detailed mechanistic models of signal transduction in MCPs 1,2 , especially within individual receptor homodimers, and more recently, multiple CheA kinasesignaling models have been put forth [4][5][6][7][8] . While these models provide numerous insights and testable predictions, they are mainly limited to specific signaling modules or domains and/or lack residue-level detail.…”

Complete structure of the core signalling unit of theE. colichemosensory array in an optimised minicell strain

“…Several lines of evidence suggest that the P1 and P2 domains of CheA undergo dynamic changes during signalling, and it has been proposed that these domains are sequestered in the kinase-OFF state. 7,34 In addition, NMR experiments have mapped a non-productive P1-P4 binding mode to this region of P4 in T. maritima CheA. 35 Therefore, the unknown density may correspond to either one or both of the CheA.P1 domains and could also include CheA.P2.…”