Biochemical characterization of the tandem HAMP domain from Natronomonas pharaonis as an intraprotein signal transducer

Abstract

Available structures of HAMP domains suggest rotation as one potential mechanism in intraprotein signal transduction. It has been proposed that in poly-HAMP modules the signal sign is inverted with each additional HAMP. We examined signal transduction through the HAMP tandem domain from the phototaxis transducer of the halophilic archaeon Natronomonas pharaonis in membrane-bound chimeras consisting of the Escherichia coli chemotaxis receptor for serine, Tsr, as an input and the mycobacterial adenylyl cyclase Rv3645 as an output domain, i.e. the basic chimera was 'Tsr-NpHAMP tandem-Rv3645 cyclase'. Neither of the NpHAMP units alone nor the NpHAMP tandem transduced a serine signal. After five targeted point mutations in the first a-helix of NpHAMP 1 , the non-functional NpHAMP modules combined into a functional HAMP tandem. 1 mM serine significantly inhibited cyclase activity (À35%; IC 50 = 30 lM) in disagreement with the structure-based predictions. Surprisingly, replacement of NpAS1 1 in the tandem by the respective AS1 from HAMP Tsr resulted in signal inversion, i.e. serine activated cyclase (+129%; EC 50 = 10 lM). Examination of 48 mutants of AS1 1 in the HAMP tandem including two residues of a putative N-terminal control cable identified five residues in NpAS1 1 which probably define different ground states of the output domain and thus affect the sign of signal output. The data question the predicted HAMP rotation as the predominant mechanism of intraprotein signal transduction and point to as yet unrecognized conformational motions of HAMP domains in intraprotein signaling.