PAS domain residues involved in signal transduction by the Aer redox sensor of Escherichia coli

Abstract

PAS domains sense oxygen, redox potential and light, and are implicated in behaviour, circadian rhythmicity, development and metabolic regulation. Although PAS domains are widespread in archaea, bacteria and eukaryota, the mechanism of signal transduction has been elucidated only for the bacterial photo sensor PYP and oxygen sensor FixL. We investigated the signalling mechanism in the PAS domain of Aer, the redox potential sensor and aerotaxis transducer in Escherichia coli. Forty‐two residues in Aer were substituted using cysteine‐replacement mutagenesis. Eight mutations resulted in a null phenotype for aerotaxis, the behavioural response to oxygen. Four of them also led to the loss of the non‐covalently bound FAD cofactor. Three mutant Aer proteins, N34C, F66C and N85C, transmitted a constant signal‐on bias. One mutation, Y111C, inverted signalling by the transducer so that positive stimuli produced negative signals and vice versa. Residues critical for signalling were mapped onto a three‐dimensional model of the Aer PAS domain, and an FAD‐binding site and ‘active site’ for signal transduction are proposed.

Crystal structure of a photoactive yellow protein from a sensor histidine kinase: Conformational variability and signal transduction

“…The three regions of Ppr-PYP that show statistically significant displacements in C␣ coordinates from E-PYP correspond to null mutants of the Aer redox sensor, a PAS domain that binds flavin adenine dinucleotide (FAD). Mutations that retain the ability to bind FAD but are unable to signal map to the ␣3-␣4, ␣5-␤4, and ␤4-␤5 loops of Ppr-PYP (48), suggesting that the structural variability observed in these loops is related to biological activity. Null mutations of Aer that lie in the ␣3-␣4 and ␤4-␤5 regions are consistent with the formation of a biologically-relevant molecular surface by those loops.…”

Charting the Signal Trajectory in a Light-Oxygen-Voltage Photoreceptor by Random Mutagenesis and Covariance Analysis

“…In addition to previously known variants (V15A, D21G, H22L, H22P, V27A, D109V) (17), we isolated several new ON variants in which aliphatic residues within AЈ␣ and the LOV ␤ sheet were exchanged, either for other hydrophobic residues (I16F, ␣ LD ϭ 1.04 Ϯ 0.36; L20F, ␣ LD ϭ 1.32 Ϯ 0.08; M111I, ␣ LD ϭ 0.84 Ϯ 0.01; V120I, ␣ LD ϭ 0.80 Ϯ 0.38) or for hydrophilic and charged residues (A19T, ␣ LD ϭ 0.78 Ϯ 0.34; L20R, ␣ LD ϭ 0.84 Ϯ 0.04; V25D, ␣ LD ϭ 0.86 Ϯ 0.44; I122N, ␣ LD ϭ 1.33 Ϯ 0.08; I122T, ␣ LD ϭ 1.16 Ϯ 0.07). We found the absolute highest number of ON and INV mutations for residues Asp 21 and His 22 at the C-terminal end of AЈ␣, which are involved in polar contacts to Asp 109 in strand H␤ and to the amide hydrogen of Gln 44 in helix C␣ (D21V, ␣ LD ϭ 9.51 Ϯ 0.81; D21Y, ␣ LD ϭ 1.27 Ϯ 0.11; H22Q, ␣ LD ϭ 0.92 Ϯ 0.61; H22R, ␣ LD ϭ 1.00 Ϯ 0.24; Q44P, ␣ LD ϭ 0.80 Ϯ 0.08; D109Y, ␣ LD ϭ 0.70 Ϯ 0.08). Of particular note and of practical utility (18), D21V displays a strong INV phenotype with low dark and high light activity, more pronounced than the previously identified D21G and en par with H22P.…”

“…Of particular note and of practical utility (18), D21V displays a strong INV phenotype with low dark and high light activity, more pronounced than the previously identified D21G and en par with H22P. Notably, replacement of Gln 44 by proline amounts to removal of the amide hydrogen atom and concomitant loss of polar bonding to Asp 21 . Certain double mutants with ON and INV phenotypes we isolated (supplemental Table S1) showed behavior that deviates from the corresponding single mutants.…”

The Streptococcus mutans vicX Gene Product Modulates gtfB / C Expression, Biofilm Formation, Genetic Competence, and Oxidative Stress Tolerance

“…In the 13 TCSTSs in S. mutans, only the VicK protein harbors a PAS domain, and PAS domains are almost exclusively sensors of oxygen and intracellular redox potential (37). In E. coli the aerotaxis transducer Aer has a PAS domain in its N-terminal region that promotes cell migration to a microenvironment with a preferred redox potential (26,27,36,41). As S. mutans is a member of the human oral microbial consortium that is usually exposed to various oxidative stresses, the ability of this organism to monitor intracellular oxygen levels is important because reactive oxygen radicals can be highly toxic to the cell.…”