Volume 18, Issue 5, Pages 507-518 (May 2005) Regulation of LuxPQ Receptor Activity by the Quorum-Sensing Signal Autoinducer-2  Matthew B. Neiditch, Michael J. Federle, Stephen T. Miller, Bonnie L. Bassler, Frederick M. Hughson  Molecular Cell  Volume 18, Issue 5, Pages 507-518 (May 2005) DOI: 10.1016/j.molcel.2005.04.020 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 Vibrio harveyi Quorum-Sensing Signal Transduction (A) AI-1 and AI-2 quorum-sensing signal transduction circuits converge to control the expression of LuxR-regulated target genes including luciferase (Mok et al., 2003). Signal transduction is mediated by phosphotransfer via two-component proteins containing conserved histidine (H) and aspartate (D) residues. Red arrows indicate the phosphoryl group flow at low cell density; at high cell density, the flow is reversed. (B) Schematic diagram of V. harveyi LuxP and LuxQ. The LuxP signal sequence (denoted SS) is proteolytically removed upon translocation into the periplasm, yielding mature LuxP (residues 22–365). Predicted LuxQ transmembrane domains (TM) flank a periplasmic domain (residues 39–280) composed of tandem PAS domains (this work). The LuxQ cytoplasmic region includes predicted HAMP (residues 299–350), PAS (365–459), coiled coil (462–491), histidine kinase (489–711), and response regulatory (736–851) domains. Colors correspond to domains depicted in (A). Molecular Cell 2005 18, 507-518DOI: (10.1016/j.molcel.2005.04.020) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 LuxQ Is a Kinase in the Absence of LuxP (A) Light production by luxN (●) and luxN, luxP (○) V. harveyi strains. Relative light units (RLU) are defined as counts min−1 cfu−1 ml−1 × 1000. (B) LuxP protein levels in V. harveyi as determined by immunoblotting. (C) Size exclusion chromatography of LuxP, LuxQp, and complexes. LuxP alone homodimerizes to a greater (apoLuxP) or lesser (holoLuxP) extent; in both cases, the homodimers lack AI-2 and equilibrate very slowly with monomers (data not shown). (D) Size exclusion chromatography of equimolar mixtures of apoLuxP (left) or holoLuxP (right) and LuxQp at various protein concentrations. Within each set of experiments, dashed lines mark the approximate elution positions of heterodimers (left) and monomers (right). Peak broadening at intermediate concentrations is presumably the result of exchange between heterodimers and monomers on the time scale of the gel filtration runs (approximately 30 min). The absorbance traces are scaled to compensate for the varying concentrations. Molecular Cell 2005 18, 507-518DOI: (10.1016/j.molcel.2005.04.020) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 ApoLuxP:LuxQp Crystal Structure (A) LuxP domains are depicted in blue and green. The membrane-proximal domain of LuxQp is colored orange, whereas the membrane-distal domain is colored yellow except for the red FG loop (residues 147–153). Dashed lines denote disordered regions (orange) or predicted transmembrane domains (blue). (B) The LuxP (cartoon):LuxQp (surface) interaction. The LuxP N terminus and residues mutated to alanine (see Figure 5) are shown in cyan. (C) Structural alignment of LuxQp membrane-distal and -proximal PAS domains. (D) Structure-based sequence alignment. Secondary structure elements were designated to correspond with PAS Kinase (1LL8) and FixL (1DRM) (Amezcua et al., 2002; Gong et al., 1998). (E) Stereoview of the apoLuxP:LuxQp interface. Bonds are color coded as in (A), except that LuxP N-terminal residues 22–26 are in cyan. Hydrogen bonds are depicted as dashed lines. Prime symbols (′) denote LuxQ residues. Molecular Cell 2005 18, 507-518DOI: (10.1016/j.molcel.2005.04.020) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 Comparison between Apo- and HoloLuxP (A) Side views (oriented along the LuxP hinge axis) of apoLuxP (left) and holoLuxP (right). The position of the hinge axis is indicated by the closed red circle. The apoLuxP conformation is that visualized in the apoLuxP:LuxQp crystal structure, with LuxQp removed for clarity, whereas the holoLuxP structure was determined previously (Chen et al., 2002). (B) Canonical front view (as in Figure 3A) of apoLuxP:LuxQp (left) and holoLuxP (right). LuxQ is not depicted bound to holoLuxP because the structure of this complex has not been determined. Molecular Cell 2005 18, 507-518DOI: (10.1016/j.molcel.2005.04.020) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 5 Activity of LuxP Mutants in the V. harveyi Bioluminescence Bioassay (A) Levels of wild-type and mutant LuxP proteins determined by immunoblotting. (B and C) Light production by V. harveyi expressing LuxP N-terminal truncation mutants (B) and alanine substitution mutants (C). Relative light units are plotted as a function of cell density. Each curve is representative of at least three independent assays. Lines were added by inspection to guide the eye. Molecular Cell 2005 18, 507-518DOI: (10.1016/j.molcel.2005.04.020) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 6 A Model for AI-2-Regulated LuxPQ Signal Transduction Both AI-2 binding and disruption of the inhibitory interactions visualized in the apoLuxP:LuxQp structure favor the high cell-density phosphatase state. Cytoplasmic coiled-coil domains (CC) and/or HAMP domains may mediate dimerization of constitutively associated LuxPQ complexes. Molecular Cell 2005 18, 507-518DOI: (10.1016/j.molcel.2005.04.020) Copyright © 2005 Elsevier Inc. Terms and Conditions