862 VOLUME 13 NUMBER 10 OCTOBER 2006 NATURE STRUCTURAL & MOLECULAR BIOLOGY

Ann M. Stock is at the Center for Advanced Biotechnology and Medicine, the Department of Biochemistry, University of Medicine and Dentistry of New Jersey–Robert Wood Johnson Medical School, and the Howard Hughes Medical Institute, Piscataway, New Jersey 08854, USA.e-mail: stock@cabm.rutgers.edu

Transmembrane signaling by asymmetryAnn M Stock

The structure of the ligand-binding domains of Vibrio harveyi LuxPQ bound to Autoinducer-2 reveals a dramatic asymmetry in quaternary structure induced by ligand binding. Structures of receptor-sensing domains in both occupied and unoccupied states provide a foundation for postulating mechanisms of transmembrane signaling.

It is often said that nature loves symmetry. Occasionally, however, nature makes important use of asymmetry. A striking example of this appears in the structure of the ligand-binding domains of a bacterial quorum-sensing receptor complex recently reported by Neiditch et al1. The authors propose that asymmetry induced by ligand binding to the periplasmic domain of the receptor is transmitted across the membrane to the intracellular kinase domain, breaking the intrinsic symmetry of the catalytic domain dimer, thus inhibiting kinase activity and thereby modulating downstream signaling.

Many bacteria use quorum sensing to assess population densities of themselves and other bacterial species2,3. Quorum sensing is fundamental to successful competition in complex environments and has been shown to be important for pathogenesis. Quorum-sensing bacteria secrete and detect small molecules or small peptides whose extracellular concentrations reflect bacterial cell density. These autoinducer signals funnel into signal- transduction pathways that regulate gene expression in a population-dependent man-ner. Autoinducer-2 (AI-2) is a furanosyl borate diester that is produced and detected by both Gram-positive and Gram-negative bacteria4. Detection of AI-2 involves two proteins: LuxP, a periplasmic protein that is a member of the extensively characterized large family of periplasmic binding proteins involved in small-molecule detection and transport5; and LuxQ, a transmembrane sensor histidine protein kinase consisting of two tandem periplasmic PAS domains (together designated LuxQp), two transmembrane helices and three cytoplasmic domains—a His- containing dimerization domain, a catalytic ATP-binding kinase domain and a receiver domain typical of two- component hybrid kinases6.

Histidine protein kinases, typi-cally the receptors in two-component phosphotransfer pathways7, are the most prevalent transmembrane signal-transduc-tion proteins in bacteria6. The activities of histidine protein kinases, including both ATP- dependent autophosphorylation and response regulator phosphatase activity, regulate the levels of phosphorylation of downstream response regulator proteins that elicit the specific output responses of the systems. Chemical ligands or physical stimuli regulate the activities of histidine protein kinases through direct or auxiliary protein-mediated interactions with the periplasmic sensor domains, transmembrane domains or cytoplasmic domains. How stimuli regulate sensor kinase activities is one of the least

understood aspects of two-component signal transduction. The great diversity in stimuli and in sensor domain structure, together with general lack of knowledge about fundamental mechanisms of transducing signals across lipid bilayers, hinders our understanding.

The structure of the Vibrio harveyi AI-2–bound LuxPQp complex reported by Neiditch et al.1 joins a very small group of structures of ligand-bound periplasmic domains of receptors involved in two-component signaling8,9. Together with structures of LuxP with4 and without1 AI-2 ligand bound and of a LuxPQp complex without ligand bound10, the latest structure provides a fairly complete picture of structural changes induced by signal binding to the sensing domain. As

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Figure 1 Schematic representation of quaternary structure asymmetry induced by AI-2 binding to the sensor histidine kinase LuxPQ. (a) LuxPQp domains, viewed perpendicular to the plane of the membrane, based on crystal structures reported by Neiditch et al1,10. In the absence of AI-2, the periplasmic binding protein LuxP (blue) in an open conformation forms a stable complex with the periplasmic domains of histidine protein kinase LuxQ (purple). In the absence of stabilizing interactions from other domains, the LuxPQp periplasmic domains do not associate with each other. Upon binding AI-2 (red), LuxP adopts a closed conformation, promoting formation of an asymmetric (LuxPQp)2 dimer. (b) Postulated arrangement of LuxQ cytoplasmic domains, viewed parallel to the plane of the membrane. In the absence of AI-2 (left), the cytoplasmic domains of the histidine protein kinase LuxQ are presumed to associate with two-fold rotational symmetry, forming a dimer that is competent for trans autophosphorylation between protomers. It is postulated that upon AI-2 binding (right), asymmetry in the periplasmic domains is transmitted through the transmembrane helices (cylinders), driving an asymmetric orientation of the cytoplasmic domains and inhibiting kinase activity.

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NATURE STRUCTURAL & MOLECULAR BIOLOGY VOLUME 13 NUMBER 10 OCTOBER 2006 863

anticipated from structures of numerous bacterial periplasmic binding proteins5, AI-2 binding to LuxP induces a closing of the two domains, much like the closing of a clam shell (Fig. 1a). Remarkably, as only one of the LuxP domains makes extensive contacts with the periplasmic domains of LuxQ, the LuxP-LuxQ interface is largely unchanged upon AI-2 binding and the hinged closure of LuxP. However, reorientation of the other domain of LuxP creates a surface that allows binding of a second LuxQp molecule, promoting dimerization of two LuxPQp units. The dimer thus formed is asymmetric, with a rotation of ~140° about an axis perpendicular to the plane of the membrane, ~40° short of the 180° rotation required for two-fold rotational symmetry. The asymmetry is substantial: one LuxQp molecule in the (LuxPQp)2 dimer contacts both LuxP pro-teins, whereas the second LuxQp protomer contacts only its cognate LuxP partner.

Data from extensive mutagenesis analyses provide strong support for the physiological relevance of the interfaces observed in the different crystal structures. On the basis of the structures and accompanying biochemical data, the authors propose a model for AI-2–induced regulation of kinase activity. They postulate that in the absence of AI-2 ligand, LuxQ periplasmic domains associate with LuxP to form LuxPQp complexes that exist independently, without appreciable binding affinity for one other. In this state, dimerization of LuxPQ complexes is driven by interactions of the LuxQ cytoplasmic dimerization domains. This generates symmetric dimers, as observed previously for other histidine kinase cytoplasmic domains11. Additional stabilization presumably is contributed by the transmembrane helices. Upon AI-2 binding, the asymmetric arrangement of the LuxPQp periplasmic domains is presumed to be propagated through the transmembrane helices. This results in asymmetric arrangement of the LuxQ cytoplasmic catalytic domains (Fig. 1b) and inhibition of kinase autophosphorylation, a reaction that must occur in trans between protomers within the kinase dimer12–14.

The model is satisfying in many respects, but it leaves some significant questions unanswered. Most importantly, how are changes in periplasmic domain arrangements coupled through transmembrane helices to cytoplasmic domain orientations? In the simplest model, such transmission would require rigid connections between the periplasmic and cytoplasmic domains within each protomer. However, data from other receptors suggest that this may not

be the case. In the extensively characterized family of bacterial chemoreceptors, ligand binding to the periplasmic domains induces extremely subtle changes in the periplasmic domains and only minor displacements (~1 Å) of one transmembrane helix rela-tive to the other15. Yet recent data suggest that chemoreceptor signaling in Escherichia coli involves large (>50 Å) displacements of cytoplasmic domain dimers within a receptor trimer of dimers16. Thus, it may be presumptuous to assume that changes that occur on one side of the membrane will be directly correlated with changes of similar magnitudes on the opposite surface.

Mechanisms of regulation need not be conserved within families of signaling proteins. There is great diversity in chemical stimuli and in the sensing domains that interact with these ligands. Data suggest that even for transmembrane receptors of two-component pathways that interact with similar ligands, such as other periplasmic binding proteins, mechanisms may not be conserved. The E. coli chemoreceptor Tar mediates responses to a number of different stimuli, including maltose bound to the periplasmic maltose-binding protein (MBP). Mutagenesis studies interpreted in relation to the structures of the Tar sensing domain dimer and maltose-bound MBP have allowed modeling of an MBP–Tarp complex17. The interaction of MBP with the Tar sensing domain is predicted to be asymmetric, but the stoichiometry of the proposed MBP–Tarp complex is 1:2, unlike the 2:2 stoichiometry of the AI-2–LuxPQp complex. In the two- component system that regulates chitin utilization in Vibrio cholerae, a chitin oligosaccharide (GlcNAc)n bound to a specific periplasmic binding protein, CBP, regulates the activ-ity of a hybrid sensor kinase, ChiS18. Although there seem to be many parallels with the components of the V. harveyi LuxQP quorum- sensing pathway, there are notable differences in the mechanism of regulation. In the chitinolytic system, it is postulated that unliganded CBP binds ChiS, maintaining the kinase in an off state; upon ligand binding, CBP is thought to dissociate from ChiS, promoting kinase activity. Thus, although significant progress is being made in the LuxPQ system, it seems unlikely that universally applicable mechanisms of regulation will emerge. Transmembrane sensory transduction, like other mechanistic aspects of signal- transduction pathways, is likely to be highly specialized and adapted to the regulatory needs of the specific system in which it operates.

The proposed modulation of LuxPQ sensor kinase activity from active to inactive states by a transition from symmetric to asymmetric quaternary structure provides an elegant mechanism for transmembrane signal transduction. As most oligomeric proteins are symmetric, the breaking of symmetry provides interesting opportunities for regulating function. Asymmetry has been observed in a number of diverse processes. Examples include negative cooperativity induced by the asymmetric binding of ligand to dimeric chemoreceptors19; the sequential assembly of flagellin subunits into flagellar filaments, facilitated by the mismatch between the 5-fold symmetry of the HAP2 cap protein and the 5.5-fold symmetry of the filament7; and DNA translocation through a hexameric ring helicase, mediated by asymmetry in nucleotides bound within individual subunits20.

It is interesting to speculate that methods of structural characterization may have an inher-ent bias toward symmetric structures and that asymmetric complexes may be under- represented in our current structural repertoire. Asymmetry in quaternary structure necessitates a larger asymmetric unit, a feature that is likely to be less favorable for both X-ray diffraction and NMR analyses. As technological advances accelerate the pace of structural characterization and macromolecular complexes are character-ized in multiple different states, we can look forward to seeing more complexes in which asymmetry modulates function.1. Neiditch, M.B. et al. Cell 126, 1095–1108 (2006).2. Camilli, A. & Bassler, B.L. Science 311, 1113–1116

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& Hughson, F.M. Mol. Cell 18, 507–518 (2005).11. Marina, A., Waldburger, C.D. & Hendrickson, W.A.

EMBO J. 24, 4247–4259 (2005).12. Pan, S.Q., Charles, T., Jin, S., Wu, Z.-L. & Nester, E.W.

Proc. Natl. Acad. Sci. USA 90, 9939–9943 (1993).13. Ninfa, E.G., Atkinson, M.R., Kamberov, E.S. & Ninfa,

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