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Review
. 2011;80:211-37.
doi: 10.1146/annurev-biochem-091008-152423.

Transmembrane Communication: General Principles and Lessons From the Structure and Function of the M2 Proton Channel, K⁺ Channels, and Integrin Receptors

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Review

Transmembrane Communication: General Principles and Lessons From the Structure and Function of the M2 Proton Channel, K⁺ Channels, and Integrin Receptors

Gevorg Grigoryan et al. Annu Rev Biochem. .
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Abstract

Signal transduction across biological membranes is central to life. This process generally happens through communication between different domains and hierarchical coupling of information. Here, we review structural and thermodynamic principles behind transmembrane (TM) signal transduction and discuss common themes. Communication between signaling domains can be understood in terms of thermodynamic and kinetic principles, and complex signaling patterns can arise from simple wiring of thermodynamically coupled domains. We relate this to functions of several signal transduction systems: the M2 proton channel from influenza A virus, potassium channels, integrin receptors, and bacterial kinases. We also discuss key features in the structural rearrangements responsible for signal transduction in these systems.

Figures

Figure 1
Figure 1
Simple linked-equilibrium models. (a) A thermodynamic cycle illustrating the coupling between activation of a two-state protein and ligand binding. (b) Activation profiles of a two-state protein as a function of an agonist ligand concentration. Ratio of dissociation constants for the inactive and active states, KI/KA, and the intrinsic energy gap, ΔGgap, determine maximal attainable activation. (c) A diagram of a simple signal-transduction molecule. Two domains, a receptor domain and a cytoplasmic functional domain, each with two states, are thermodynamically coupled, with the receptor modulated by an activating ligand. All eight states of the system are explicitly shown. Free-energy differences between ligand-bound states (back side of the cube) are omitted for simplicity, but follow simply from thermodynamic cycles. L indicates the concentration of the cytoplasmic ligand. In a and c, equilibrium free-energy differences correspond to the direction of the arrow with which they are shown. (d ) Activation profiles of the system in panel c as a function of the energy gap of the cytoplasmic domain, ΔGgapC, and coupling free energy of the receptor to the cytoplasmic domain, ΔΔGcR. KA and KI are set to 10 nM and 20 μM, respectively, and the energy gap of the receptor domain, ΔGgapR, is set to 3.0 kcal/mol. The effective energy gap for this system can be shown to be ΔGgapeff=ΔGgapC+RTln(e(ΔGgapRΔΔGcR)RT(1+LKI)+(1+LKA)e(ΔGgapR+ΔΔGcR)RT(1+LKI)+(1+LKA))ΔΔGcR. Thus, if ΔΔGcR is zero (i.e., there is no coupling between the receptor and cytoplasmic domains), the overall gap is just ΔGgapC, but as long as ΔΔGcR is negative (i.e., activation of the receptor promotes the active state of the functional domain), higher values of ΔGgapR contribute to a higher overall energy gap. Similarly, tighter coupling (lower ΔΔGcR) leads to a higher overall gap and larger degree of maximal activation.
Figure 2
Figure 2
A more complex model of a signaling molecule. (a) In addition to the receptor and functional cytoplasmic domains as in Figure 1, here the transmembrane (TM) domain is explicitly modeled as having two states. The intrinsic fraction of TM domain in the active state is defined by ΔGgapTM, and it is coupled to the cytoplasmic domain via ΔGcTM. The 16 states of the system along with the corresponding free energies are shown in the table on the right. (b) The dependency of activation profiles on ΔGgapTM and ΔGcTM. KA and KI are set to 10 nM and 20 μM, respectively, and values of other parameters are shown. The line with circles demonstrates the case where no two-state TM domain is present, and the receptor domain couples directly to the cytoplasmic domain with the same ΔGgapR as in other models.
Figure 3
Figure 3
A realistic model of signal transduction, inspired by the VirA protein of the VirA/VirG two-component system. (a) In addition to the domains and states in the model presented in Figure 2, there is a cytoplasmic linker domain, with its own active and inactive states, which is modulated by the signaling ligand C. A different ligand, L, modulates the receptor. The total number of states in this system is 64, and for clarity only the fully inactive and the fully active states are diagrammed. (b) Activation, relative to the resting state, is plotted against an increasing concentration of ligand L in the presence of different concentrations of C. This illustrates the ability to integrate signals from multiple ligands. Though this model does not explicitly treat the receptor as being dimeric (as is the case for the VirA protein), it can be easily extended to do so. In this case, the binding of a ligand may or may not be coupled with receptor dimerization, and there may exist cooperativity (negative or positive) between the two ligand-binding sites. Potential scenarios that can be explored include the following: Binding of a single ligand may push the system toward the active state, whereas binding two ligands symmetrically may favor the inactive state; alternatively, the receptor may signal only when two ligands are bound; or binding of the second ligand may proceed independently of the first one.
Figure 4
Figure 4
The ion-conducting pores of M2 and KcsA. Both are drawn with their cytoplasmic domain oriented downward. The physiological flow of ions is inward (down) for M2 versus outward (up) for KcsA. (a) A high-resolution crystal structure of M2 [Protein Data Bank (PDB) code 3LBW] showing the overall structure, and (b) a blow-up of the ordered pore-lining side chains and water molecules. In all five panels, water molecules are shown as small magenta spheres, and the identity of the various side chains are annotated in panel b. (c) The structure of KcsA (PDB code 1K4C) with the potassium ions in the selectivity filter shown in dark gray. Additional potassium ions are shown in gray, and waters of hydration in magenta. (d and e) The structures of amantadine and tetrabutylammonium (TBA) bound to M2 and KcsA (PDB code 2HVK for the latter), respectively. The structure of M2 in panel d is from the solid-state NMR structure (2KQT) with the water from 3LBW docked in to show the approximate locations of water molecules.
Figure 5
Figure 5
(a,b) Parametric views of structural changes in the pore-forming helices of the M2 protein channel and (c,d ) the KcsA channel. Pore-forming helix bundles (residues 25–46 for M2 and 86–122 in KcsA; for the latter, some structures lacked the N-terminal section of this region) were described via the arched bundle parameterization (outlined in Reference 86). In total, 8 structures were analyzed for M2 [Protein Data Bank (PDB) codes 2RLF, 3C9J, 2KQT, 2L0J, 3LBW, and 3 symmetric structures derived from 3 different helices of the asymmetric structure 3BKD], and 10 structures were analyzed for KcsA (PDB codes 1R3J, 2HVK, 3EFF, 3F5W, 3F7V, 3F7Y, 3FB5, 3FB6, 3FB7, and 3FB8). Essential features were identified by forcing progressively more of the parameters to have the same values for all structures. Panels a and c illustrate the accuracy of the parametric representation by comparing true backbone structures (cyan) with idealized templates (green) for representative bundles from M2 and KcsA, respectively. An advantage of the mathematically modeled structures is the unambiguous alignment frame they provide. (b) Axial and side views (for the latter, only two opposing helices shown) of superposed parameterized models of all eight M2 structures clearly illustrate the dominant modes of motion (illustrated with arrows). Essential parameters of variation for these structures are the radius of curvature of helical arches, helix tilt angle, and bundle radius (86), with the possible exception of the solution NMR structure 2RLF [whereas all structures were fit within 1.0-Å root mean square deviation (RMSD) with just these three parameters, the first model in 2RLF produced an RMSD of 1.66 Å]. (d ) Axial and side views (for the latter, only two opposing helices are shown) of superposed parameterized models of all 10 KcsA structures. The essential parameters of variation for these structures are radius and direction of curvature of helical arches, and helix pitch angle (86).
Figure 6
Figure 6
Thermodynamic model of integrin signaling. (a) At the coarsest level, extracellular, transmembrane (TM), and intracellular domains of integrins can be thought of as being either active or inactive, giving rise to eight coupled states as illustrated. The various effector molecules modulate integrin state by preferentially binding to select domain conformations. “Inside-out” signaling refers to modulation of the extracellular domain conformation via interactions between an integrin's cytosolic tails and intracellular signaling molecules. “Outside-in” events sometimes refer to the activation of oligomerization-dependent signaling pathways subsequent to binding polyvalent ligands. However, keeping with our model, here it refers to the ability of extracellular ligands to affect the conformation and function of intracellular domains. (b) Using reasonable values for the energy gaps and coupling constants associated with these states as well as equilibrium binding constants for the active and inactive states of different effector molecules, one can interrogate the thermodynamics of integrin signaling in a coarse, semiapproximate manner. Shown here is the level of integrin activation (separation of TM/tail regions) in response to increasing amounts of an activating antibody as a function of Mn2+ concentration. This illustrates outside-in signaling (the level of activation is shown on the z-axis and also indicated by the pseudocolor). (c,d ) Inside-out signaling as illustrated by the fraction of extracellular domain bound to native ligand as a function of different amounts of active intracellular talin and kindlin. The binding affinity of the ligand as well as the amounts of talin/kindlin all couple to determine the activation state of the integrin.
Figure 7
Figure 7
Coupled equilibria linking integrin transmembrane (TM) interactions to cytosolic events. Integrin αβ TM heterodimers (left) stabilize inactive ectodomain conformations, and TM separation (right) stabilizes active ectodomain conformations. The αβ heterodimer stabilizes interactions between the β-cytoplasmic domain and the inner leaflet of the bilayers (top). Cytosolic proteins talin and kindlin shift the equilibrium toward an active conformation by stabilizing the extended cytoplasmic domain and separated TM domains (bottom right). Loss of either talin-1 or kindlin-3 in mouse platelets results in bleeding and nearly complete loss of inside-out integrin activation (138, 139). Similarly, patients who lack kindlin-3 suffer from bleeding and immune impairments due to loss of αIIbβ3 and β2 integrin functions, respectively (–144). Although platelets that lack talin and kindlin-3 are physiologically impaired, they are capable of partial integrin activation in the presence of very strong agonists. Also, large excesses of talin can activate integrins in the absence of kindlins (145), coexpression of kindlins and talin result in significantly increased activation (146, 147), and overexpression of kindlins alone does not significantly activate integrins. This equilibrium can further be expanded via phosphorylation of integrin interaction motifs (148, 149) or competition with other signaling molecules, such as filamins (150, 151).

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