Broad disorder and the allosteric mechanism of myosin II regulation by phosphorylation

Double electron electron resonance EPR methods was used to measure the effects of the allosteric modulators, phosphorylation, and ATP, on the distances and distance distributions between the two regulatory light chain of myosin (RLC). Three different states of smooth muscle myosin (SMM) were studied: monomers, the short-tailed subfragment heavy meromyosin, and SMM filaments. We reconstituted myosin with nine single cysteine spin-labeled RLC. For all mutants we found a broad distribution of distances that could not be explained by spin-label rotamer diversity. For SMM and heavy meromyosin, several sites showed two heterogeneous populations in the unphosphorylated samples, whereas only one was observed after phosphorylation. The data were consistent with the presence of two coexisting heterogeneous populations of structures in the unphosphorylated samples. The two populations were attributed to an on and off state by comparing data from unphosphorylated and phosphorylated samples. Models of these two states were generated using a rigid body docking approach derived from EM [Wendt T, Taylor D, Trybus KM, Taylor K (2001) Proc Natl Acad Sci USA 98:4361-4366] (PNAS, 2001, 98:4361-4366), but our data revealed a new feature of the off-state, which is heterogeneity in the orientation of the two RLC. Our average off-state structure was very similar to the Wendt model reveal a new feature of the off state, which is heterogeneity in the orientations of the two RLC. As found previously in the EM study, our on-state structure was completely different from the off-state structure. The heads are splayed out and there is even more heterogeneity in the orientations of the two RLC.     

Transmembrane allosteric coupling of the gates in a potassium channel

It has been hypothesized that transmembrane allostery is the basis for inactivation of the potassium channel KcsA: opening the intracellular gate is spontaneously followed by ion expulsion at the extracellular selectivity filter. This suggests a corollary: following ion expulsion at neutral pH, a spontaneous global conformation change of the transmembrane helices, similar to the motion involved in opening, is expected. Consequently, both the low potassium state and the low pH state of the system could provide useful models for the inactivated state. Unique NMR studies of full-length KcsA in hydrated bilayers provide strong evidence for such a mutual coupling across the bilayer: namely, upon removing ambient potassium ions, changes are seen in the NMR shifts of carboxylates E118 and E120 in the pH gate in the hinges of the inner transmembrane helix (98–103), and in the selectivity filter, all of which resemble changes seen upon acid-induced opening and inhibition and suggest that ion release can trigger channel helix opening.Potassium channel activation and inactivation is fundamental to many physiological functions including muscle contraction and the generation of synaptic action potentials (1). KcsA is a 160-residue pH-activated homotetrameric K⁺ channel isolated from the soil bacterium Streptomyces lividans (2, 3) with high sequence homology and functional similarity to mammalian potassium channels (4). It has provided an excellent model for studies of ion-conduction by X-ray crystallography (3, 5, 6), electrophysiology (7, 8), and NMR (9–21). Like many potassium channels, it exhibits (4, 6, 22, 23) slow, spontaneous inactivation involving the residues near the extracellular selectivity filter subsequent to channel activation. Recent results from X-ray crystallography and molecular dynamics suggest that the gates are coupled and that inactivation is prompted by channel opening, mediated via a series of intrasubunit steric contacts involving F103 with T74, T75, and M96 and an intersubunit contact with the neighboring I100 side chain (4–6, 24, 25). In separate experiments, the extracellular gate has been observed to respond directly to ambient [K⁺]: at high [K⁺] it exists in a conductive form, and at low K⁺ it collapses into a nonconductive state (3). Our NMR studies suggest that the low [K⁺] state and the low pH inactivated state may be similar; this conclusion is supported by the effect of the mutation E71A and the pattern of chemical shift perturbations in the selectivity filter when the ion is depleted (9, 19). Meanwhile, X-ray crystallography studies suggest that mutants (E71A) unable to undergo inactivation are also unable to expel ions (26).An established similarity of the low pH and the low [K⁺] states would clarify the importance of allosteric coupling and have the practical consequence that the well-behaved low K⁺ state could serve as a useful structural proxy for the otherwise fleeting inactivated state. For these reasons we tested this correspondence using NMR experiments. If the low K⁺ state is similar to the inactivated state of KcsA achieved by lowering the pH, it is expected that structural changes indicative of channel opening observed at low [K⁺] would occur not only in the selectivity filter but also in the pH gate and the hinge region. However, some studies imply that these two gates might be uncoupled or weakly coupled. For example, X-ray crystallographic studies of KcsA, where K⁺ sensitivity was largely isolated to the selectivity filter (3). In this work, we asked whether, by contrast, full-length wild-type KcsA (160 aa) reconstituted into hydrated lipid bilayers exhibits global structural changes upon ion expulsion suggestive of channel opening. To accomplish this, nearly complete ¹³C and ¹⁵N chemical shift assignments were obtained for the transmembrane and loop regions from four-dimensional (4D) solid-state nuclear magnetic resonance (SSNMR) (27), providing numerous reporters for conformational change during ion binding. In low [K⁺] ion conditions at neutral pH, not only does KcsA expel the K⁺ ions from the inner selectivity filter sites, but the channel also exhibits chemical shift perturbations at the pH gate and the hinge of the inner transmembrane helix, suggesting features akin to the inhibited state that is present at low pH and high [K⁺]. That these two distinct conditions result in a nearly identical state of the channel offers strong evidence for transmembrane allostery in the inactivation process.     

Evidence for an allosteric mechanism of substrate release from membrane-transporter accessory binding proteins

Numerous membrane importers rely on accessory water-soluble proteins to capture their substrates. These substrate-binding proteins (SBP) have a strong affinity for their ligands; yet, substrate release onto the low-affinity membrane transporter must occur for uptake to proceed. It is generally accepted that release is facilitated by the association of SBP and transporter, upon which the SBP adopts a conformation similar to the unliganded state, whose affinity is sufficiently reduced. Despite the appeal of this mechanism, however, direct supporting evidence is lacking. Here, we use experimental and theoretical methods to demonstrate that an allosteric mechanism of enhanced substrate release is indeed plausible. First, we report the atomic-resolution structure of apo TeaA, the SBP of the Na(+)-coupled ectoine TRAP transporter TeaBC from Halomonas elongata DSM2581(T), and compare it with the substrate-bound structure previously reported. Conformational free-energy landscape calculations based upon molecular dynamics simulations are then used to dissect the mechanism that couples ectoine binding to structural change in TeaA. These insights allow us to design a triple mutation that biases TeaA toward apo-like conformations without directly perturbing the binding cleft, thus mimicking the influence of the membrane transporter. Calorimetric measurements demonstrate that the ectoine affinity of the conformationally biased triple mutant is 100-fold weaker than that of the wild type. By contrast, a control mutant predicted to be conformationally unbiased displays wild-type affinity. This work thus demonstrates that substrate release from SBPs onto their membrane transporters can be facilitated by the latter through a mechanism of allosteric modulation of the former.     

Selective allosteric coupling in core chemotaxis signaling complexes

Bacterial chemotaxis is mediated by signaling complexes that sense chemical gradients and direct bacteria to favorable environments by controlling a histidine kinase as a function of chemoreceptor ligand occupancy. Core signaling complexes contain two trimers of transmembrane chemoreceptor dimers, each trimer binding a coupling protein CheW and a protomer of the kinase dimer. Core complexes assemble into hexagons, and these form hexagonal arrays. The notable cooperativity and amplification in bacterial chemotaxis is thought to reflect allosteric interactions in cores, hexagons, and arrays, but little is known about this presumed allostery. We investigated allostery in core complexes assembled with two chemoreceptor species, each recognizing a different ligand. Chemoreceptors were inserted in Nanodiscs, which rendered them water soluble and allowed isolation of individual complexes. Neighboring dimers in receptor trimers influenced one another’s operational ligand affinity, indicating allosteric coupling. However, this coupling did not include the key function of kinase inhibition. Our data indicated that only one receptor dimer could inhibit kinase as a function of ligand occupancy. This selective allosteric coupling corresponded with previously identified structural asymmetry: only one dimer in a trimer contacts kinase and only one CheW. We suggest one of these dimers couples ligand occupancy to kinase inhibition. Additionally, we found that kinase protomers are allosterically coupled, conveying inhibition across the dimer interface. Because kinase dimers connect core complex hexagons, allosteric communication across dimer interfaces provides a pathway for receptor-generated kinase inhibition in one hexagon to spread to another, providing a crucial step for the extensive amplification characteristic of chemotactic signaling.Bacterial chemotaxis is mediated by signaling complexes that sense specific chemicals and direct bacterial cells to favorable chemical environments. They do so by controlling autophosphorylation of a chemotaxis histidine kinase and thereby phosphorylation of the chemotaxis response regulator CheY (reviewed in refs. 1 and 2). The cellular concentration of phospho-CheY determines the pattern of cellular motility and thus directional movement. In Escherichia coli and many other bacteria, signaling complexes involve stable noncovalent interactions among transmembrane bacterial chemoreceptors, histidine kinase CheA and coupling protein CheW. In vivo, signaling complexes appear as arrays that can contain tens to thousands of these components (3, 4). In the extensively characterized systems of E. coli and Salmonella enterica, arrays include several different kinds of chemoreceptors, each able to recognize one or a few specific attractants and repellents.Chemoreceptors are homodimers organized as a series of helical bundles and coiled coils extending ∼300 Å (1, 2). Receptor homodimers interact at their cytoplasmic, membrane-distal tips to form trimers of dimers (5, 6). Because the amino acid sequence of that tip region is essentially identical among the five chemoreceptors of E. coli, trimers can contain more than one type of receptor (6). Trimers interact with CheA and CheW to form signaling complexes. The minimal structural and functional core signaling complex contains two trimers of chemoreceptor homodimers, with each trimer in contact with a monomeric CheW and one of the two protomers in a CheA homodimer (Fig. 1 C and D) (7–10). Using Nanodiscs, individual core complexes can be reconstituted in vitro as water-soluble, active units (Fig. 1A) and their specific features characterized (7, 8, 11, 12). Nanodiscs are small, ∼100-nm-diameter, plugs of lipid bilayer with the hydrophobic region shielded from the aqueous environment by a protective belt of membrane scaffold protein (13), thereby rendering that small plug of bilayer water soluble. Nanodiscs made with natural E. coli lipids provide a native environment for purified transmembrane chemoreceptors (14, 15). Water-soluble core signaling complexes assembled with purified Nanodisc-inserted receptors, CheA, and CheW activate the kinase ∼750-fold, almost as well as complexes formed in vitro with chemoreceptors in their natural membranes (7). As in native arrays, the enhanced activity is controlled by receptors; specifically, it is reduced as a function of receptor ligand occupancy (7).Open in a separate windowFig. 1.The core chemotaxis signaling complex and its components. (A) Cartoon of an isolated, water-soluble core signaling complex, assembled with two Nanodisc-inserted trimers of chemoreceptor homodimers, one CheA homodimer, and two CheWs (7). (B) Domain organization of the dimer of the chemotaxis histidine kinase CheA. The two protomers of the dimer are distinguished by shading for one and a prime on the domain names of the other. P1 carries the autophosphorylated histidine, P2 binds substrate proteins CheY and CheB, P3 is the four-helix bundle dimerization domain, P4 is the catalytic domain, and P5 is the receptor-binding domain and a structural homolog of CheW. (C and D) Molecular models, derived from fitting X-ray structures to tomographic densities, of interactions among components of the core signaling complex modified from figure P1 in ref. 10. (C) View parallel to the membrane. (D) view from the membrane toward CheA and CheW.Control of kinase activity by receptor ligand occupancy exhibits notable signal amplification. In vivo it can be as much as 35-fold (16), implying that occupancy of one receptor can inhibit up to 35 kinase active sites. Such amplification is thought to involve allosteric spread of inhibition from a single ligand-occupied receptor to multiple kinases through the network of physical contacts in core signaling complexes and their arrays (1). We were interested in investigating allosteric influences in the simplest structural and functional unit, the core signaling complex. An experimental approach was suggested by studies in vivo (17) and in vitro (18) documenting that the presence of two kinds of receptors altered sensitivity and cooperativity of attractant responses mediated by each receptor type, indicating that chemoreceptors were affected by their neighbors, presumably through allosteric interactions. Thus, we characterized ligand control of kinase activity in Nanodisc-based, isolated core complexes in which both trimers contained two kinds of receptors, each recognizing a different ligand.     

Large-scale allosteric conformational transitions of adenylate kinase appear to involve a population-shift mechanism

Large-scale conformational changes in proteins are often associated with the binding of a substrate. Because conformational changes may be related to the function of an enzyme, understanding the kinetics and energetics of these motions is very important. We have delineated the atomically detailed conformational transition pathway of the phosphotransferase enzyme adenylate kinase (AdK) in the absence and presence of an inhibitor. The computed free energy profiles associated with conformational transitions offer detailed mechanistic insights into, as well as kinetic information on, the ligand binding mechanism. Specifically, potential of mean force calculations reveal that in the ligand-free state, there is no significant barrier separating the open and closed conformations of AdK. The enzyme samples near closed conformations, even in the absence of its substrate. The ligand binding event occurs late, toward the closed state, and transforms the free energy landscape. In the ligand-bound state, the closed conformation is energetically most favored with a large barrier to opening. These results emphasize the underlying dynamic nature of the enzyme and indicate that the conformational transitions in AdK are more intricate than a mere two-state jump between the crystal-bound and -unbound states. Based on the existence of the multiple conformations of the enzyme in the open and closed states, a different viewpoint of ligand binding is presented. Our estimated activation energy barrier for the conformational transition is also in reasonable accord with the experimental findings.     

Experimental basis for a new allosteric model for multisubunit proteins

Monod, Wyman, and Changeux (MWC) explained allostery in multisubunit proteins with a widely applied theoretical model in which binding of small molecules, so-called allosteric effectors, affects reactivity by altering the equilibrium between more reactive (R) and less reactive (T) quaternary structures. In their model, each quaternary structure has a single reactivity. Here, we use silica gels to trap protein conformations and a new kind of laser photolysis experiment to show that hemoglobin, the paradigm of allostery, exhibits two ligand binding phases with the same fast and slow rates in both R and T quaternary structures. Allosteric effectors change the fraction of each phase but not the rates. These surprising results are readily explained by the simplest possible extension of the MWC model to include a preequilibrium between two tertiary conformations that have the same functional properties within each quaternary structure. They also have important implications for the long-standing question of a structural explanation for the difference in hemoglobin oxygen affinity of the two quaternary structures.Small molecules can regulate protein reactivity by binding to residues distant from the active site, a phenomenon known as allostery. The classic theoretical model proposed by Monod, Wyman, and Changeux (MWC) (1, 2) for explaining this phenomenon considered only proteins with multiple subunits, and the two-state allosteric model of MWC was originally used by them to explain the functional properties of the hemoglobin tetramer, the “honorary enzyme” (1, 3). This famous model has since been applied to a wide variety of other systems in biology, including ligand-gated ion channels, G-protein–coupled receptors, nuclear receptors, and supramolecular assemblies such as chaperonins (4–7). In the case of hemoglobin, the paradigm of allostery, MWC makes two key postulates that have been extensively tested by experiments. Oxygen binding to the deoxy quaternary structure (T) is noncooperative; cooperativity arises from a shift in the population from the low-affinity T quaternary structure to the high-affinity R quaternary structure as the oxygen concentration is increased. The second postulate is that allosteric effectors regulate oxygen affinity by altering only the R ⇄ T preequilibrium. Investigations of hemoglobin focused primarily on the first postulate, which was finally confirmed by single-crystal oxygen-binding measurements that ruled out a sequential model (8) and ended a 25-y controversy (9–12). The second is of more general interest because it applies to all multisubunit proteins exhibiting allosteric behavior and has been known for many years to be inconsistent with the fact that allosteric effectors markedly affect oxygen affinity without changing the quaternary preequilibrium [see data summaries by Imai, Yonetani, and coworkers (13, 14)]. The change in oxygen affinity constants, K_T and K_R, with conditions was interpreted as indicating that tertiary conformations must be affected or that more than two quaternary states exist. Consequently, many variations of the MWC model have appeared over the years to explain this result, as well as a wide range of equilibrium, kinetic, and spectroscopic data for hemoglobin (see, for example, refs. 11, 12, and 15–20).Now that regulating protein reactivity by altering tertiary conformations is recognized as widespread in the protein world (21–26), there has been a need to extend the MWC model for multisubunit proteins to include tertiary as well as quaternary conformational changes. The tertiary two-state (TTS) model of Henry et al. (27) is the simplest possible extension and is capable of providing a quantitative explanation for a vast array of experimental data for hemoglobin. In this work, we test a key prediction of the TTS model by using a new kind of kinetic measurement in which carbon monoxide rebinding to R-state hemoglobin is measured following millisecond-to-kilosecond continuous-wave (cw) photodissociation of silica gel-encapsulated hemoglobin.Silica gels slow conformational changes in hemoglobin by many orders of magnitude without altering equilibria (28–30), as shown by identical oxygen binding curves in the gel and in solution (Fig. 1). Consequently, it is possible to interrogate properties of protein conformations that are undetectable in both kinetic and equilibrium experiments in solution, but are key for testing theoretical models. In a previous study, we carried out nanosecond pulsed laser photolysis experiments on HbCO encapsulated and thereby trapped in either the T or R quaternary structures, corresponding to the arrangement of the four subunits in fully unliganded and fully liganded hemoglobin, respectively (Fig. 2B) (34). We discovered that, although the R and T quaternary structures show a single fast and slow CO rebinding phase in the presence of allosteric effectors, respectively, in the absence of allosteric effectors the T quaternary structure exhibits two phases—the expected slow phase and, surprisingly, a much faster phase with a rate identical to that found in R (Fig. 2B). This fast rate was interpreted in terms of the TTS model (Fig. 3) as arising from subunit conformations of liganded T trapped by the gel in the r tertiary conformation that is functionally identical to unliganded r subunits in the R quaternary structure. Because there is no exchange of tertiary conformations during ligand rebinding, the experiment measures the equilibrium fraction of r subunits in liganded T.Open in a separate windowFig. 1.Hill plots for oxygen binding to hemoglobin in solution, gel, crystal, and sickle fiber (y is fractional saturation with oxygen and p is oxygen pressure). (Inset) Comparison of p50 (the oxygen pressure at half fractional saturation of the four hemes) in the absence of allosteric effectors in solution and gel. T⁺, R⁺ and T⁻, R⁻ refer to the T, R quaternary structures in the presence and absence of the allosteric effectors, inositol hexaphosphate (IHP) and bezafibrate (Bzf). The striking identity of the oxygen affinities in solution, gel, and crystal show that both the gel and the crystal trap unstable tertiary and quaternary structures, but do not alter their equilibrium properties. The extreme low affinity (highest p50) occurs because the liganded molecule (in the sickle fiber also) remains in the t tertiary conformation of the tertiary two-state model. Detailed solvent conditions: gray circles (R⁻ gel): Hb encapsulated in gel as R from Shibayama: 100 mM phosphate, pH 7, 20 °C, p50 = 0.16 torr, n = 0.86 (28). Violet circles (R⁻ crystal): Hb C crystals in R quaternary structure, 0.8 M NaH₂PO₄, 1.7 M K₂HPO₄, pH 7.2, 21–22 °C, p50 = 0.32 torr, n = 1.03 (31). Magenta dashed line [R⁻ solution (soln)]: binding of fourth oxygen, K₄, in solution, 100 mM Hepes, pH 7.0, 15 °C, 1/K₄ = 0.18 (14). Orange continuous line (T⁻ soln): binding of first oxygen, K₁, in solution, 20 mM Bis-Tris, 5 mM Cl⁻, 1 μM EDTA, pH 7.6, 25 °C, 1/K₁ = 7.6 torr (32) [data normalized to 15 °C through correction factor from Imai (13)]. Dark blue dashed line (T⁻ gel): Hb encapsulated in gel in the absence of allosteric effectors, 100 mM Hepes, pH 7.6, 15 °C, p50 = 7.9 torr, n = 1 (33). Green dashed line (T⁺ gel): Hb encapsulated in gel as T in the presence of allosteric effectors, 100 mM Hepes, 10 mM IHP, 2 mM Bzf, 200 mM Cl⁻, 1 mM EDTA, pH 7.0, 15 °C (34). Red dashed line (T crystal): Hb crystals in 10 mM potassium phosphate, 54% (wt/vol) PEG 1000, 2 mM IHP, 1 mM EDTA, 15 °C, pH 7.0, p50 = 135 torr, n = 0.97 (35). Open circles (T sickle fiber): sickle cell fibers, 23.5 °C (36). Light blue continuous line (T⁺ soln): binding of first oxygen in solution in the presence of allosteric effectors, 100 mM Hepes, 2 mM IHP, 10 mM Bzf, 100 mM M Cl⁻, pH 7.0, 15 °C, 1/K₁ = 139 torr (14). (Inset) p50 dependence on pH. T⁻ gel, dark blue circles (33); T⁻ soln, orange circles (32).Open in a separate windowFig. 2.Nanosecond pulsed laser photolysis experiments of CO rebinding with cartoon explanation. (A) CO rebinding in solution. Ligand rebinding is characterized by three phases—unimolecular geminate rebinding, bimolecular rebinding to the R quaternary structure, and a slower bimolecular phase corresponding to rebinding to molecules that have switched from the R to the T quaternary structure (37). (B) CO rebinding in gel to hemoglobin in the R quaternary structure (red curve; labeled R⁻), to the T quaternary structure in the absence of allosteric effectors (cyan curve; labeled T⁻), to the T quaternary structure in the presence of the allosteric effectors IHP and BZF (blue curve; labeled T⁺). Solution and gel experiments were performed at 20 °C and at 0.2 and 1 atm CO, respectively. The black curve is a linear combination of the CO rebinding curves for the T⁺ and R gels that optimally superimposes on the T⁻ curve. (C) Cartoon interpretation of experiments in gels, where squares represent slow binding (t) subunit conformations, and circles, fast binding (r) subunits. The open symbols signify unliganded, and closed symbols, liganded. The gel traps both tertiary and quaternary structures for the duration of the experiment. The fast rebinding r conformation is not observed in pulsed laser photolysis of HbCO in the T quaternary structure in solution (38), because it relaxes too fast to the slow rebinding (t) conformation characteristic of the fully unliganded T quaternary structure before any significant geminate rebinding occurs (27).Open in a separate windowFig. 3.Schematic structures of the MWC and TTS models for a dimer with equivalent subunits. The subset of MWC is enclosed with a green dashed line. In both T and R quaternary structures, the empty and filled symbols correspond to unliganded and liganded subunits; squares, to less reactive (t) tertiary conformations; and circles, to more reactive (r) tertiary conformations of the TTS model. For clarity, degenerate states (which introduce statistical factors in the partition function) are not shown. The relative lengths of the arrows indicate that r has a higher reactivity than t, that the reactivity of t is the same in both quaternary structures, as is the case for r, that the T quaternary structure biases the tertiary equilibrium toward t, and that the R quaternary structure biases the tertiary conformational equilibrium toward r. Although the partition function for a tetramer can be as mathematically compact as that of a dimer (SI Text), a similar diagram for a tetramer is much too complex.The TTS model is the simplest extension of the MWC model that includes a tertiary as well as a quaternary preequilibrium (27) (Fig. 3). The model had previously explained the appearance of a T-like optical absorption spectrum as a tertiary conformational relaxation from r to t within unliganded R before the R → T transition following nanosecond photodissociation of R-HbCO in solution (27). The TTS model has also been used by Spiro and coworkers (39) to explain the kinetics of structural changes observed by time-resolved resonance Raman experiments. To distinguish between the TTS model and other theoretical models for hemoglobin allostery (12), it became essential to determine the functional properties of the t subunits of unliganded R. We therefore designed a new kind of laser photolysis experiment that consists of extended cw illumination of HbCO encapsulated in the R quaternary structure to create an equilibrium population of unliganded subunits. It was expected that the gel would also prevent tertiary conformational exchange during ligand rebinding, so that the rebinding kinetics of individual conformations could be measured in the absence of any tertiary conformational exchange dynamics.     

Direct analysis of cooperativity in multisubunit allosteric proteins

Allosteric regulation of protein function is a fundamental phenomenon of major importance in many cellular processes. Such regulation is often achieved by ligand-induced conformational changes in multimeric proteins that may give rise to cooperativity in protein function. At the heart of allosteric mechanisms offered to account for such phenomenon, involving either concerted or sequential conformational transitions, lie changes in intersubunit interactions along the ligation pathway of the protein. However, structure–function analysis of such homooligomeric proteins by means of mutagenesis, although it provides valuable indirect information regarding (allosteric) mechanisms of action, it does not define the contribution of individual subunits nor interactions thereof to cooperativity in protein function, because any point mutation introduced into homooligomeric proteins will be present in all subunits. Here, we present a general strategy for the direct analysis of cooperativity in multisubunit proteins that combines measurement of the effects on protein function of all possible combinations of mutated subunits with analysis of the hierarchy of intersubunit interactions, assessed by using high-order double-mutant cycle-coupling analysis. We show that the pattern of high-order intersubunit coupling can serve as a discriminative criterion for defining concerted versus sequential conformational transitions underlying protein function. This strategy was applied to the particular case of the voltage-activated potassium channel protein (Kv) to provide compelling evidence for a concerted all-or-none activation gate opening of the Kv channel pore domain. An direct and detailed analysis of the contribution of high-order intersubunit interactions to cooperativity in the function of an allosteric protein has not previously been presented.     

Intrinsic disorder as a generalizable strategy for the rational design of highly responsive,allosterically cooperative receptors

Control over the sensitivity with which biomolecular receptors respond to small changes in the concentration of their target ligand is critical for the proper function of many cellular processes. Such control could likewise be of utility in artificial biotechnologies, such as biosensors, genetic logic gates, and “smart” materials, in which highly responsive behavior is of value. In nature, the control of molecular responsiveness is often achieved using “Hill-type” cooperativity, a mechanism in which sequential binding events on a multivalent receptor are coupled such that the first enhances the affinity of the next, producing a steep, higher-order dependence on target concentration. Here, we use an intrinsic-disorder–based mechanism that can be implemented without requiring detailed structural knowledge to rationally introduce this potentially useful property into several normally noncooperative biomolecules. To do so, we fabricate a tandem repeat of the receptor that is destabilized (unfolded) via the introduction of a long, unstructured loop. The first binding event requires the energetically unfavorable closing of this loop, reducing its affinity relative to that of the second binding event, which, in contrast occurs at a preformed site. Using this approach, we have rationally introduced cooperativity into three unrelated DNA aptamers, achieving in the best of these a Hill coefficient experimentally indistinguishable from the theoretically expected maximum. The extent of cooperativity and thus the steepness of the binding transition are, moreover, well modeled as simple functions of the energetic cost of binding-induced folding, speaking to the quantitative nature of this design strategy.The ability to control the shape and midpoint of binding curves is critical to nature’s ability to optimize many cellular processes (1). One of the most widely used mechanisms by which nature so tunes the behavior of her receptors is allostery, in which the binding of one ligand alters the affinity with which subsequent ligands bind. Allostery comes in two “flavors.” Heterotropic allostery, in which the two ligands differ, provides a means of shifting the midpoint of a binding curve to higher or lower target concentrations without changing the curve’s intrinsically hyperbolic shape and thus without altering its sensitivity to small changes in the relative concentration of its molecular target (Fig. 1, Left). An example is the binding of bisphosphoglycerate to mammalian hemoglobin, which decreases the protein’s affinity for oxygen, thus pushing its binding curve to higher concentrations and enhancing oxygen transport efficiency, while leaving the intrinsic shape of its binding curve unaltered. Homotropic allostery, in contrast, occurs when the ligands are the same; that is, when the binding of one copy of a ligand changes the affinity with which subsequent copies of the same molecule bind. This mechanism, commonly referred to as “cooperativity,” changes not only the placement but also the shape of the binding curve, producing either a more responsive, higher-order dependence on ligand concentration (positive cooperativity) (Fig. 1, Right) or a less responsive, lower-order dependence (negative cooperativity). Like heterotropic allostery, cooperativity is also seen in the function of hemoglobin; the protein uses this mechanism to bind four oxygen molecules in a positively cooperative, approximately “all-or-nothing” fashion, steepening its binding curve and enhancing its ability to deliver oxygen over the rather modest concentration gradient present between the lungs and the peripheral tissues.Open in a separate windowFig. 1.Nature often controls the shape and position of ligand–response curves via allostery. (Left) In heterotropic allostery, the binding of one ligand to a receptor increases or decreases the affinity with which a second, different ligand binds, shifting the placement of the binding curve without altering its shape and thus without altering the width of its useful dynamic range (shaded boxes) or, in turn, its sensitivity to small changes in target concentration. (Right) In homotropic allostery, in contrast, the binding of one copy of target ligand changes the affinity with which additional copies of the same ligand bind, altering both the placement and the shape of the binding curve. The latter effect allows the system to respond more (positive cooperativity) or less (negative cooperativity) sensitively to changes in target ligand concentration. For positive cooperativity, receptor occupancy is a higher (than unity) order function of target concentration, with the exponent, n_H, being known as the Hill coefficient.The ubiquity with which nature exploits homotropic and heterotropic allostery has motivated efforts to rationally engineer these mechanisms into biomolecular receptors normally lacking them, both to test our understanding of the principles underlying these effects and to harness them to improve the utility of artificial biotechnologies. The rational introduction of heterotropic allostery into otherwise nonallosteric receptors, for example, has seen significant prior exploration (e.g., refs. 2–8), with both mechanical coupling (e.g., refs. 2 and 5–8) and mutually exclusive folding (e.g., refs. 3 and 4) approaches all having been used to successfully introduce this useful mechanism into a range of protein- and nucleic acid-based receptors. The design of allosterically cooperative receptors, in contrast, has seen far less success. That is, although a handful of examples of rationally designed cooperativity have been reported to date (9–12), no general approach has previously been reported by which such behavior can be rationally introduced into any arbitrarily complex biomolecule. This failure has limited the extent to which cooperativity, which could provide a powerful means of improving the ability of artificial biotechnologies to respond to small changes in molecular concentration (9, 13), can be applied in applications, such as biosensing (14, 15), “smart” drug delivery materials (16, 17), and molecular (18) and genetic (19) logic gates, in which such enhanced responsiveness would be of value.Two reasons account for why, despite its underlying simplicity and elegance, achieving the rational design of positive cooperativity has proven far from straightforward. First, to achieve the effect requires the creation of systems in which a higher affinity site is occupied only after a lower affinity site (which would normally be filled only at higher ligand concentrations) that binds the same ligand is already filled. This contrasts sharply with heterotropic allostery, in which the two binding sites typically exhibit little if any cross-reactivity. Second, all of the binding sites of a cooperative receptor recognize copies of the same ligand, rendering it more difficult to alter the affinity of one independently of that of the others. This is again in contrast to heterotropic allostery, in which each binding site is chemically distinct, allowing each to be independently optimized. Given these difficulties, and given the relative infancy of biomolecular design efforts (20–22), the ability to perform the structure-based design of cooperativity appears beyond current capabilities except for the simplest, most well-understood receptors (9–12). Here, however, we use an approach to the rational engineering of allosterically cooperative receptors that does not require detailed, structure-based design. Indeed, our approach is so simple that it can be performed, as demonstrated here, even in the absence of detailed knowledge of the parent receptor’s structure.Our design approach is inspired by intrinsically disordered proteins, proteins that are normally unfolded and only fold upon binding their target ligand. Specifically, both theoretical (23) and experimental (24, 25) studies have demonstrated that the global conformation change these proteins undergo upon an initial ligand binding event provides a convenient means of preorganizing a second, distal ligand binding site. This improves the affinity of the second binding event (because binding need no longer pay the unfavorable cost associated with folding), leading to positive allosteric behavior. Ferreon et al. (24), for example, have shown that the intrinsically disordered oncoprotein adenovirus early region 1A (E1A) folds upon binding either of its two (different) target ligands (CREB binding protein or retinoblastoma protein), thus increasing the affinity with which the second ligand binds and rendering the system heterotropically allosteric. In addition, Furukawa et al. (25) have shown that the partially intrinsically disordered protein STIM 1 exhibits strongly homotropic allosteric binding to calcium. Here, we use this same mechanism to rationally introduce cooperativity into a number of normally noncooperative aptamers (DNA-based receptors often adopting complex tertiary folds), thus producing steeper, more responsive binding curves than those seen for the unmodified parent molecule.     

A common allosteric site and mechanism in caspases

We present a common allosteric mechanism for control of inflammatory and apoptotic caspases. Highly specific thiol-containing inhibitors of the human inflammatory caspase-1 were identified by using disulfide trapping, a method for site-directed small-molecule discovery. These compounds became trapped by forming a disulfide bond with a cysteine residue in the cavity at the dimer interface approximately 15 A away from the active site. Mutational and structural analysis uncovered a linear circuit of functional residues that runs from one active site through the allosteric cavity and into the second active site. Kinetic analysis revealed robust positive cooperativity not seen in other endopeptidases. Recently, disulfide trapping identified a similar small-molecule site and allosteric transition in the apoptotic caspase-7 that shares only a 23% sequence identity with caspase-1. Together, these studies show a general small-molecule-binding site for functionally reversing the zymogen activation of caspases and suggest a common regulatory site for the allosteric control of inflammation and apoptosis.     

Intrinsic disorder in the C-terminal domain of the Shaker voltage-activated K+ channel modulates its interaction with scaffold proteins

The interaction of membrane-embedded voltage-activated potassium channels (Kv) with intracellular scaffold proteins, such as the postsynaptic density 95 (PSD-95) protein, is mediated by the channel C-terminal segment. This interaction underlies Kv channel clustering at unique membrane sites and is important for the proper assembly and functioning of the synapse. In the current study, we address the molecular mechanism underlying Kv/PSD-95 interaction. We provide experimental evidence, based on hydrodynamic and spectroscopic analyses, indicating that the isolated C-terminal segment of the archetypical Shaker Kv channel (ShB-C) is a random coil, suggesting that ShB-C belongs to the recently defined class of intrinsically disordered proteins. We show that isolated ShB-C is still able to bind its scaffold protein partner and support protein clustering in vivo, indicating that unfoldedness is compatible with ShB-C activity. Pulldown experiments involving C-terminal chains differing in flexibility or length further demonstrate that intrinsic disorder in the C-terminal segment of the Shaker channel modulates its interaction with the PSD-95 protein. Our results thus suggest that the C-terminal domain of the Shaker Kv channel behaves as an entropic chain and support a "fishing rod" molecular mechanism for Kv channel binding to scaffold proteins. The importance of intrinsically disordered protein segments to the complex processes of synapse assembly, maintenance, and function is discussed.     

Transplanting allosteric control of enzyme activity by protein-protein interactions: coupling a regulatory site to the conserved catalytic core

Glycerol kinase from Escherichia coli, but not Haemophilus influenzae, is inhibited allosterically by phosphotransferase system protein IIA(Glc). The primary structures of these related kinases contain 501 amino acids, differing at 117. IIA(Glc) inhibition is transplanted from E. coli glycerol kinase into H. influenzae glycerol kinase by interconverting only 11 of the differences: 8 residues that interact with IIA(Glc) at the allosteric binding site and 3 residues in the conserved ATPase catalytic core that do not interact with IIA(Glc) but the solvent accessible surface of which decreases when it binds. The three core residues are crucial for coupling the allosteric site to the conserved catalytic core of the enzyme. The site of the coupling residues identifies a regulatory locus in the sugar kinase/heat shock protein 70/actin superfamily and suggests relations between allosteric regulation and the active site closure that characterizes the family. The location of the coupling residues provides empirical validation of a computational model that predicts a coupling pathway between the IIA(Glc)-binding site and the active site [Luque, I. & Freire, E. (2000) Proteins Struct. Funct. Genet. Suppl. 4, 63-71]. The requirement for changes in core residues to couple the allosteric and active sites and switching from inhibition to activation by a single amino acid change are consistent with a postulated mechanism for molecular evolution of allosteric regulation.     

Principles underlying energetic coupling along an allosteric communication trajectory of a voltage-activated K+ channel

The information flow between distal elements of a protein may rely on allosteric communication trajectories lying along the protein's tertiary or quaternary structure. To unravel the underlying features of energy parsing along allosteric pathways in voltage-gated K(+) channels, high-order thermodynamic coupling analysis was performed. We report that such allosteric trajectories are functionally conserved and delineated by well defined boundaries. Moreover, allosteric trajectories assume a hierarchical organization whereby increasingly stronger layers of cooperative residue interactions act to ensure efficient and cooperative long-range coupling between distal channel regions. Such long-range communication is brought about by a coupling of local and global conformational changes, suggesting that the allosteric trajectory also corresponds to a pathway of physical deformation. Supported by theoretical analyses and analogy to studies analyzing the contribution of long-range residue coupling to protein stability, we propose that such experimentally derived trajectory features are a general property of allosterically regulated proteins.     

Paired moving charge model of energy coupling. III. Intrinsic ionophores in energy coupling systems.

The experimental basis for the postulated role of intrinsic ionophores in mitochondrial ion transport and energy coupling is summarized. Intrinsic ionophores appear to be linked to, or contained within, specific ionophoroproteins localized in the inner membrane, and the isolation of these ionophores requires their release from the ionophoroproteins. At least ten different species of ionophores have been isolated from the mitochondrion, five of which have been wholly or in part chemically identified. Intrinsic ionophores have been implicated in the activation of inorganic phosphate in ATP synthesis and hydrolysis, and in the contol of the coupling modes. The presence of ionophores in soluble proteins such as troponin and in ATP-energized kinases has been demonstrated.