Intersubunit Interfaces Research Articles

Assessing potentiation of the (α4)3(β2)2 nicotinic acetylcholine receptor by the allosteric agonist CMPI

The extracellular domain of the nicotinic acetylcholine receptor isoforms formed by three α4 and two β2 subunits ((α4)3(β2)2 nAChR) harbors two high-affinity “canonical” acetylcholine (ACh)-binding sites located in the two α4:β2 intersubunit interfaces and a low-affinity “noncanonical” ACh-binding site located in the α4:α4 intersubunit interface. In this study, we used ACh, cytisine, and nicotine (which bind at both the α4:α4 and α4:β2 interfaces), TC-2559 (which binds at the α4:β2 but not at the α4:α4 interface), and 3-(2-chlorophenyl)-5-(5-methyl-1-(piperidin-4-yl)-1H-pyrrazol-4-yl)isoxazole (CMPI, which binds at the α4:α4 but not at the α4:β2 interface), to investigate the binding and gating properties of CMPI at the α4:α4 interface. We recorded whole-cell currents from Xenopus laevis oocytes expressing (α4)3(β2)2 nAChR in response to applications of these ligands, alone or in combination. The electrophysiological data were analyzed in the framework of a modified Monod–Wyman–Changeux allosteric activation model. We show that CMPI is a high-affinity, high-efficacy agonist at the α4:α4 binding site and that its weak direct activating effect is accounted for by its inability to productively interact with the α4:β2 sites. The data presented here enhance our understanding of the functional contributions of ligand binding at the α4:α4 subunit interface to (α4)3(β2)2 nAChR-channel gating. These findings support the potential use of α4:α4 specific ligands to increase the efficacy of the neurotransmitter ACh in conditions associated with decline in nAChRs activity in the brain.

The general anesthetic etomidate and fenamate mefenamic acid oppositely affect GABAAR and GlyR: a structural explanation.

GABA and glycine act as inhibitory neurotransmitters in the CNS. Inhibitory neurotransmission is mediated via activation of ionotropic GABAA and glycine receptors. We used a modeling approach to explain the opposite effects of the general anesthetic etomidate (ETM) and fenamate mefenamic acid (MFA) on GABA- and glycine-activated currents recorded in isolated cerebellar Purkinje cells and hippocampal pyramidal neurons, respectively. These drugs potentiated GABAARs but blocked GlyRs. We built a homology model of α1β GlyR based on the cryo-EM structure of open α1 GlyR, used the α1β3γ2 GABAAR structure from the PDB, and applied Monte-Carlo energy minimization to optimize models of receptors and ligand-receptor complexes. In silico docking suggests that ETM/MFA bind at the transmembrane β( +)/α( -) intersubunit interface in GABAAR. Our models predict that the bulky side chain of the highly conserved Arg19' residue at the plus interface side wedges the interface and maintains the conducting receptor state. We hypothesized that MFA/ETM binding at the β( +)/α( -) interface leads to prolongation of receptor life-time in the open state. Having analyzed different GABAAR and GlyR structures available in the PDB, we found that mutual arrangement of the Arg19' and Gln-26' side chains at the plus and minus interface sides, respectively, plays an important role when the receptor switches from the open to closed state. We show that this process is accompanied by narrowing of the intersubunit interfaces, leading to extrusion of the Arg19' side chain from the interface. Our models allow us to explain the lack of GlyR potentiation in our electrophysiological experiments.

Selective Elimination of the Key Subunit Interfaces Facilitates Conversion of Native 24-mer Protein Nanocage into 8-mer Nanorings.

Living systems utilize proteins as building blocks to construct a large variety of self-assembled nanoscale architectures. Yet, creating protein-based assemblies with specific geometries in the laboratory remains challenging. Here, we present a new approach that completely eliminates one natural intersubunit interface of multisubunit protein architecture with high symmetry, resulting in reassembly of the protein architecture into one with lower symmetry. We have applied this approach to the conversion of the 24-mer cage-like ferritin into non-native 8-mer protein nanorings in solution. In the crystal structure, such newly formed nanorings connect with each other through hydrogen bonding in a repeating head-to-tail pattern to form nanotubes, and adjacent nanotubes are staggered relative to one another to create three-dimensional porous protein assemblies. The above strategy allows the study of conversion between protein architectures with different geometries by adjusting the interactions at the intersubunit interfaces, and the fabrication of novel bio-nanomaterials with different geometries.

Mutational Analysis at Intersubunit Interfaces of an Anionic Glutamate Receptor Reveals a Key Interaction Important for Channel Gating by Ivermectin.

The broad-spectrum anthelmintic drug ivermectin (IVM) activates and stabilizes an open-channel conformation of invertebrate chloride-selective glutamate receptors (GluClRs), thereby causing a continuous inflow of chloride ions and sustained membrane hyperpolarization. These effects suppress nervous impulses and vital physiological processes in parasitic nematodes. The GluClRs are pentamers. Homopentameric receptors assembled from the Caenorhabditis elegans (C. elegans) GluClα (GLC-1) subunit can inherently respond to IVM but not to glutamate (the neurotransmitter). In contrast, heteromeric GluClα/β (GLC-1/GLC-2) assemblies respond to both ligands, independently of each other. Glutamate and IVM bind at the interface between adjacent subunits, far away from each other; glutamate in the extracellular ligand-binding domain, and IVM in the ion-channel pore periphery. To understand the importance of putative intersubunit contacts located outside the glutamate and IVM binding sites, we introduced mutations at intersubunit interfaces, between these two binding-site types. Then, we determined the effect of these mutations on the activation of the heteromeric mutant receptors by glutamate and IVM. Amongst these mutations, we characterized an α-subunit point mutation located close to the putative IVM-binding pocket, in the extracellular end of the first transmembrane helix (M1). This mutation (αF276A) moderately reduced the sensitivity of the heteromeric GluClαF276A/βWT receptor to glutamate, and slightly decreased the receptor subunits’ cooperativity in response to glutamate. In contrast, the αF276A mutation drastically reduced the sensitivity of the receptor to IVM and significantly increased the receptor subunits’ cooperativity in response to IVM. We suggest that this mutation reduces the efficacy of channel gating, and impairs the integrity of the IVM-binding pocket, likely by disrupting important interactions between the tip of M1 and the M2-M3 loop of an adjacent subunit. We hypothesize that this physical contact between M1 and the M2-M3 loop tunes the relative orientation of the ion-channel transmembrane helices M1, M2 and M3 to optimize pore opening. Interestingly, pre-exposure of the GluClαF276A/βWT mutant receptor to subthreshold IVM concentration recovered the receptor sensitivity to glutamate. We infer that IVM likely retained its positive modulation activity by constraining the transmembrane helices in a preopen orientation sensitive to glutamate, with no need for the aforementioned disrupted interactions between M1 and the M2-M3 loop.

Allosteric HIV-1 integrase inhibitors promote aberrant protein multimerization by directly mediating inter-subunit interactions: Structural and thermodynamic modeling studies.

Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) bind at the dimer interface of the IN catalytic core domain (CCD), and potently inhibit HIV-1 by promoting aberrant, higher-order IN multimerization. Little is known about the structural organization of the inhibitor-induced IN multimers and important questions regarding how ALLINIs promote aberrant IN multimerization remain to be answered. On the basis of physical chemistry principles and from our analysis of experimental information, we propose that inhibitor-induced multimerization is mediated by ALLINIs directly promoting inter-subunit interactions between the CCD dimer and a C-terminal domain (CTD) of another IN dimer. Guided by this hypothesis, we have built atomic models of inter-subunit interfaces in IN multimers by incorporating information from hydrogen-deuterium exchange (HDX) measurements to drive protein-protein docking. We have also developed a novel free energy simulation method to estimate the effects of ALLINI binding on the association of the CCD and CTD. Using this structural and thermodynamic modeling approach, we show that multimer inter-subunit interface models can account for several experimental observations about ALLINI-induced multimerization, including large differences in the potencies of various ALLINIs, the mechanisms of resistance mutations, and the crucial role of solvent exposed R-groups in the high potency of certain ALLINIs. Our study predicts that CTD residues Tyr226, Trp235 and Lys266 are involved in the aberrant multimer interfaces. The key finding of the study is that it suggests the possibility of ALLINIs facilitating inter-subunit interactions between an external CTD and the CCD-CCD dimer interface.

Subunit stoichiometry and arrangement in a heteromeric glutamate-gated chloride channel

The invertebrate glutamate-gated chloride-selective receptors (GluClRs) are ion channels serving as targets for ivermectin (IVM), a broad-spectrum anthelmintic drug used to treat human parasitic diseases like river blindness and lymphatic filariasis. The native GluClR is a heteropentamer consisting of α and β subunit types, with yet unknown subunit stoichiometry and arrangement. Based on the recent crystal structure of a homomeric GluClαR, we introduced mutations at the intersubunit interfaces where Glu (the neurotransmitter) binds. By electrophysiological characterization of these mutants, we found heteromeric assemblies with two equivalent Glu-binding sites at β/α intersubunit interfaces, where the GluClβ and GluClα subunits, respectively, contribute the "principal" and "complementary" components of the putative Glu-binding pockets. We identified a mutation in the IVM-binding site (far away from the Glu-binding sites), which significantly increased the sensitivity of the heteromeric mutant receptor to both Glu and IVM, and improved the receptor subunits' cooperativity. We further characterized this heteromeric GluClR mutant as a receptor having a third Glu-binding site at an α/α intersubunit interface. Altogether, our data unveil heteromeric GluClR assemblies having three α and two β subunits arranged in a counterclockwise β-α-β-α-α fashion, as viewed from the extracellular side, with either two or three Glu-binding site interfaces.

Interrogation of the intersubunit interface of the open Hv1 proton channel with a probe of allosteric coupling

The Hv1 voltage-gated proton channel is a dimeric complex consisting of two voltage-sensing domains (VSDs), each containing a gated proton permeation pathway. Dimerization is controlled by a cytoplasmic coiled-coil domain. The transitions from the closed to the open state in the two VSDs are known to occur cooperatively; however, the underlying mechanism is poorly understood. Intersubunit interfaces play a critical role in allosteric processes; but, such interfaces have not been determined in the open Hv1 channel. Here we show that 2-guanidinothiazole derivatives block the two Hv1 VSDs in a cooperative way, and use one of the compounds as a probe of allosteric coupling between open subunits. We find that the extracellular ends of the first transmembrane segments of the VSDs form the intersubunit interface that mediates coupling between binding sites, while the coiled-coil domain does not directly participate in the process. We also find strong evidence that the channel’s proton selectivity filter controls blocker binding cooperativity.

Allosteric Modulation of GABA-A Receptors by Different Drugs

Understanding how drugs allosterically modulate GABA-A receptor (GABAAR) channel function remains a challenge. Anesthetic drugs like etomidate, propofol and pentobarbital bind at transmembrane domain (TMD) inter-subunit interfaces at different locations. Benzodiazepines bind at the αγ interface in the extracellular domain (ECD) and neurosteroids are believed to bind within the α-subunit TMD helix bundle. A common feature of these drugs is that they potentiate GABA-induced currents. Here, we examined whether structural mechanisms underlying their functional effects are distinct. We monitored motions induced individually by these drugs at each inter-subunit TMD interface and also tested whether modulation caused by pairs of drugs binding to distinct sites are additive or super-additive. We individually introduced a cysteine in the M1 helices of each subunit at α1I227, β2L223 and γ2LI238 to probe the different inter-subunit interfaces. We expressed wild-type and mutant α1β2γ2L GABAARs in Xenopus oocytes and measured rates of modification of the substituted cysteines in the absence and presence of GABA and different allosteric modulators: the anesthetics pentobarbital (PB) and etomidate, the neurosteroid THDOC and the benzodiazepine flurazepam. GABA activation significantly increased MTSEA modification of αI227C, βL223C and γI238C. PB activation increased modification of αI227C and γI238C but not βL223C. Modulating concentrations of PB or etomidate had no effects. THDOC significantly increased the rate of modification of βL223C whereas flurazepam increased γI238C modification. We found that potentiation of EC10 GABA currents by combining modulating concentrations of THDOC and flurazepam was the sum of the potentiation produced by the two drugs individually. Combining PB and THDOC produced potentiation more than the sum of the potentiation produced by the two drugs individually. Our data suggests that drugs binding to different sites act via distinct mechanisms. Further analysis is underway to determine if these mechanisms are independent or coupled.

Origin of Asymmetry at the Intersubunit Interfaces of V1-ATPase from Thermusthermophilus

V-type ATPase (V-ATPase) is one of the rotary ATPase complexes that mediate energy conversion between the chemical energy of ATP and the ion gradient across the membrane through a rotary catalytic mechanism. Because V-ATPase has structural features similar to those of well-studied F-type ATPase, the structure is expected to highlight the common essence of the torque generation of rotary ATPases. Here, we report a complete model of the extra-membrane domain of the V-ATPase (V1-ATPase) of a thermophilic bacterium, Thermus thermophilus, consisting of three A subunits, three B subunits, one D subunit, and one F subunit. The X-ray structure at 3.9Å resolution provides detailed information about the interactions between A3B3 and DF subcomplexes as well as interactions among the respective subunits, which are defined by the properties of side chains. Asymmetry at the intersubunit interfaces was detected from the structural differences among the three AB pairs in the different reaction states, while the large interdomain motion in the catalytic A subunits was not observed unlike F1 from various species and V1 from Enterococcus hirae. Asymmetry is mainly realized by rigid-body rearrangements of the relative position between A and B subunits. This is consistent with the previous observations by the high-resolution electron microscopy for the whole V-ATPase complexes. Therefore, our result plausibly implies that the essential motion for the torque generation is not the large interdomain movement of the catalytic subunits but the rigid-body rearrangement of subunits.

The NTD-CTD intersubunit interface plays a critical role in assembly and stabilization of the HIV-1 capsid

BackgroundLentiviruses exhibit a cone-shaped capsid composed of subunits of the viral CA protein. The intrinsic stability of the capsid is critical for HIV-1 infection, since both stabilizing and destabilizing mutations compromise viral infectivity. Structural studies have identified three intersubunit interfaces in the HIV-1 capsid, two of which have been previously studied by mutational analysis. In this present study we analyzed the role of a third interface, that which is formed between the amino terminal domain (NTD) and carboxyl terminal domain (CTD) of adjacent subunits.ResultsWe provided evidence for the presence of the NTD-CTD interface in HIV-1 particles by engineering intersubunit NTD-CTD disulfide crosslinks, resulting in accumulation of disulfide-linked oligomers up to hexamers. We also generated and characterized a panel of HIV-1 mutants containing substitutions at this interface. Some mutants showed processing defects and altered morphology from that of wild type, indicating that the interface is important for capsid assembly. Analysis of these mutants by transmission electron microscopy corroborated the importance of this interface in assembly. Other mutants exhibited quantitative changes in capsid stability, many with unstable capsids, and one mutant with a hyperstable capsid. Analysis of the mutants for their capacity to saturate TRIMCyp-mediated restriction in trans confirmed that the unstable mutants undergo premature uncoating in target cells. All but one of the mutants were markedly attenuated in replication owing to impaired reverse transcription in target cells.ConclusionsOur results demonstrate that the NTD-CTD intersubunit interface is present in the mature HIV-1 capsid and is critical for proper capsid assembly and stability.

Interactions among Positions in the Third and Fourth Membrane-associated Domains at the Intersubunit Interface of the N-Methyl-d-aspartate Receptor Forming Sites of Alcohol Action

The N-methyl-D-aspartate (NMDA) glutamate receptor is a major target of ethanol in the brain. Previous studies have identified positions in the third and fourth membrane-associated (M) domains of the NMDA receptor GluN1 and GluN2A subunits that influence alcohol sensitivity. The predicted structure of the NMDA receptor, based on that of the related GluA2 subunit, indicates a close apposition of the alcohol-sensitive positions in M3 and M4 between the two subunit types. We tested the hypothesis that these positions interact to regulate receptor kinetics and ethanol sensitivity by using dual substitution mutants. In single-substitution mutants, we found that a position in both subunits adjacent to one previously identified, GluN1(Gly-638) and GluN2A(Phe-636), can strongly regulate ethanol sensitivity. Significant interactions affecting ethanol inhibition and receptor deactivation were observed at four pairs of positions in GluN1/GluN2A: Gly-638/Met-823, Phe-639/Leu-824, Met-818/Phe-636, and Leu-819/Phe-637; the latter pair also interacted with respect to desensitization. Two interactions involved a position in M4 of both subunits, GluN1(Met-818) and GluN2A(Leu-824), that does not by itself alter ethanol sensitivity, whereas a previously identified ethanol-sensitive position, GluN2A(Ala-825), did not unequivocally interact with any other position tested. These results also indicate a shift by one position of the predicted alignment of the GluN1 M4 domain. These findings have allowed for the refinement of the NMDA receptor M domain structure, demonstrate that this region can influence apparent agonist affinity, and support the existence of four sites of alcohol action on the NMDA receptor, each consisting of five amino acids at the M3-M4 domain intersubunit interfaces.

The Ryanodine Receptor N-Terminal Disease Hot Spot Intersubunit Interface is Disrupted by Channel Opening and Affected by Disease Mutations Acting via Long-Range Structural Changes

Ryanodine Receptors (RyRs) are intracellular calcium-release channels acting as one of the key regulatory elements in the excitation-contraction coupling. More than 350 mutations have been found in RyRs that are known to underlie severe genetic diseases. Mutations in the skeletal muscle isoform (RyR1) are associated with malignant hyperthermia (MH) and central core disease (CCD), while mutations in the cardiac isoform (RyR2) cause catecholaminergic polymorphic ventricular tachycardia (CPVT) and arrhythmogenic right ventricular dysplasia (ARVD). Most mutations confer a gain of function, but the precise mechanisms that explain enhanced channel opening up to the molecular scale have remained elusive. Here we present pseudo-atomic models of the N-terminal disease hot spot in the open and closed states of the RyR, along with crystal structures of several disease mutants. The data show that the intersubunit interfaces formed by tetrameric N-terminal disease hot spots are disrupted upon channel opening in wild-type RyRs. This intersubunit interface harbors 19 disease mutations, the largest cluster within the N-terminal region, indicating the vulnerability of this interface in channel regulation. We present crystal structures and thermal stabilities of nine disease mutants located at other interfaces. The effect of most mutations are destabilizing to the protein, with decreases in melting temperatures as large as ∼10°C. Buried disease mutations cause structural changes to the intersubunit interface, while mutations affecting ionic pairing at the intra-subunit interface significantly alter relative domain orientations. Mutations far away from the intersubunit interface can thus affect these contacts via long-range conformational changes. These results illuminate the intersubunit interface between N-terminal disease hot spots as a prime target for disease mutations through direct or indirect conformational changes.

Properties and Crystal Structure of Methylenetetrahydrofolate Reductase from Thermus thermophilus HB8

BackgroundMethylenetetrahydrofolate reductase (MTHFR) is one of the enzymes involved in homocysteine metabolism. Despite considerable genetic and clinical attention, the reaction mechanism and regulation of this enzyme are not fully understood because of difficult production and poor stability. While recombinant enzymes from thermophilic organisms are often stable and easy to prepare, properties of thermostable MTHFRs have not yet been reported.Methodology/Principal FindingsMTHFR from Thermus thermophilus HB8, a homologue of Escherichia coli MetF, has been expressed in E. coli and purified. The purified MTHFR was chiefly obtained as a heterodimer of apo- and holo-subunits, that is, one flavin adenine dinucleotide (FAD) prosthetic group bound per dimer. The crystal structure of the holo-subunit was quite similar to the β8α8 barrel of E. coli MTHFR, while that of the apo-subunit was a previously unobserved closed form. In addition, the intersubunit interface of the dimer in the crystals was different from any of the subunit interfaces of the tetramer of E. coli MTHFR. Free FAD could be incorporated into the apo-subunit of the purified Thermus enzyme after purification, forming a homodimer of holo-subunits. Comparison of the crystal structures of the heterodimer and the homodimer revealed different intersubunit interfaces, indicating a large conformational change upon FAD binding. Most of the biochemical properties of the heterodimer and the homodimer were the same, except that the homodimer showed ≈50% activity per FAD-bound subunit in folate-dependent reactions.Conclusions/SignificanceThe different intersubunit interfaces and rearrangement of subunits of Thermus MTHFR may be related to human enzyme properties, such as the allosteric regulation by S-adenosylmethionine and the enhanced instability of the Ala222Val mutant upon loss of FAD. Whereas E. coli MTHFR was the only structural model for human MTHFR to date, our findings suggest that Thermus MTHFR will be another useful model for this important enzyme.

An Epilepsy-Related Region in the GABAA Receptor Mediates Long-Distance Effects on GABA and Benzodiazepine Binding Sites

The GABA(A) receptor mutation gamma(2)R43Q causes absence epilepsy in humans. Homology modeling suggests that gamma(2)Arg43, gamma(2)Glu178, and beta(2)Arg117 participate in a salt-bridge network linking the gamma(2) and beta(2) subunits. Here we show that several mutations at these locations exert similar long-distance effects on other intersubunit interfaces involved in GABA and benzodiazepine binding. These mutations alter GABA-evoked receptor kinetics by slowing deactivation, enhancing desensitization, or both. Kinetic modeling and nonstationary noise analysis for gamma(2)R43Q reveal that these effects are due to slowed GABA unbinding and slowed recovery from desensitization. Both gamma(2)R43Q and beta(2)R117K also speed diazepam dissociation from the receptor's benzodiazepine binding interface, as assayed by the rate of decay of diazepam-induced potentiation of GABA-evoked currents. These data demonstrate that gamma(2)Arg43 and beta(2)Arg117 similarly regulate the stability of both the GABA and benzodiazepine binding sites at the distant beta/alpha and alpha/gamma intersubunit interfaces, respectively. A simple explanation for these results is that gamma(2)Arg43 and beta(2)Arg117 participate in interactions between the gamma(2) and beta(2) subunits, disruptions of which alter the neighboring intersubunit binding sites in a similar fashion. In addition, gamma(2)Arg43 and gamma(2)Glu178 regulate desensitization, probably mediated within the transmembrane domains near the pore. Therefore, mutations at the gamma/beta intersubunit interface have specific long-distance effects that are propagated widely throughout the GABA(A) receptor protein.