Subunit Interface Research Articles

Contrasting Effects of the γ-Aminobutyric Acid Type A Receptor β3 Subunit N265M Mutation on Loss of Righting Reflexes Induced by Etomidate and the Novel Anesthetic Barbiturate R-mTFD-MPAB.

Previous studies have shown that etomidate modulates γ-aminobutyric acid type A receptors by binding at the β-α subunit interface within the transmembrane domain of receptors that incorporate β2 or β3 subunits. Introducing an asparagine-to-methionine (N265M) mutation at position 265 of the β3 subunit, which sits within the etomidate-binding site, attenuates the hypnotic effect of etomidate in vivo. It was reported recently that the photoactivatable barbiturate R-mTFD-MPAB also acts on γ-aminobutyric acid type A receptors primarily by binding to a homologous site at the γ-β interface. Given this difference in drug-binding sites established by the in vitro experiments, we hypothesized that the β3-N265M-mutant mice would not be resistant to the anesthetic effects of R-mTFD-MPAB in vivo, whereas the same mutant mice would be resistant to the anesthetic effects of R-etomidate. We measured the effects of IV injection of etomidate and R-mTFD-MPAB on loss and recovery of righting reflex in wild-type mice and in mice carrying the β3-N265M mutation. Etomidate-induced hypnosis, as measured by the duration of loss of righting reflex, was attenuated in the N265M knock-in mice, confirming prior results. By contrast, recovery of balance and coordinated movement, as measured by the ability to maintain all 4 paws on the ground, was unaffected by the mutation. Neither hypnosis nor impairment of coordinated movement produced by the barbiturate R-mTFD-MPAB was affected by the mutation. The findings confirmed our hypothesis that mutating the etomidate-binding site would not alter the response to the barbiturate R-mTFD-MPAB. Furthermore, we confirmed previous studies indicating that etomidate-induced hypnosis is mediated in part by β3-containing receptors. We also extended previous findings by showing that etomidate-impaired balance and coordinated movement are not mediated by β3-containing receptors, thus implicating β2-containing receptors in this end point.

Dispersed gold nanoparticles potentially ruin gold barley yellow dwarf virus and eliminate virus infectivity hazards

Gold nanoparticles (AuNPs) application melted barley yellow dwarf virus-PAV (BYDV-PAV) spherical nanoparticle capsids. Synergistic therapeutic effects for plant virus resistance were induced by interaction with binding units of prepared AuNPs in a water solution which was characterized and evaluated by zeta sizer, zeta potential and transmission electron microscopy (TEM). The yield of purified nanoparticles of BYDV-PAV was obtained from Hordeum vulgare (Barley) cultivars, local and Giza 121/Justo. It was 0.62 mg/ml from 27.30 g of infected leaves at an A260/A280 ratio. Virus nanoparticle has a spherical shape 30 nm in size by TEM. BYDV-PAV combined with AuNPs to challenge virus function in vivo and in vitro. Dual AuNPs existence in vivo and in vitro affected compacted configuration of viral capsid protein in the interior surface of capsomers, the outer surface, or between the interface of coat protein subunits for 24 and 48 h incubation period in vitro at room temperature. The sizes of AuNPs that had a potentially dramatic deteriorated effect are 3.151 and 31.67 nm with a different intensity of 75.3% for the former and 24.7% for the latter, which enhances optical sensing applications to eliminate virus infectivity. Damages of capsid protein due to AuNPs on the surface of virus subunits caused variable performance in four different types of TEM named puffed, deteriorated and decorated, ruined and vanished. Viral yield showed remarkably high-intensity degree of particle symmetry and uniformity in the local cultivar greater than in Giza 121/Justo cultivar. A high yield of ruined VLPs in the local cultivar than Justo cultivar was noticed. AuNPs indicated complete lysed VLPs and some deteriorated VLPs at 48 h.

Mapping of the Communication-Mediating Interface in Nonribosomal Peptide Synthetases Using a Genetically Encoded Photocrosslinker Supports an Upside-Down Helix-Hand Motif

Nonribosomal peptide synthetases (NRPSs) are large modular protein templates that assemble bioactive peptides, many of which possess therapeutic importance. Protein–protein interactions between subunits of bacterial NRPSs are essential for proper template formation. The structural basis of the typical subunit interface between epimerization (E) and condensation domains is only poorly understood. Conflicting helix-helix and helix-hand models were previously proposed. Here, the genetically encoded photocrosslinker p-benzoylphenylalanine (BpF) was incorporated into the C-terminal communication-mediating domain (COM) of GrsA. Using the partner elongation module TycB1 to form a dipeptide product, we could correlate the ability to form covalent crosslinks with the functional module interaction. Perturbation of the module interaction with the large side chain of BpF in a scan at 19 positions demonstrated the importance of three hydrophobic residues in an α-helical arrangement. Mapping of covalent crosslinks using tandem mass spectrometry revealed the residues from the interior of the condensation domain as part of the protein interface; a finding not predicted by the helix–helix model. The epimerization domain of GrsA was found to be important for the interaction. Together with multiple sequence analyses and structural modeling, our results suggest an upside-down helix-hand model in which the C-terminal COM-helix is embedded in a hand motif with a hydrophobic core in a reversed orientation compared to a previous proposal. Our results provide a more detailed and the first direct structural understanding of the COM domain interaction and will contribute to successful biocombinatorial engineering attempts in the design of artificial NRPS templates.

Mutations at the Subunit Interface of Yeast Proliferating Cell Nuclear Antigen Reveal a Versatile Regulatory Domain.

Proliferating cell nuclear antigen (PCNA) plays a key role in many cellular processes and due to that it interacts with a plethora of proteins. The main interacting surfaces of Saccharomyces cerevisiae PCNA have been mapped to the interdomain connecting loop and to the carboxy-terminal domain. Here we report that the subunit interface of yeast PCNA also has regulatory roles in the function of several DNA damage response pathways. Using site-directed mutagenesis we engineered mutations at both sides of the interface and investigated the effect of these alleles on DNA damage response. Genetic experiments with strains bearing the mutant alleles revealed that mutagenic translesion synthesis, nucleotide excision repair, and homologous recombination are all regulated through residues at the subunit interface. Moreover, genetic characterization of one of our mutants identifies a new sub-branch of nucleotide excision repair. Based on these results we conclude that residues at the subunit boundary of PCNA are not only important for the formation of the trimer structure of PCNA, but they constitute a regulatory protein domain that mediates different DNA damage response pathways, as well.

Computational studies of human class V alcohol dehydrogenase - the odd sibling.

BackgroundAll known attempts to isolate and characterize mammalian class V alcohol dehydrogenase (class V ADH), a member of the large ADH protein family, at the protein level have failed. This indicates that the class V ADH protein is not stable in a non-cellular environment, which is in contrast to all other human ADH enzymes. In this report we present evidence, supported with results from computational analyses performed in combination with earlier in vitro studies, why this ADH behaves in an atypical way.ResultsUsing a combination of structural calculations and sequence analyses, we were able to identify local structural differences between human class V ADH and other human ADHs, including an elongated β-strands and a labile α-helix at the subunit interface region of each chain that probably disturb it. Several amino acid residues are strictly conserved in class I–IV, but altered in class V ADH. This includes a for class V ADH unique and conserved Lys51, a position directly involved in the catalytic mechanism in other ADHs, and nine other class V ADH-specific residues.ConclusionsIn this study we show that there are pronounced structural changes in class V ADH as compared to other ADH enzymes. Furthermore, there is an evolutionary pressure among the mammalian class V ADHs, which for most proteins indicate that they fulfill a physiological function. We assume that class V ADH is expressed, but unable to form active dimers in a non-cellular environment, and is an atypical mammalian ADH. This is compatible with previous experimental characterization and present structural modelling. It can be considered the odd sibling of the ADH protein family and so far seems to be a pseudoenzyme with another hitherto unknown physiological function.Electronic supplementary materialThe online version of this article (doi:10.1186/s12858-016-0072-y) contains supplementary material, which is available to authorized users.

Molecular mode of action of CGS 9895 at α1 β2 γ2 GABAA receptors.

γ-aminobutyric type A (GABAA ) receptors are the main inhibitory neurotransmitter receptors in the brain and are targets for numerous clinically important drugs such as benzodiazepines, anxiolytics, and anesthetics. Previously, pyrazoloquinoline 2-p-methoxyphenylpyrazolo [4,3-c] quinolin-3(5H)-one (CGS 9895) was described as a positive allosteric modulator acting through the α+/β- interface in the extracellular domain of GABAA receptors. The localization of the binding site was based on a steric hindrance approach, rather than on direct effects of point mutations. In this study we further characterized modulation by this compound which seems to have multiple sites of action. We investigated GABAA receptors expressed in Xenopus laevis oocytes using voltage-clamp electrophysiology. We have identified the α1 Y209 residue at the α+/β- interface as a key residue for CGS 9895 modulation. In addition, the interaction between this residue and various structural analogs was characterized, allowing tentative positioning of CGS 9895 versus α1 Y209 (rat sequence). Not all compounds were found to be sensitive to mutations at the α1 Y209 residue. In addition, the interaction of CGS 9895 with flurazepam was characterized. Flurazepam is hypothesized to act at the same subunit interface in the extracellular domain. We also provide evidence that the GABAA receptor harbors additional modulatory sites for CGS 9895 at each of the subunit interfaces in the transmembrane domain. GABAA receptors are the main inhibitory neurotransmitter receptors in the brain and are targets for numerous clinically important drugs such as benzodiazepines, anxiolytics and anesthetics. We have identified the α1 Y209 residue present at the extracellular α+/β- subunit interface as a key residue for the positive allosteric modulation of the GABAA receptor by CGS 9895. This receptor harbors additional modulatory sites for this compound at subunit interfaces in the transmembrane domain.

An asparagine residue mediates intramolecular communication in nucleotide-regulated pyrophosphatase.

Many prokaryotic soluble PPases (pyrophosphatases) contain a pair of regulatory adenine nucleotide-binding CBS (cystathionine β-synthase) domains that act as 'internal inhibitors' whose effect is modulated by nucleotide binding. Although such regulatory domains are found in important enzymes and transporters, the underlying regulatory mechanism has only begun to come into focus. We reported previously that CBS domains bind nucleotides co-operatively and induce positive kinetic co-operativity (non-Michaelian behaviour) in CBS-PPases (CBS domain-containing PPases). In the present study, we demonstrate that a homodimeric ehPPase (Ethanoligenens harbinense PPase) containing an inherent mutation in an otherwise conserved asparagine residue in a loop near the active site exhibits non-co-operative hydrolysis kinetics. A similar N312S substitution in 'co-operative' dhPPase (Desulfitobacterium hafniense PPase) abolished kinetic co-operativity while causing only minor effects on nucleotide-binding affinity and co-operativity. However, the substitution reversed the effect of diadenosine tetraphosphate, abolishing kinetic co-operativity in wild-type dhPPase, but restoring it in the variant dhPPase. A reverse serine-to-asparagine replacement restored kinetic co-operativity in ehPPase. Molecular dynamics simulations revealed that the asparagine substitution resulted in a change in the hydrogen-bonding pattern around the asparagine residue and the subunit interface, allowing greater flexibility at the subunit interface without a marked effect on the overall structure. These findings identify this asparagine residue as lying at the 'crossroads' of information paths connecting catalytic and regulatory domains within a subunit and catalytic sites between subunits.

Phosphorylation state-dependent modulation of spinal glycine receptors alleviates inflammatory pain.

Diminished inhibitory neurotransmission in the superficial dorsal horn of the spinal cord is thought to contribute to chronic pain. In inflammatory pain, reductions in synaptic inhibition occur partially through prostaglandin E2- (PGE2-) and PKA-dependent phosphorylation of a specific subtype of glycine receptors (GlyRs) that contain α3 subunits. Here, we demonstrated that 2,6-di-tert-butylphenol (2,6-DTBP), a nonanesthetic propofol derivative, reverses inflammation-mediated disinhibition through a specific interaction with heteromeric αβGlyRs containing phosphorylated α3 subunits. We expressed mutant GlyRs in HEK293T cells, and electrophysiological analyses of these receptors showed that 2,6-DTBP interacted with a conserved phenylalanine residue in the membrane-associated stretch between transmembrane regions 3 and 4 of the GlyR α3 subunit. In native murine spinal cord tissue, 2,6-DTBP modulated synaptic, presumably αβ heteromeric, GlyRs only after priming with PGE2. This observation is consistent with results obtained from molecular modeling of the α-β subunit interface and suggests that in α3βGlyRs, the binding site is accessible to 2,6-DTBP only after PKA-dependent phosphorylation. In murine models of inflammatory pain, 2,6-DTBP reduced inflammatory hyperalgesia in an α3GlyR-dependent manner. Together, our data thus establish that selective potentiation of GlyR function is a promising strategy against chronic inflammatory pain and that, to our knowledge, 2,6-DTBP has a unique pharmacological profile that favors an interaction with GlyRs that have been primed by peripheral inflammation.

Identification of New Mutations at the PCNA Subunit Interface that Block Translesion Synthesis

Proliferating cell nuclear antigen (PCNA) plays an essential role in DNA replication and repair by interacting with a large number of proteins involved in these processes. Two amino acid substitutions in PCNA, both located at the subunit interface, have previously been shown to block translesion synthesis (TLS), a pathway for bypassing DNA damage during replication. To better understand the role of the subunit interface in TLS, we used random mutagenesis to generate a set of 33 PCNA mutants with substitutions at the subunit interface. We assayed the full set of mutants for viability and sensitivity to ultraviolet (UV) radiation. We then selected a subset of 17 mutants and measured their rates of cell growth, spontaneous mutagenesis, and UV-induced mutagenesis. All except three of these 17 mutants were partially or completely defective in induced mutagenesis, which indicates a partial or complete loss of TLS. These results demonstrate that the integrity of the subunit interface of PCNA is essential for efficient TLS and that even conservative substitutions have the potential to disrupt this process.

Photochemical Generation of a Tryptophan Radical within the Subunit Interface of Ribonucleotide Reductase.

The Escherichia coli class Ia ribonucleotide reductase (RNR) achieves forward and reverse proton-coupled electron transfer (PCET) over a pathway of redox active amino acids (β-Y122 ⇌ β-Y356 ⇌ α-Y731 ⇌ α-Y730 ⇌ α-C439) spanning ∼35 Å and two subunits every time it turns over. We have developed photoRNRs that allow radical transport to be phototriggered at tyrosine (Y) or fluorotyrosine (FnY) residues along the PCET pathway. We now report a new photoRNR in which photooxidation of a tryptophan (W) residue replacing Y356 within the α/β subunit interface proceeds by a stepwise ET/PT (electron transfer then proton transfer) mechanism and provides an orthogonal spectroscopic handle with respect to radical pathway residues Y731 and Y730 in α. This construct displays an ∼3-fold enhancement in photochemical yield of W(•) relative to F3Y(•) and a ∼7-fold enhancement relative to Y(•). Photogeneration of the W(•) radical occurs with a rate constant of (4.4 ± 0.2) × 10(5) s(-1), which obeys a Marcus correlation for radical generation at the RNR subunit interface. Despite the fact that the Y → W variant displays no enzymatic activity in the absence of light, photogeneration of W(•) within the subunit interface results in 20% activity for turnover relative to wild-type RNR under the same conditions.

Mechanism of Action of the Benzimidazole Fungicide on Fusarium graminearum: Interfering with Polymerization of Monomeric Tubulin But Not Polymerized Microtubule.

Tubulins are the proposed target of clinically relevant anticancer drugs, anthelmintic, and fungicide. β2-tubulin of the plant pathogen Fusarium graminearum was considered as the target of benzimidazole compounds by homology modeling in our previous work. In this study, α1-, α2-, and β2-tubulin of F. graminearum were produced in Escherichia coli. Three benzimidazole compounds (carbendazim, benomyl, and thiabendazole) interacted with the recombinant β2-tubulin and reduced the maximum fluorescence intensity of 2 μM β2-tubulin 47, 50, and 25%, respectively, at saturation of compound-tubulin complexes. Furthermore, carbendazim significantly inhibited the polymerization of α1-/β2-tubulins and α2-/β2-tubulins 90.9 ± 0.4 and 93.5 ± 0.05%, respectively, in vitro. A similar result appeared with benomyl on the polymerization of α1-/β2-tubulins and α2-/β2-tubulins at 89.9 ± 0.1% and 92.6 ± 1.2% inhibition ratios, respectively. In addition, thiabendazole inhibited 81.6 ± 1% polymerization of α1-/β2-tubulins, whereas it had less effect on α2-/β2-tubulin polymerization, with 20.1 ± 1.9% inhibition ratio. However, the three compounds cannot destabilize the polymerized microtubule. To illuminate the issue, mapping the carbendazim binding sites and β/α subunit interface on β/α-tubulin complexes by homology modeling showed that the two domains were closed to each other. Understanding the nature of the interaction between benzimidazole compounds and F. graminearum tubulin is fundamental for the development of tubulin-specific anti-F. graminearum compounds.

Murine startle mutant Nmf11 affects the structural stability of the glycine receptor and increases deactivation.

Key points Hyperekplexia or startle disease is a serious neurological condition affecting newborn children and usually involves dysfunctional glycinergic neurotransmission.Glycine receptors (GlyRs) are major mediators of inhibition in the spinal cord and brainstem.A missense mutation, replacing asparagine (N) with lysine (K), at position 46 in the GlyR α1 subunit induced hyperekplexia following a reduction in the potency of the transmitter glycine; this resulted from a rapid deactivation of the agonist current at mutant GlyRs.These effects of N46K were rescued by mutating a juxtaposed residue, N61 on binding Loop D, suggesting these two asparagines may interact.Asparagine 46 is considered to be important for the structural stability of the subunit interface and glycine binding site, and its mutation represents a new mechanism by which GlyR dysfunction induces startle disease. Dysfunctional glycinergic inhibitory transmission underlies the debilitating neurological condition, hyperekplexia, which is characterised by exaggerated startle reflexes, muscle hypertonia and apnoea. Here we investigated the N46K missense mutation in the GlyR α1 subunit gene found in the ethylnitrosourea (ENU) murine mutant, Nmf11, which causes reduced body size, evoked tremor, seizures, muscle stiffness, and morbidity by postnatal day 21. Introducing the N46K mutation into recombinant GlyR α1 homomeric receptors, expressed in HEK cells, reduced the potencies of glycine, β‐alanine and taurine by 9‐, 6‐ and 3‐fold respectively, and that of the competitive antagonist strychnine by 15‐fold. Replacing N46 with hydrophobic, charged or polar residues revealed that the amide moiety of asparagine was crucial for GlyR activation. Co‐mutating N61, located on a neighbouring β loop to N46, rescued the wild‐type phenotype depending on the amino acid charge. Single‐channel recording identified that burst length for the N46K mutant was reduced and fast agonist application revealed faster glycine deactivation times for the N46K mutant compared with the WT receptor. Overall, these data are consistent with N46 ensuring correct alignment of the α1 subunit interface by interaction with juxtaposed residues to preserve the structural integrity of the glycine binding site. This represents a new mechanism by which GlyR dysfunction induces startle disease.

Abundance and Temperature Dependency of Protein-Protein Interaction Revealed by Interface Structure Analysis and Stability Evolution.

Protein complexes are major forms of protein-protein interactions and implement essential biological functions. The subunit interface in a protein complex is related to its thermostability. Though the roles of interface properties in thermal adaptation have been investigated for protein complexes, the relationship between the interface size and the expression level of the subunits remains unknown. In the present work, we studied this relationship and found a positive correlation in thermophiles rather than mesophiles. Moreover, we found that the protein interaction strength in complexes is not only temperature-dependent but also abundance-dependent. The underlying mechanism for the observed correlation was explored by simulating the evolution of protein interface stability, which highlights the avoidance of misinteraction. Our findings make more complete the picture of the mechanisms for protein complex thermal adaptation and provide new insights into the principles of protein-protein interactions.

Protein translation machinery holds a key for transition of planktonic cells to biofilm state in Enterococcus faecalis: A proteomic approach

Enterococcus faecalis is a member of human gut microflora causing nosocomial infection involving biofilm formation. Ethyl methyl sulfonate induced mutants were analysed using crystal violet assay, SEM and CLSM microscopy which confirmed AK-E12 as biofilm efficient and AK-F6 as biofilm deficient mutants. Growth curve pattern revealed AK-E12 was fast growing whereas, AK-F6 was found slow growing mutant. 2D-Electrophorosis and MALDI-TOF analysis revealed over and underexpression of many translation-elongation associated proteins in mutants compared to wild type. Protein translation elongation factor G, translation elongation factor Tu and ribosomal subunit interface proteins were underexpressed and UTP–glucose-1-phosphate uridylyl transferase and cell division protein divIVA were overexpressed in AK-E12 as compared to wild type. In AK-F6, except 10 kDa chaperonin which was over-expressed other selected proteins were found to be suppressed. RT-PCR confirmed proteomic data except for the translation elongation factor G which showed contradictory data of proteome expression in AK-E12. Protein-protein interaction networks were constructed using STRING 10.0 which demonstrated strong connection of translation-elongation proteins with other proteins. Hence, it concludes from the data that translation elongation factors are important in transition of planktonic cells to biofilm cells in Enterococcus faecalis.

Polymerase Acidic Protein-Basic Protein 1 (PA-PB1) Protein-Protein Interaction as a Target for Next-Generation Anti-influenza Therapeutics.

The limited therapeutic options against the influenza virus (flu) and increasing challenges in drug resistance make the search for next-generation agents imperative. In this context, heterotrimeric viral PA/PB1/PB2 RNA-dependent RNA polymerase is an attractive target for a challenging but strategic protein-protein interaction (PPI) inhibition approach. Since 2012, the inhibition of the polymerase PA-PB1 subunit interface has become an active field of research following the publication of PA-PB1 crystal structures. In this Perspective, we briefly discuss the validity of flu polymerase as a drug target and its inhibition through a PPI inhibition strategy, including a comprehensive analysis of available PA-PB1 structures. An overview of all of the reported PA-PB1 complex formation inhibitors is provided, and approaches used for identification of the inhibitors, the hit-to-lead studies, and the emerged structure-activity relationship are described. In addition to highlighting the strengths and weaknesses of all of the PA-PB1 heterodimerization inhibitors, we analyze their hypothesized binding modes and alignment with a pharmacophore model that we have developed.

The Role of Subunit Interfaces of Malate Dehydrogenase in Protein Folding & Unfolding

Malate dehydrogenase (MDH) is an essential enzyme for several metabolic pathways including the production of NADH as a precursor for ATP synthesis in the mitochondrion and glyoxysome. In the cytosol MDH is involved in the reduction of oxaloacetate as part of the aspartate‐malate shuttle.. The enzyme is a dimer of chemically identical subunits and while much is known about the enzymatic steps, little is known about the folding mechanism of MDH and the link between folding and regulation or catalysis. The goal of the current project is to understand the folding process and explore the role of subunit interactions to test the hypothesis that the native dimer conformation of MDH governs the correct folding of the enzyme subunits in order to impose the observed spatial asymmetry of the subunits and facilitate active site communication across the interface thought to be involved in both overall catalysis and regulation. Clustal analysis identified a number of interface conserved residues and 4 mutants, H90Q, E256Q, S266A and L269A were constructed. The wild type protein and various mutants were expressed in E.Coli and purified using NiNTA Chromotography using the engineered His‐Tag. Purity was established by SDS‐Page or MALDI‐tof. Quaternary structure was established by size exclusion chromatography. Reversible denaturation of wild type and mutants was studied by fluorescence spectroscopy and CD to determine tertiary and secondary structures respectively, with activity loss and regain followed by enzymatic assay. The protein unfolding/folding dynamics were also probed using limited proteolysis in conjunction with mass spectrometry.

Probing the Subunit Interface of Malate Dehydrogenase: Effects of S266A and L269A Mutations

Malate Dehydrogenase is thought to involve a reciprocating subunit mechanism involving ligand induced interactions across the subunit interface in the dimer and such a mechanism is thought to be involved in both catalysis and citrate regulation of this important enzyme. Clustal analysis of organelle forms of the enzyme and HINT analysis of interactions across the interface have identified two residues, S266 and L269 that could be involved in communication between the active site and the subunit interface. Two mutants, S266A and L269A have been constructed, expressed and purified by Nickel‐NTA affinity chromatography. Initial rate kinetic studies of each mutant reveal unique non‐ Michaelis‐Menten behavior. S266A shows linear Michaelis menten plots with Oxaloacetate as varied substrate but normal saturation with NADH while L269A shows non‐Michaelis Menten behavior with NADH as varied substrate but normal saturation with Oxaloacetate. To construct a heterodimer with one subunit containing the mutant while the other subunit is normal at the interface but unable to effectively bind Oxaloacetate as a result of mutation of upto three of the active site arginines, we have explored reversible dissociation of each relevant homo dimer to create heterodimers. Preliminary results suggest that while heterodimers can be separated by ion exchange chromatography the dynamic nature of the dimer formation creates problems. To overcome this complication we are exploring chemical cross‐linking of the dimer form of the enzyme with bifunctional imidates.

Cooperative protein structural dynamics of homodimeric hemoglobin linked to water cluster at subunit interface revealed by time-resolved X-ray solution scattering.

Homodimeric hemoglobin (HbI) consisting of two subunits is a good model system for investigating the allosteric structural transition as it exhibits cooperativity in ligand binding. In this work, as an effort to extend our previous study on wild-type and F97Y mutant HbI, we investigate structural dynamics of a mutant HbI in solution to examine the role of well-organized interfacial water cluster, which has been known to mediate intersubunit communication in HbI. In the T72V mutant of HbI, the interfacial water cluster in the T state is perturbed due to the lack of Thr72, resulting in two less interfacial water molecules than in wild-type HbI. By performing picosecond time-resolved X-ray solution scattering experiment and kinetic analysis on the T72V mutant, we identify three structurally distinct intermediates (I1, I2, and I3) and show that the kinetics of the T72V mutant are well described by the same kinetic model used for wild-type and F97Y HbI, which involves biphasic kinetics, geminate recombination, and bimolecular CO recombination. The optimized kinetic model shows that the R-T transition and bimolecular CO recombination are faster in the T72V mutant than in the wild type. From structural analysis using species-associated difference scattering curves for the intermediates, we find that the T-like deoxy I3 intermediate in solution has a different structure from deoxy HbI in crystal. In addition, we extract detailed structural parameters of the intermediates such as E-F distance, intersubunit rotation angle, and heme-heme distance. By comparing the structures of protein intermediates in wild-type HbI and the T72V mutant, we reveal how the perturbation in the interfacial water cluster affects the kinetics and structures of reaction intermediates of HbI.

Pulsed Electron Paramagnetic Resonance of Spin Labeled Human GABA(A)Rs Functionally Reconstituted in Lipid Bilayers Suggests there is Space for Lipids between the Subunits

GABA(A)Rs are members of the Cys-loop superfamily of ion channel receptors. They are pentamers containing up to three different homologous subunits arranged pseudo symmetrically around a central anion–conducting pore. The binding sites of GABA and many drugs are located between specific subunits. Human α1β3 GABA(A)Rs, expressed in HEK cells, were spin labeled during purification in the etomidate binding site on the third transmembrane helix of the β3-subunit (β3 M3 M286C) and functionally reconstituted into detergent–free asolectin bilayers. The separation between the tethered nitroxides, measured by double electron electron resonance (DEER), is related to the intersubunit separation and the local conformation of the spin label. In the desensitized state, two pairs of nitroxide separations were observed. Each pair contains a nearest and a next nearest neighbor subunit separation that obeys pentameric symmetry constraints. Rotamer analysis was performed on homology models based on published crystal structures of several homologous anion–conducting, Cys-loop homo-pentamers. These predicted well the longer of the two pentameric pairs, but not the shorter. However, both pairs of separations can be accommodated by models, in which the subunits are systematically moved radially away from the ion pore, so increasing the subunit spacing (side of the pentamer). An increase of 0.75 to 1 A is optimal. This analysis suggested that one pair of pentamerically–related nitroxide separations corresponds to a population of spin labels in the lipid-protein interface and the other to that between the subunits. The optimal increase in subunit spacing is the minimum needed to dock lipids into the subunit interface, suggesting that this might occur in the bilayer state but not in the crystal structures. Supported by GM58448 and GM35215.

Functional Mechanisms of Desensitization in AMPA Receptors

Ionotropic glutamate receptors (iGluRs) are ligand gated ion channels that mediate excitatory synaptic transmission in the brain. Desensitization of AMPA receptors following glutamate binding is critical for brain function, and involves rearrangement of the ligand binding domains (LBDs). Recently, a couple of full-length structures of desensitized iGluRs were revealed. One of them, the cryo-EM structure of the GluK2 kainate receptor showed that the ligand binding domains (LBDs) adopt a quasi four-fold symmetric arrangement1. Contrastingly, the GluA2 full-length crystal structure with Fluorowillardine bound showed a disrupted, asymmetric orientation of the LBDs and disrupted ATDs2. We reasoned that these LBD arrangements should be detectable by crosslinking experiments during receptor desensitization. We noticed that cysteine mutants at the interface between subunits A and C, which cross-link receptors in partially-active states when desensitization is blocked3, can also trap desensitized receptors. When we exposed receptors to 100 µM glutamate, crosslinks formed that were much more stable than those seen in the absence of desensitization. Consistent with stabilizing a desensitized state, introduction of bulky amino acids at this subunit interface produced a receptor with slow recovery from desensitization. Using a mutation that strongly stabilized the desensitized state (E713T, Y768R), we were able to change the stability of crosslinks or, in one case, block their formation, indicating that there are multiple desensitized arrangements. Homology modelling guided by these functional data suggests that the stable trapping arises from an arrangement distinct from that observed in the partially-active state. Bibliography 1. Meyerson, J. R. et al. Structural mechanism of glutamate receptor activation and desensitization. Nature (2014). 2. Dürr, K. L. et al. Structure and Dynamics of AMPA Receptor GluA2 in Resting, Pre-Open, and Desensitized States. Cell (2014). 3. Lau, A. Y. et al. A Conformational Intermediate in Glutamate Receptor Activation. Neuron (2013).