Intrasubunit Salt Bridge Research Articles

New look at hemoglobin allostery.

New look at hemoglobin allostery.

GABAA receptor biogenesis is impaired by the γ2 subunit febrile seizure-associated mutation, GABRG2(R177G)

A missense mutation in the GABAA receptor γ2L subunit, R177G, was reported in a family with complex febrile seizures (FS). To gain insight into the mechanistic basis for these genetic seizures, we explored how the R177G mutation altered the properties of recombinant α1β2γ2L GABAA receptors expressed in HEK293T cells. Using a combination of electrophysiology, flow cytometry, and immunoblotting, we found that the R177G mutation decreased GABA-evoked whole-cell current amplitudes by decreasing cell surface expression of α1β2γ2L receptors. This loss of receptor surface expression resulted from endoplasmic reticulum (ER) retention of mutant γ2L(R177G) subunits, which unlike wild-type γ2L subunits, were degraded by ER-associated degradation (ERAD). Interestingly, when compared to the condition of homozygous γ2L(R177G) subunit expression, disproportionately low levels of γ2L(R177G) subunits reached the cell surface with heterozygous expression, indicating that wild-type γ2L subunits possessed a competitive advantage over mutant γ2L(R177G) subunits for receptor assembly and/or forward trafficking. Inhibiting protein synthesis with cycloheximide demonstrated that the R177G mutation primarily decreased the stability of an intracellular pool of unassembled γ2L subunits, suggesting that the mutant γ2L(R177G) subunits competed poorly with wild-type γ2L subunits due to impaired subunit folding and/or oligomerization. Molecular modeling confirmed that the R177G mutation could disrupt intrasubunit salt bridges, thereby destabilizing secondary and tertiary structure of γ2L(R177G) subunits. These findings support an emerging body of literature implicating defects in GABAA receptor biogenesis in the pathogenesis of genetic epilepsies (GEs) and FS.

Structure of the NADH-dependent thermostable alcohol dehydrogenase TADH from Thermus sp. ATN1 provides a platform for engineering specificity and improved compatibility with inorganic cofactor-regeneration catalysts

Abstract Alcohol dehydrogenases (ADHs) of the medium chain reductase (MDR) subfamily are valuable biocatalysts for the production of optically active alcohols and are now used routinely in industry for the preparation of synthetic intermediates. TADH from Thermus sp. ATN1 combines the advantages of both thermostability and dependence on the less expensive, non-phosphoryated, cofactor NADH as the hydride donor. The structure of TADH in complex with NADH has been determined and refined to a resolution of 2.74 A. The structure reveals structural features commensurate with known stabilising factors of thermostable MDRs, including shorter peripheral loops and an increased amount of inter- and intra-subunit salt bridges and hydrogen bonds compared to mesophilic MDRs of known structure. A study of the active site reveals the molecular determinants of NADH and substrate binding in TADH that help to shed light on observed differences in cofactor specificity and stereoselectivity when compared to its thermophilic NADPH-dependent homolog from Thermoanaerobium brockii (TbADH). The structure also provides a basis for investigating the enzyme-associated deactivation of the catalyst ([Cp*Rh(bpy)(H 2 O)] 2+ ), which has been used in TADH-catalysed reactions for the recycling of the nicotinamide cofactor.

Molecular Interactions Involved in KCSA pH Gating

The bacterial potassium channel KcsA is gated by high concentrations of intracellular protons, allowing the channel to open at pH < 5.5. Replacing key ionizable residues from the N and C termini of KcsA with residues mimicking their protonated counterparts with respect to charge renders the channel open up to pH 9.0 (Thompson et al. 2008). We proposed that these residues function as the proton-binding sites. At neutral pH they form a complex network of inter- and intrasubunit salt bridges and hydrogen bonds near the bundle crossing, stabilizing the closed state. At acidic pH, these residues change their ionization state, thereby disrupting this network, favoring channel opening. While our previous work identified a network of residues involved in pH sensing, it did not rigorsously dissect the interactions that govern channel opening. To this end, we performed a series of single and pairwise mutations of the residues in the pH sensor. Using electrophysiology and X-ray crystallography we hope to gain a deeper insight into the mechanism of proton dependent gating in KcsA.

A conserved arginine near the filter of Kir1.1 controls Rb/K selectivity

ROMK (Kir1.1) channels are important for K secretion and recycling in the collecting duct, connecting tubule and thick ascending limb of the mammalian nephron. We have identified a highly conserved Arg in the P loop of the channel near the selectivity filter that controls Rb/K selectivity. Mutation of this Arg to a Tyr (R128Y-Kir1.1b, R147Y-Kir1.1a) increased the macroscopic conductance ratio, GRb/GK by 17 ± 3 fold and altered the selectivity sequence from NH4 > K > Tl > Rb >> Cs in wt-Kir1.1 to: Rb > Cs > Tl > NH4 >> K in R128Y, without significant change in the high K/Na permeability ratio of Kir1.1. R128M produced similar, but smaller, increases in Rb, Tl, NH4 and Cs conductance relative to K. R128Y remained susceptible to block by both external Ba and the honeybee toxin, TPNQ, although R128Y had a reduced affinity for TPNQ, relative to wild-type. The effect of R128Y-Kir1.1b on the GRb/GK ratio can be partly explained by a larger single-channel Rb conductance (12.4 ± 0.5 pS) than K conductance (< 1.5 pS ) in this mutant. The kinetics of R128Y gating at -120 mV with Rb as the permeant ion were similar to those of wt-Kir1.1 conducting Rb, but with a longer open time (129 ms vs. 6 ms for wt) and two closed states (13 ms, 905 ms), resulting in an open probability (Po) of 0.5, compared to a Po of 0.9 for wt-Kir1.1, which had a single closed state of 1 ms at -120mV. Single-channel R128Y rectification was eliminated in excised, inside-out patches by removal of internal Mg and polyamines. The large increase in the Rb/K conductance ratio, with no change in K/Na permeability or rectification, is consistent with R128Y-Kir1.1b causing a subtle change in the selectivity filter, perhaps by disruption of an intra-subunit salt bridge (R128-E118) near the filter.

Energetic Coupling Between Amino Acids in the pH-Sensing Region of the KcsA Channel

The bacterial potassium channel KcsA is gated by high concentrations of intracellular protons, allowing the channel to open at pH < 5.5. Replacing key ionizable residues from the N and C termini of KcsA with residues mimicking their protonated counterparts with respect to charge renders the channel open up to pH 9.0 (Thompson et al., 2008). We proposed that these residues function as the proton-binding sites. At neutral pH they form a complex network of inter- and intrasubunit salt bridges and hydrogen bonds near the bundle crossing, stabilizing the closed state.

A Conserved Arginine Near the Selectivity Filter of Kir1.1 Controls Rb/K Selectivity

ROMK (Kir1.1) channels are important for K secretion and recycling in the collecting duct, connecting tubule and thick ascending limb of the mammalian nephron. We have identified a highly conserved Arg in the P loop of the channel near the selectivity filter that controls Rb/K selectivity. Mutation of this Arg to a Tyr (R128Y-Kir1.1b, R147Y-Kir1.1a) increased Rb/K selectivity by 11 ± 1 fold, NH4/K selectivity by 6 ±1 fold, and Cs/K selectivity by 32 ± 5 fold over wild-type (wt), with no significant change in K/Na selectivity. R128Y remained susceptible to block by both external Ba and the honey bee toxin, TPNQ, although it had a 100 fold lower affinity for TPNQ than wild-type. Single-channel R128Y-Kir1.1b conductance averaged 12 ± 0.5 pS in 100mM RbCl, 0 Mg, 0 Ca solutions, compared to 18 ± 2 pS for wt-Kir1.1b in the same Rb solutions. In excised, inside-out patches (polyamine-free, 0 Mg solutions), R128Y-Kir1.1b had a non-rectifying Rb conductance of 10 ± 0.6 pS, but no visible K current, consistent with its low K selectivity. The kinetics of Rb permeation through R128Y were similar to the kinetics of K permeation through wt-Kir1.1b, but with a longer open time (245 ms vs. 19ms for wt); and two closed states (2.8ms, 25ms) yielding an average open probability (Po) of 0.7 at −100mV, compared to a Po > 0.9 for wt-Kir1.1, which has a single closed state of 1.3ms. The observed 11 fold increase in Rb/K selectivity with no change in K/Na selectivity or rectification is consistent with R128Y-Kir1.1b causing a subtle change in the selectivity filter, perhaps by disruption of an intra-subunit salt bridge (R128-E118) near the filter.

Salt bridges in the miniature viral channel Kcv are important for function

The viral potassium channel Kcv comprises only 94 amino acids, which represent the pore module of more complex K(+) channels. As for Kir-type channels, Kcv also has a short N-terminal helix exposed to the cytoplasm, upstream of the first transmembrane domain. Here we show that this helix is relevant for Kcv function. The presence of charged amino acids, which form dynamic inter- and intra-subunit salt bridges is crucial. Electrophysiological measurements, yeast rescue experiments and molecular dynamics simulations show that mutants in which the critical salt bridge formation is impaired have no or reduced channel activity. We conclude that these salt bridges destabilise the complexation of K(+) ions by negative charges on the inner transmembrane domain at the entrance into the cavity. This feature facilitates a continuous and coordinated transfer of ions between the cavity and the cytoplasm for channels without the canonical bundle crossing.

A conserved salt bridge critical for GABA A receptor function and loop C dynamics

Chemical signaling in the brain involves rapid opening and closing of ligand gated ion channels (LGICs). LGICs are allosteric membrane proteins that transition between multiple conformational states (closed, open, and desensitized) in response to ligand binding. While structural models of cys-loop LGICs have been recently developed, our understanding of the protein movements underlying these conformational transitions is limited. Neurotransmitter binding is believed to initiate an inward capping movement of the loop C region of the ligand-binding site, which ultimately triggers channel gating. Here, we identify a critical intrasubunit salt bridge between conserved charged residues (betaE153, betaK196) in the GABA(A) receptor (GABA(A)R) that is involved in regulating loop C position. Charge reversals (E153K, K196E) increased the EC(50) for GABA and for the allosteric activators pentobarbital (PB) and propofol indicating that these residues are critical for channel activation, and charge swap (E153K-K196E) significantly rescued receptor function suggesting a functional electrostatic interaction. Mutant cycle analysis of alanine substitutions indicated that E153 and K196 are energetically coupled. By monitoring disulfide bond formation between cysteines substituted at these positions (E153C-K196C), we probed the mobility of loop C in resting and ligand-bound states. Disulfide bond formation was significantly reduced in the presence of GABA or PB suggesting that agonist activation of the GABA(A)R proceeds via restricting loop C mobility.

Molecular mechanism of pH sensing in KcsA potassium channels

The bacterial potassium channel KcsA is gated by high concentrations of intracellular protons, allowing the channel to open at pH < 5.5. Despite prior attempts to determine the mechanism responsible for pH gating, the proton sensor has remained elusive. We have constructed a KcsA channel mutant that remains open up to pH 9.0 by replacing key ionizable residues from the N and C termini of KcsA with residues mimicking their protonated counterparts with respect to charge. A series of individual and combined mutations were investigated by using single-channel recordings in lipid bilayers. We propose that these residues are the proton-binding sites and at neutral pH they form a complex network of inter- and intrasubunit salt bridges and hydrogen bonds near the bundle crossing that greatly stabilize the closed state. In our model, these residues change their ionization state at acidic pH, thereby disrupting this network, modifying the electrostatic landscape near the channel gate, and favoring channel opening.

Crystallographic analysis of the interaction of nitric oxide with quaternary-T human hemoglobin.

In addition to interacting with hemoglobin as a heme ligand to form nitrosylhemoglobin, NO can react with cysteine sulfhydryl groups to form S-nitrosocysteine or cysteine oxides such as cysteinesulfenic acid. Both modes of interaction are very sensitive to the quaternary structure of hemoglobin. To directly view the interaction of NO with quaternary-T deoxyhemoglobin, crystallographic studies were carried out on crystals of deoxyhemoglobin that were exposed to gaseous NO under a variety of conditions. Consistent with previous spectroscopic studies in solution, these crystallographic studies show that the binding of NO to the heme groups of crystalline wild-type deoxyhemoglobin ruptures the Fe-proximal histidine bonds of the alpha-subunits but not the beta-subunits. This finding supports Perutz's theory that ligand binding induces tension in the alpha Fe-proximal histidine bond. To test Perutz's theory, deoxy crystals of the mutant hemoglobin betaW37E were exposed to NO. This experiment was carried out because previous studies have shown that this mutation greatly reduces the quaternary constraints that oppose the ligand-induced movement of the alpha-heme Fe atom into the plane of the porphyrin ring. As hypothesized, the Fe-proximal histidine bonds in both the beta- and the alpha-subunits remain intact in crystalline betaW37E after exposure to NO. With regard to S-nitrosocysteine or cysteine oxide formation, no evidence for the reaction of NO with any cysteine residues was detected under anaerobic conditions. However, when deoxyhemoglobin crystals are first exposed to air and then to NO, the appearance of additional electron density indicates that Cys93(F9)beta has been modified, most likely to cysteinesulfenic acid. This modification of Cys93(F9)beta disrupts the intrasubunit salt bridge between His146(HC3)beta and Asp94(FG1)beta, a key feature of the quaternary-T hemoglobin structure. Also presented is a reanalysis of our previous crystallographic studies [Chan, N.-L., et al. (1998) Biochemistry 37, 16459-16464] of the interaction of NO with liganded hemoglobin in the quaternary-R2 structure. These studies showed additional electron density at Cys93(F9)beta that was consistent with an NO adduct. However, for reasons discussed in this paper, we now believe that this adduct may be the Hb-S-N.-O-H radical intermediate and not Hb-S-N=O as previously suggested.

Structural basis for the enhanced thermal stability of alcohol dehydrogenase mutants from the mesophilic bacterium Clostridium beijerinckii: contribution of salt bridging.

Previous research in our laboratory comparing the three-dimensional structural elements of two highly homologous alcohol dehydrogenases, one from the mesophile Clostridium beijerinckii (CbADH) and the other from the extreme thermophile Thermoanaerobacter brockii (TbADH), suggested that in the thermophilic enzyme, an extra intrasubunit ion pair (Glu224-Lys254) and a short ion-pair network (Lys257-Asp237-Arg304-Glu165) at the intersubunit interface might contribute to the extreme thermal stability of TbADH. In the present study, we used site-directed mutagenesis to replace these structurally strategic residues in CbADH with the corresponding amino acids from TbADH, and we determined the effect of such replacements on the thermal stability of CbADH. Mutations in the intrasubunit ion pair region increased thermostability in the single mutant S254K- and in the double mutant V224E/S254K-CbADH, but not in the single mutant V224E-CbADH. Both single amino acid replacements, M304R- and Q165E-CbADH, in the region of the intersubunit ion pair network augmented thermal stability, with an additive effect in the double mutant M304R/Q165E-CbADH. To investigate the precise mechanism by which such mutations alter the molecular structure of CbADH to achieve enhanced thermostability, we constructed a quadruple mutant V224E/S254K/Q165E/M304R-CbADH and solved its three-dimensional structure. The overall results indicate that the amino acid substitutions in CbADH mutants with enhanced thermal stability reinforce the quaternary structure of the enzyme by formation of an extended network of intersubunit ion pairs and salt bridges, mediated by water molecules, and by forming a new intrasubunit salt bridge.

Allostery in rabbit pyruvate kinase: development of a strategy to elucidate the mechanism.

Isozymes of pyruvate kinase (PK) expressed in rabbit muscle and kidney show different allosteric kinetics. The only amino acid changes in the two isozymes, originating from alternative RNA splicing, occur at a stretch of 55 amino acids in the C domain near the subunit interface. The self-correcting distance geometry (SECODG) program DIAMOD was used to calculate a homology model of these interfacial contacts in the four helix bundle of the kidney PK dimer, based on the X-ray structure of the tetrameric rabbit muscle PK [Larsen et al. (1994) Biochemistry 33, 6301-6309]. Energy refinement with the program FANTOM, using the ECEPP/2 force field to assess packing and electrostatic interactions between the two subunits, yielded two groups of energetically favorable conformations. The primary difference in the two groups is the loop conformation of residue Pro 402, which is serine in muscle PK. In one loop conformation, the conserved Lys 421 can form an intersubunit salt bridge as observed in the muscle PK crystal structure. The other loop conformation favors an alternative intrasubunit salt bridge, similar to that found in the Escherichia coli PK structure, which was not used for generating the model. The intersubunit salt bridge leads to an intersubunit hydrogen bonding between Lys 421 of one subunit and Tyr 443 of the other. To provide direct evidence on the roles of these residues, site-directed mutagenesis of the muscle PK gene was conducted. Converting Ser 402 to a proline and Tyr 443 to a phenylalanine changed neither the secondary nor the tetrameric structure, as measured by far UV-CD and sedimentation velocity, respectively. However, the S402P mutant exhibits steady-state kinetics, indicating that the mutant is more reponsive to regulation by effectors, while the mutant Y443F was essentially equivalent to wild-type muscle PK protein except for a lower affinity to phosphoenolpyruvate. These findings suggest a pivotal role for a few key residues in the allosteric regulation in PK.

Detection of changes in pairwise interactions during allosteric transitions: coupling between local and global conformational changes in GroEL.

A protein engineering approach for detecting and measuring local conformational changes that accompany allosteric transitions in proteins is described. Using this approach, we can identify interactions that are made or broken during allosteric transitions. The method is applied to probe for changes in pairwise interactions in the chaperonin GroEL during its ATP-induced allosteric transitions. Two pairwise interactions are investigated: one between subunits (Asp-41 with Thr-522) and the other within subunits (Glu-409 with Arg-501). We find that the intraring intersubunit interaction between Asp-41 and Thr-522 changes little during the allosteric transitions of GroEL, indicating that the hydrogen bond between these residues is maintained. In contrast, the intrasubunit salt bridge between Glu-409 and Arg-501 becomes significantly weaker during the ATP-induced allosteric transitions of GroEL. Our results are consistent with the electron microscopy observations of an ATP-induced hinge movement of the apical domains relative to the equatorial domains.

Crystal structure of the ASP-199.fwdarw.asparagine mutant of chloramphenicol acetyltransferase to 2.35.ANG. resolution: structural consequences of disruption of a buried salt bridge

The crystal structure of the Asp-199----Asn mutant of chloramphenicol acetyltransferase (CAT) has been determined to 2.35-A resolution. In wild-type CAT Asp-199 is involved in a fully buried intrasubunit salt bridge with Arg-18, an interaction that is adjacent to the active site. Replacement of aspartate with asparagine by site-directed mutagenesis disrupts this salt bridge and causes extensive conformational changes within the active site. The imidazole group of the catalytically essential His-195 is reoriented, with the loss of interactions thought to stabilize the preferred tautomer of this residue. Arg-18 and Asn-199 form three new intersubunit interactions as a result of large side-chain torsion angle changes which cause the movement of two polypeptide loops, some residues of which are up to 20 A away from the site of the mutation. The new interactions of Arg-18 and Asn-199 compensate for the loss of the buried salt bridge and afford near-wild-type thermostability to Asn-199 CAT, albeit with a greatly reduced activity.

Role of the beta 146 histidyl residue in the alkaline Bohr effect of hemoglobin.

High-resolution proton nuclear magnetic resonance spectroscopy has been used to investigate the effects of inorganic anions, such as phosphate or chloride, on the alkaline Bohr effect of normal human adult hemoglobin. By monitoring the chemical shift of the C2 proton of the beta 146 histidyl residue as a function of pH, we have determined its pK values in both ligated and unligated forms. In the presence of 0.1 M Bis-Tris buffer (with chloride ion concentration ranging from 0.005 to 0.06 M) in D2O at 27 degrees C, the pK value of the beta 146 histidine of deoxyhemoglobin is 7.98 +/- 0.03 and that of (carbon monoxy)hemoglobin is 7.85 +/- 0.03. However, in the presence of 0.2 M phosphate and 0.2 M NaCl in D2O at 27 degrees C, the corresponding pK values are 8.08 and 7.14, as previously reported by this laboratory [Kilmartin, J. V., Breen, J. J., Roberts, G. C. K., & Ho, C. (1973) Proc. Natl. Acad. Sci. U.S.A. 70, 1246-1249]. This large difference in the pK value between the deoxy and carbon monoxy forms in the presence of 0.2 M phosphate and 0.2 M NaCl was interpreted as direct support for (1) the breaking of an intrasubunit salt bridge between beta 146 histidine and beta 94 aspartate when the hemoglobin molecule undergoes the quaternary structural transition as proposed by Perutz [Perutz, M. F. (1970) Nature (London) 228, 726-739] and (2) Perutz's suggestion that the beta 146 histidine is one of the amino acid residues responsible for the alkaline Bohr effect. The absence of a large change in the pK value of the beta 146 histidine in the presence of 0.1 M Bis-Tris buffer implies that (1) the above-mentioned intrasubunit salt bridge is not broken in going from the deoxy to the carbon monoxy form and (2) the beta 146 histidyl residue does not contribute significantly to the alkaline Bohr effect under these conditions. We have also found that in measuring the oxygen affinity of hemoglobin as a function of pH in the presence of 0.1 M Bis-Tris or 0.2 M phosphate plus 0.2 M NaCl (both in D2O), there is no significant difference in the alkaline Bohr effect in these two media. Hence, our results suggest that the detailed molecular mechanism for the Bohr effect depends on the experimental conditions.

X-ray and functional studies of hemoglobins Nancy and Cochin-Port-Royal.

The mutations in hemoglobin Nancy beta145(HC2) Tyr leads to Asp and hemoglobin Cochin-Portal-Royal beta146(HC3) His leads to Arg involve residues which are thought to be essential for the full expression of allosteric action in hemoglobin. Relative to the structure of deoxyhemoglobin A, our x-ray study of deoxyhemoglobin Nancy shows severe disordering of the beta chain COOH-terminal tetrapeptide and a possible movement of the beta heme iron atom toward the plane of the porphyrin ring. These structural perturbations result in a high oxygen affinity, reduced Bohr effect, and lack of cooperatively in hemoglobin Nancy. In the presence of inositol hexaphosphate (IHP), the Hill constant for hemoglobin Nancy increases from 1.1 to 2.0. But relative to its action on hemoglobin A, IHP is much less effective in reducing the oxygen affinity and in increasing the Bohr effect of hemoglobin Nancy. This indicates that IHP does not influence the R in equilibrium T equilibrium as much in hemoglobin Nancy as in hemoglobin A, and this probably is due to the disordering of His 143beta which is known to be part of the IHP binding site. IHP is also known to produce large changes in the absorption spectrum of methemoglobin A, but we find that it has no effect on the spectrum of methemoglobin Nancy. In contrast to the large structural changes in deoxyhemoglobin Nancy, the structure of deoxyhemoglobin Cochin-Port-Royal differs from deoxyhemoglobin A only in the position of the side chain of residue 146beta. The intrasubunit salt bridge between His 146beta and Asp 94beta in deoxyhemoglobin A is lost in deoxyhemoglobin Cochin-Portal-Royal with the guanidinium ion of Arg 146beta floating freely in solution. This small difference in structure results in a reduced Bohr effect, but does not cause a change in the Hill coefficient, the response to 2,3-diphosphoglycerate, or the oxygen affinity at physiological pH.