Carboxylate Pairs Research Articles

Interplay between local protein interactions and water bridging of a proton antenna carboxylate cluster

Proteins that bind protons at cell membrane interfaces often expose to the bulk clusters of carboxylate and histidine sidechains that capture protons transiently and, in proton transporters, deliver protons to an internal site. The protonation-coupled dynamics of bulk-exposed carboxylate clusters, also known as proton antennas, is poorly described. An essential open question is how water-mediated bridges between sidechains of the cluster respond to protonation change and facilitate transient proton storage. To address this question, here I studied the protonation-coupled dynamics at the proton-binding antenna of PsbO, a small extrinsinc subunit of the photosystem II complex, with atomistic molecular dynamics simulations and systematic graph-based analyses of dynamic protein and protein-water hydrogen-bond networks. The protonation of specific carboxylate groups is found to impact the dynamics of their local protein-water hydrogen-bond clusters. Regardless of the protonation state considered for PsbO, carboxylate pairs that can sample direct hydrogen bonding, or bridge via short hydrogen-bonded water chains, anchor to nearby basic or polar protein sidechains. As a result, carboxylic sidechains of the hypothesized antenna cluster are part of dynamic hydrogen bond networks that may rearrange rapidly when the protonation changes.

Ion Selectivity in Acid-Sensing Ion Channels and Epithelial Sodium Channels

Members of the epithelial Na+ channel/degenerin (ENaC/DEG) superfamily are Na+-selective channels with a common overall structure. Each of the three subunits within these trimeric channels has two transmembrane domains (M1 and M2), of which M2 forms the pore. Included in this family are the proton-gated acid-sensing ion channels (ASICs), which are formed by identical or homologous subunits. ASICs mediate excitatory Na+ currents (relative Na+/K+ permeability of ∼ 10/1) and play key roles in many physiological processes, including nociception and cell death following ischemic stroke. ENaCs are obligate heterotrimers comprised of the principal α subunits, as well as β and γ subunits and display significantly higher Na+ selectivity than ASICs (relative Na+/K+ permeability of ∼ 100/1). Previous work had indicated that the selectivity filter in both ENaCs and ASICs is formed by a conserved G-X-S motif in the pore. However, we have recently shown the mASIC1a selectivity filter to be composed of two carboxylate pairs in the lower part of the pore, namely M2 E18’ and D21’. As both the G-X-S motif and the carboxylates in 18’ and 21’ are conserved between ASICs and ENaCs, there are likely other factors contributing to the stark differences in Na+ selectivity. We have thus probed the contribution of residues in M1 to ion selectivity in ASICs and ENaCs. By introducing point-mutations, bulky side chains of the ENaC M1 were replaced by less bulky side chains, and the converse was performed in ASIC1a. We show that the bulky residues in M1 are important for Na+ selectivity, conceivably by influencing the size of the pore.

Gating Mechanism and Movements in Acid Sensing Ion Channel 1A

Acid Sensing Ion Channels (ASICs) are proton sensitive ion channels found in central and peripheral nervous system. They are involved in a variety of physiological processes including sensory perception, synaptic transmission and nociception. It is also involved in pathological processes like stroke and hypoxia leading to cell death. Thus understanding the mechanism of working of these ion channels are of importance in order to be able to design modulators. Here we present results from a combination of biophysical, molecular, functional and computational studies that looks at various aspects of ASIC functions. Earlier studies pointed towards possible involvement of carboxylate residues in the extracellular domain, in pH sensing. Our results indicate that there are three carboxylate pairs that are involved in gating. The carboxylates upon proton binding, induce a conformational change that causes the finger and thumb domains of ASIC come closer to each other. Our results indicate that these carboxylate pairs and the conformational change are key factors that mediate gating of the ion channel. We then proceeded to study the inter subunit conformational changes that might be involved in desensitization process. Biophysical investigations indicate that the subunits move away from each other upon desensitization. This movement is not global between the subunits. The segments closer to the central vestibule stay relatively in the same position whereas the residues towards the periphery of the ion channel moves away from each other more prominently. These results show that the ion channel undergoes a lateral flowering-like motion during desensitization in comparison with the resting state of the ion channel.

Cleft Closure Mechanism in Gating of Acid Sensing Ion Channel 1A

Acid sensing ion channels (ASICs) are trimeric proton activated channels which share sequence similarities with Degenerin/Epithelial sodium channels. They are responsible for proton evoked currents in central nervous system of most vertebrates. Their physiological functions include synaptic transmission, sensory perception, learning, and pathophysiology of stroke. Crystal structure of the low pH state of chicken ASIC shows thumb and palm like domains in the extracellular part of each subunit connected through hydrogen bonds between carboxylate residue pairs, thought to be part of proton sensors in ASIC. Based on the low pH structure it's been hypothesized that these domains would be further apart at high pH due to negative charges and protonation would allow the two domains to move closer, which in turn could trigger changes in the transmembrane segments, causing channel opening. We have used Fluorescence Resonance Energy Transfer(FRET) to study the high pH resting structure of ASIC and determined conformational changes between the thumb and palm domains. Cysteine residues were introduced on these domains and tagged with thiol reactive donor and acceptor fluorophores. The recognition site for protease Xa was introduced on either side of the cysteine on the palm domain. This allowed us to perform the FRET measurements in oocytes and HEK-293 cells without purification, as addition of the protease allowed us to quantitively characterize the background FRET. The FRET constructs were characterized using electrophysiology to establish that they were functional. The FRET based distances showed 3A decrease in distance between thumb domain residue 340 and palm domain residues 130 and 139 upon changing the pH from 8 to 6. This decrease in distance provides evidence for the hypothesized movement in the extracellular domain of ASIC and also supports the protonation of the carboxylate pairs across these domains.

On the reliability of the corrected semiempirical quantum chemical method (PM6-DH2) for assigning the protonation states in HIV-1 protease/inhibitor complexes

A novel computational protocol for determining the most probable protonation states in protein/ligand complexes is presented. The method consists in treating large parts of the enzyme using the corrected semiempirical quantum chemical (QM) method – PM6-D2 for optimization and PM6-DH2 for single-point energies – while the rest is calculated using molecular mechanics (MM) within a hybrid QM/MM fashion. The surrounding solvent is approximated by an implicit model. This approach is applied to two model systems, two different carboxylate pairs in one general and one unique HIV-1 protease/inhibitor complex. The effect of the size of the movable QM part is investigated in a series of several sizes, 3-, 6-, 8- and 10-Å regions surrounding the inhibitor. For the smallest region (< 450 atoms) the computationally more costly DFT QM/MM optimizations are performed as a check of the correctness. Proton transfer (PT) phenomena occur at both the PM6-D2 and DFT levels, which underlines the requirement for a QM approach. The barriers of PT are checked in model carboxylic acid pairs using the highly accurate MP2 and CCSD(T) values. An important result of this study is the fine-tuning of the protocol which can be used in further applications; its limitations are also shown, pointing to future developments. The calculations reveal which protonation variants of the active site are the most stable. In conclusion, the presented protocol can also be utilized for defining probable isomers in biomolecular systems. It can also serve as a preparatory step for further interaction-energy and binding-score calculations.

4-(2-Benzoylethyl)benzoic acid

The title compound, C16H14O3, adopts a conformation in which each functional group is almost coplanar with its adjacent ring, while the two aromatic rings are twisted with respect to one another with a dihedral angle of 78.51 (3)°. The compound dimerizes by standard centrosymmetric hydrogen-bonded carboxyl pairing [O⋯O = 2.6218 (11) Å and O—H⋯O = 176 (2)°]. The packing includes two inter­molecular C—H⋯O close contacts with the ketone group.

4-Acetylbenzoic acid: hydrogen bonding and packing in a simple aromatic ∊-keto acid

The title compound, C9H8O3, adopts a conformation in which both substituents lie nearly coplanar with the ring. Asymmetric units aggregate by centrosymmetric carboxyl pairing. Close offset stacking of rings in parallel planes creates inter­molecular C⋯C π–π contacts of 3.322 (3) and 3.352 (3) Å, with an accompanying C⋯O π–π contact of 3.147 (3) Å and similarly close inter­actions between O atoms and aromatic π systems. One close inter­molecular C—H⋯O contact is also present.

Physical Regulation of the Self-Assembly of Tobacco Mosaic Virus Coat Protein

We present a statistical mechanical model based on the principle of mass action that explains the main features of the in vitro aggregation behavior of the coat protein of tobacco mosaic virus (TMV). By comparing our model to experimentally obtained stability diagrams, titration experiments, and calorimetric data, we pin down three competing factors that regulate the transitions between the different kinds of aggregated state of the coat protein. These are hydrophobic interactions, electrostatic interactions, and the formation of so-called “Caspar” carboxylate pairs. We suggest that these factors could be universal and relevant to a large class of virus coat proteins.

(−)-3,7-Dioxo-5β-cholanic acid: dual hydrogen-bonding modes in a diketonic bile-acid derivative

The asymmetric unit of the title compound, C(24)H(36)O(4), contains three molecules, all differing in their side-chain conformations and all linked by hydrogen bonding confined entirely within a three-molecule block. One connection is of the acid-to-ketone type [O...O = 2.7055 (19) Angstrom and O-H...O = 180 degrees ] and the other involves carboxyl pairing [O...O = 2.6485 (18) and 2.6598 (18) Angstrom, and O-H...O = 168 and 174 degrees ]. Numerous intermolecular C-H...O close contacts connect neighbouring molecules.

(+)-3,12-Dioxo-5β-cholanic acid: hydrogen bonding in a diketo bile-acid derivative

The asymmetric unit of the title crystal structure, C24H36O4, contains two independent molecules, differing only in their side-chain conformations and linked though O—H...O hydrogen bonds by carboxyl pairing [O...O = 2.6715 (17) and 2.6544 (17) Å; O—H...O = 170 and 179°]. Six intermolecular C—H...O=C close contacts were found.

2-Hydroxyisophthalic acid: hydrogen-bonding patterns in the monohydrate and the tetraphenylphosphonium salt. An instance of dramatic acidity enhancement by symmetric, internally hydrogen-bonded anion stabilization.

The monohydrate of the title phenolic diacid (C8H6O5.H2O, 2-hydroxybenzene-1,3-dioic acid or 3-carboxysalicylic acid) adopts a planar conformation, with the phenol hydrogen internally hydrogen-bonded to the carbonyl of one highly ordered carboxyl, which, in turn, donates a hydrogen bond to the oxygen of water. The second carboxyl is disordered and hydrogen-bonded both to water and to the disordered carboxyl of a centrosymmetrically related neighbor in a static disorder arrangement extending over two full asymmetric units. The water accepts either one or two hydrogen bonds and donates a long bifurcated hydrogen bond shared equally by O atoms of the phenol and the disordered carboxyl. The hydrogen bonding includes no standard carboxyl pairing and is entirely two-dimensional. The resulting planar ribbons stack translationally at a distance of 3.413 (8) A, in an offset arrangement having non-translational interplanar distances of 0.821 (5) and 2.592 (6) A. This structure is compared with two previously reported for this compound. The title compound forms a monoanion, whose tetraphenylphosphonium salt is described (C32H25O5P, tetraphenylphosphonium 2,6-dicarboxyphenolate, tetraphenylphosphonium 2-oxidoisophthalic acid or tetraphenylphosphonium 3-carboxysalicylate). The phenol oxygen is the site of formal negative charge on the anion, which is stabilized in a planar arrangement by symmetrical hydrogen bonds from both ortho-carboxyl groups. The energetics of this arrangement, the phenol and carboxyl acidities, and factors affecting those acidities and providing anion stabilization are discussed.

Structural and mutational studies of the carboxylate cluster in iron-free ribonucleotide reductase R2.

The R2 protein of ribonucleotide reductase features a di-iron site deeply buried in the protein interior. The apo form of the R2 protein has an unusual clustering of carboxylate side chains at the empty metal-binding site. In a previous study, it was found that the loss of the four positive charge equivalents of the diferrous site in the apo protein appeared to be compensated for by the protonation of two histidine and two carboxylate side chains. We have studied the consequences of removing and introducing charged residues on the local hydrogen-bonding pattern in the region of the carboxylate cluster of Corynebacterium ammoniagenes and Escherichia coli protein R2 using site-directed mutagenesis and X-ray crystallography. The structures of the metal-free forms of wild-type C. ammoniagenes R2 and the mutant E. coli proteins D84N, S114D, E115A, H118A, and E238A have been determined and their hydrogen bonding and protonation states have been structurally assigned as far as possible. Significant alterations to the hydrogen-bonding patterns, protonation states, and hydration is observed for all mutant E. coli apo proteins as compared to wild-type apo R2. Further structural variations are revealed by the wild-type apo C. ammoniagenes R2 structure. The protonation and hydration effects seen in the carboxylate cluster appear to be due to two major factors: conservation of the overall charge of the site and the requirement of electrostatic shielding of clustered carboxylate residues. Very short hydrogen-bonding distances between some protonated carboxylate pairs are indicative of low-barrier hydrogen bonding.

Close pairs of carboxylates: a possibility of multicenter hydrogen bonds in proteins.

Covalent attachment of hydrogen to the donor atom may be not an essential characteristic of stable hydrogen bonds. A positively charged particle (such as a proton), located between the two negatively charged residues, may lead to a stable interaction of the two negative residues. This paper analyzes close Asp-Glu pairs of residues in a large set of protein chains; 840 such pairs of residues were identified, of which 28% were stabilized by a metal ion, 12% by a positive residue nearby and 60% are likely to be stabilized by a proton. The absence of apparent structural constraints, secondary structure preferences, somewhat lower B-factors and a distinct correlation between pH and the minimal O-O distance in carboxylate pairs suggest that most of the abnormally close pairs could indeed be stabilized by a shared proton. Implications for protein stability and modeling are discussed.

N?H bond stretching in histidine complexes: a solid-state NMR study

N—H bond lengths in imidazolium–carboxylate pairs were studied as models for enzyme active site motifs. The bond lengths were measured using ‘2D 2ϕ-DipShift,’ a solid-state NMR method wherein the N,H dipolar coupling is determined using heteronuclear dipolar spinning sideband patterns. Like the situation for compressed O—H···O systems, some complexes exhibit very short N···O distances and weaker N,H dipolar coupling. The weaker time-average N,H dipolar coupling for systems with short N···O hydrogen bonds can be most likely associated with an altered potential well for the proton, or a ‘stretched’ covalent bond, although other interpretations involving a double well potential are discussed. The range or variation in average bond lengths seen in this study is much narrower than that previously reported for O—H···O systems (1.01–1.07 Å for N—H vs 1.0–1.3 Å for O—H bonds). Copyright © 2001 John Wiley & Sons, Ltd.

Design, synthesis and evaluation of synthetic receptors for the recognition of aspartate pairs in an α-helical conformation

The specific targeting of protein surface functional groups remains a largely unexplored aspect in molecular recognition. In this study, a series of zwitterionic, 16-mer peptides serve as models for the recognition of carboxylate pairs in proteins. A receptor is described that contains two guanidinium groups separated by 4–5 Å by a rigid bicyclo[3.3.0]octane spacer. Modeling studies indicate that such a receptor would be suitable for binding with two aspartate carboxylates when the amino acids are separated by two ( i+3) or three ( i+4) other amino acids in an α-helical peptide. Studies employing circular dichroism spectroscopy demonstrated that the addition of the receptor to the i+3 peptide substrate caused a 23% enhancement of helical structure in 15% water/methanol at 25°C. Other substrate peptides [( i+1), ( i+4), ( i+7), ( i+10)] showed lower helical induction. Similar, but weaker binding and helical induction were observed under buffered conditions (10 mM Tris-Mes, pH 7.0). These results, along with studies employing a series of related di-cationic receptors, suggest a 1:1 binding model composed of specific hydrogen interactions between each receptor guanidinium with each substrate carboxylate when the peptide adopts a helical conformation.

The structure of concanavalin A and its bound solvent determined with small-molecule accuracy at 0.94 [Aring ]resolution

The structure of concanavalin A, a saccharide-binding metalloprotein of subunit molecular weight 25000 Da§, is reported with precision and accuracy equivalent to those of small-molecule crystal structures. An X-ray data-to-parameter ratio of 6.1:1 has allowed the first unrestrained anisotropic refinement of a protein structure and, indeed, one of large molecular weight. This has been achieved by the combined power of synchrotron radiation, a highly sensitive CCD-based area detector and cryogenic techniques on crystals of exceptional quality. The high precision of the atomic coordinates and the direct observation of many hydrogen atom positions, on both the protein and bound solvent, provide new insights into the protein structure, including precise geometry of the metal-binding sites, the role of water molecules in the extended saccharide-binding site, the structure and dynamics of bound and buried water, and direct evidence that proximal carboxylic acid side chains can exist as carboxyl–carboxylate pairs. The biophysical chemistry of this protein is thereby elucidated in detail in this and the companion paper.

The amino acid sequence of the cowpea strain of tobacco mosaic virus protein

The amino acid sequence of the coat protein of the cowpea strain of tobacco mosaic virus (cowpea virus) has been determined. The tryptic peptide overlaps were obtained by digesting the protein with chymotrypsin and separating and analysing the lysine- and arginine-containing chymotryptic peptides. The primary structure of cowpea virus protein has been found to differ markedly from that of any other known strain of tobacco mosaic virus, and contains 3 amino acid residues more and 96 amino acid changes from the type strain. The significance of the distribution of those areas of the protein in which the amino acid residues are the same for all naturally occurring strains and chemically induced mutants of tobacco mosaic virus so far studied and the residues that form the important carboxyl · carboxylate pairs are discussed.