Structural consequences of mutations in interfacial Tyr residues of a protein antigen-antibody complex. The case of HyHEL-10-HEL

Tyrosine is an important amino acid in protein-protein interaction hot spots. In particular, many Tyr residues are located in the antigen-binding sites of antibodies and endow high affinity and high specificity to these antibodies. To investigate the role of interfacial Tyr residues in protein-protein interactions, we performed crystallographic studies and thermodynamic analyses of the interaction between hen egg lysozyme (HEL) and the anti-HEL antibody HyHEL-10 Fv fragment. HyHEL-10 has six Tyr residues in its antigen-binding site, which were systematically mutated to Phe and Ala using site-directed mutagenesis. The crystal structures revealed several critical roles for these Tyr residues in the interaction between HEL and HyHEL-10 as follows: 1) the aromatic ring of Tyr-50 in the light chain (LTyr-50) was important for the correct ternary structure of variable regions of the immunoglobulin light chain and heavy chain and of HEL; 2) deletion of the hydroxyl group of Tyr-50 in the heavy chain (HTyr-50) resulted in structural changes in the antigen-antibody interface; and 3) the side chains of HTyr-33 and HTyr-53 may help induce fitting of the antibody to the antigen. Hot spot Tyr residues may contribute to the high affinity and high specificity of the antigen-antibody interaction through a diverse set of structural and thermodynamic interactions.     

Involvement of water molecules in the association of monoclonal antibody HyHEL-5 with bobwhite quail lysozyme.

Fluorescence polarization spectroscopy and isothermal titration calorimetry were used to study the influence of osmolytes on the association of the anti-hen egg lysozyme (HEL) monoclonal antibody HyHEL-5 with bobwhite quail lysozyme (BWQL). BWQL is an avian species variant with an Arg-->Lys mutation in the HyHEL-5 epitope, as well as three other mutations outside the HyHEL-5 structural epitope. This mutation decreases the equilibrium association constant of HyHEL-5 for BWQL by over 1000-fold as compared to HEL. The three-dimensional structure of this complex has been obtained recently. Fluorescein-labeled BWQL, obtained by labeling at pH 7.5 and purified by hydrophobic interaction chromatograpy, bound HyHEL-5 with an equilibrium association constant close to that determined for unlabeled BWQL by isothermal titration calorimetry. Fluorescence titration, stopped-flow kinetics, and isothermal titration calorimetry experiments using various concentrations of the osmolytes glycerol, ethylene glycol, and betaine to perturb binding gave a lower limit of the uptake of approximately 6-12 water molecules upon formation of the HyHEL-5/BWQL complex.     

Interchain hydrogen bonds via bound water molecules in the collagen triple helix

If the collagen triple helix is so built as to have one set of NH ? O hydrogen bonds of the type N₃H₃(A) ? O₂(B), then it is possible to have a linkage between N₁H₁(B) and O₁(A) through the intermediary of a water molecule with an oxygen O leading to the formation of the hydrogen bonds N₁(B) ? O and O (A). In the same configuration, another water molecule with an oxygen O can link two earbonyl oxygens of chains A and B forming the hydrogen bonds O O₁(A) and O O₀ (B). The two water oxygens also become receptors at the same time for CH ? O hydrogen bonds. Thus, the neighboring chains in the triple helix are held together by secondary valence bond linkages occurring regularly sit intervals of about 3 Å along the length of the protofibril. The additional water molecules occur on the periphery of the proto-fibril and will contribute their full share towards stabilizing the structure in the solid state. In solution, they will be disturbed by the medium unless they are protected by long side groups. It appears that this type of two-bonded structure, in which one NH ? O bond is to a water molecule, can explain several observations on the stability and hydrogen exchange properties of collagen itself and related synthetic polypeptides. The nature of the water bonds and their strength are found to be better in the one-bonded structure proposed from Madras than in the one having the coordinates of Rich and Crick.     

The importance of hydrogen bonding in the complexation of lanthanide ions by polymer-bound malonamide-type ligands

Polymer-bound malonate ligands modified with diethylenetriamine (DETA-MAm) are prepared and the lanthanide ion affinities from solutions of 0.001-8 M HCl are quantified. A mechanism is proposed. The affinities are not due to the triamine ligand alone or the adjacent carbonyl sites alone: protonation of the carbonyl oxygen triggers formation of an iminium ion and it acts as an ion-exchange site. Two competing reactions occur during binding: electrostatic attraction of [Ln(H₂O)_xCl₄]⁻ by the protonated ligand and (partial) loss of the waters of hydration. The affinity and selectivity are affected by substituents on the iminium (CNRR′(+)) ion. Research with tetramethylmalonamide showed that its two methyl groups at the iminium site weaken the positive charge and decrease its affinity for the chlorocomplexes of the later lanthanides; DETA-MAm has at its amide nitrogen only one −CH₂− (and one H) moiety and therefore is a stronger but less selective ligand since electrostatic attraction is more dominant in the overall mechanism. The higher affinities of malonate monoamidated with ethylenediamine (EDA-MAm) and decreased affinities for those amidated with ethanolamine (EA-MAm) suggest that the protonated -NH- stabilizes the lanthanide chlorocomplex.     

Association and dissociation kinetics of anti-hen egg lysozyme monoclonal antibodies HyHEL-5 and HyHEL-10.

The immunoglobulin G1 (IgG1) kappa antibodies HyHEL-5 and HyHEL-10 interact with nonoverlapping epitopes on hen egg lysozyme (HEL); the HyHEL-5/HEL interface has two energetically and structurally important salt links, whereas the HyHEL-10/HEL interface involves predominantly hydrogen bonds and van der Waals interactions. The kinetics of association and dissociation of antibodies HyHEL-5 and HyHEL-10 with HEL under a variety of conditions were investigated in this study. The association of each antibody with HEL follows second-order kinetics. The association process is significantly diffusion-limited, as indicated by the viscosity dependence of the interaction of both antibodies with HEL, although detailed energetics suggest that the association process may be more complex. The association rate constant for the HyHEL-5/HEL system is within a factor of 2 of the modified Smoluchowski estimate for proteins of this size, whereas HyHEL-10 interacts with HEL with an association rate an order of magnitude lower. The association reactions are insensitive to ionic strength, showing only a twofold decrease in the association rate constant when the ionic strength was increased from 27 mM to 500 mM. Interestingly, the association rate constant for the interaction of HyHEL-5 with HEL varies with pH in the range 6.0-10.0, whereas HyHEL-10/HEL association is not affected by pH in the same range. The dissociation of the HyHEL-5/HEL and HyHEL-10/HEL complexes follow first-order kinetics with half-lives at 25 degrees C of approximately 3,150 s and approximately 21,660 s, respectively.     

Investigation of the haem-nicotinate interaction in leghaemoglobin. Role of hydrogen bonding.

A strategic assessment of the contributions of two active-site hydrogen bonds in the binding of nicotinate to recombinant ferric soybean leghaemoglobin a (rLb) was carried out by mutagenic replacement of the hydrogen-bonding residues (H61A and Y30A variants) and by complementary chemical substitution of the carboxylate functionality on the nicotinate ligand. Dissociation constants, Kd (pH 5.5, mu = 0.10 M, 25.0 +/- 0.1 degrees C), for binding of nicotinate to ferric rLb, H61A and Y30A were 1.4 +/- 0.3 microM, 19 +/- 1 microM and 11 +/- 1 microM, respectively; dissociation constants for binding of nicotinamide were, respectively, 38 +/- 1 mM, 50 +/- 2 mM and 12 +/- 1 mM, and for binding of pyridine were 260 +/- 50 microM, 4.5 +/- 0.5 microM and 66 +/- 8 microM, respectively. Binding of cyanide and azide to the H61A and Y30A variants was unaffected by the mutations. The pH-dependence of nicotinate binding for rLb and Y30A was consistent with a single titration process (pKa values 6.9 +/- 0.1 and 6.7 +/- 0.2, respectively); binding of nicotinate to H61A was independent of pH. Reduction potentials for the rLb and rLb-nicotinate derivatives were 29 +/- 2 mV (pH 5.40, 25.0 degrees C, mu = 0.10 M) and - 65 +/- 2 mV (pH 5.42, 25.0 degrees C, mu = 0.10 M), respectively. The experiments provide a quantitative assessment of the role of individual hydrogen bonds in the binding process, together with a definitive determination of the pKa of His61 and unambiguous evidence that titration of His61 controls binding in the neutral to alkaline region.     

The role of hydrogen bonding in the cuticular wax of Hordeum vulgare L.

The role of hydrogen bonding in the cuticular wax of Hordeum vulgare L. has been investigated by comparing differential scanning calorimetry and X-ray powder diffraction results of the wax with those of n-alkane mixtures with chain-length distributions resembling that of the wax. It is concluded that hydrogen bonding prevents separation of the short and long chain-length distributions and results in the formation of an amorphous component which is large compared with that of a typical paraffinic wax. It seems that the longer ester chains (39 ≥n≥ 50) bridge the amorphous zone containing chain-ends between two adjacent layers of shorter chains (20 ≥n≥ 33), where n is the number of carbon atoms per chain. In contrast to a paraffinic wax, which has a monolayered structure, this plant wax has a bilayered structure.     

The Phe-X-Glu DNA binding motif of MutS. The role of hydrogen bonding in mismatch recognition

The crystal structures of MutS protein from Thermus aquaticus and Escherichia coli in a complex with a mismatch-containing DNA duplex reveal that the Glu residue in a conserved Phe-X-Glu motif participates in a hydrogen-bonded contact with either an unpaired thymidine or the thymidine of a G-T base-base mismatch. Here, the role of hydrogen bonding in mismatch recognition by MutS is assessed. The relative affinities of MutS for DNA duplexes containing nonpolar shape mimics of A and T, 4-methylbenzimidazole (Z), and difluorotoluene (F), respectively, that lack hydrogen bonding donors and acceptors, are determined in gel mobility shift assays. The results provide support for an induced fit mode of mismatch binding in which duplexes destabilized by mismatches are preferred substrates for kinking by MutS. Hydrogen bonding between the O epsilon 2 group of Glu and the mismatched base contributes only marginally to mismatch recognition and is significantly less important than the aromatic ring stack with the conserved Phe residue. A MutS protein in which Ala is substituted for Glu(38) is shown to be defective for mismatch repair in vivo. DNA binding studies reveal a novel role for the conserved Glu residue in the establishment of mismatch discrimination by MutS.     

Understanding interaction and dynamics of water molecules in the epoxy via molecular dynamics simulation

The robust structural integrity of the epoxy plays an important role in ensuring the long-term service life of its applications, which is affected by the absorbed moisture. In order to understand the mechanism of the moisture effect, the knowledge of the interaction and dynamics of the water molecules inside the epoxy is of great interest. Molecular dynamics simulation is used in this work to investigate the structure and bonding behaviour of the water molecules in the highly cross-linked epoxy network. When the moisture concentration is low, the water molecules are well dispersed in the cross-linked structure and located in the vicinity of the epoxy functional groups, which predominantly form the hydrogen bond (H-bond) with the epoxy network, resulting in the low water mobility in the epoxy. At the high concentration, the water favourably forms the large cluster due to the predominant water–water H-bond interaction, and the water molecules diffuse primarily inside the cluster, which leads to the high water mobility and the accelerated H-bond dynamics. The variation of the bonding behaviour and dynamics of the water molecules reported here could be exploited to understand the material change and predict the long-term performance of the epoxy-based products during the intended service life.     

The role of protein-bound water molecules in microbial rhodopsins

Protein-bound internal water molecules are essential features of the structure and function of microbial rhodopsins. Besides structural stabilization, they act as proton conductors and even proton storage sites. Currently, the most understood model system exhibiting such features is bacteriorhodopsin (bR). During the last 20 years, the importance of water molecules for proton transport has been revealed through this protein. It has been shown that water molecules are as essential as amino acids for proton transport and biological function. In this review, we present an overview of the historical development of this research on bR. We furthermore summarize the recently discovered protein-bound water features associated with proton transport. Specifically, we discuss a pentameric water/amino acid arrangement close to the protonated Schiff base as central proton-binding site, a protonated water cluster as proton storage site at the proton-release site, and a transient linear water chain at the proton uptake site. We highlight how protein conformational changes reposition or reorient internal water molecules, thereby guiding proton transport. Last, we compare the water positions in bR with those in other microbial rhodopsins to elucidate how protein-bound water molecules guide the function of microbial rhodopsins. This article is part of a Special Issue entitled: Retinal Proteins — You can teach an old dog new tricks.     

The role of a hydrogen bonding network in the transmembrane beta-barrel OMPLA

The hydrogen bonding of polar side-chains has emerged as an important theme for membrane protein interactions. The crystal structure of the dimeric state of the transmembrane beta-barrel protein outer membrane phospholipase A (OMPLA) revealed an intermolecular hydrogen bond mediated by a highly conserved glutamine side-chain (Q94). It has been shown that the introduction of a polar residue can drive the association of model helices, and by extension it was presumed that the glutamine hydrogen bond played a key role in stabilizing the OMPLA dimer. However, a thermodynamic investigation using sedimentation equilibrium ultracentrifugation in detergent micelles reveals that the hydrogen bond plays only a very modest role in stabilizing the dimer. The Q94 side-chain is hydrogen bonded intramolecularly to residues Y92 and S96, but amino acid substitutions at these positions suggest these intramolecular interactions are not responsible for attenuating the strength of the intermolecular Q94 hydrogen bond. Other substitutions suggested that hydration of the local environment around Q94 may be responsible for the modest strength of the hydrogen bond. Heat inactivation experiments with the variants suggest that the Y92-Q94-S96 network may instead be important for thermal stability of the monomer. These results highlight the context dependence and broad range of interactions that can be mediated by polar residues in membrane proteins.     

The role of interfacial structured water on the glycoprotein arrangement in liposomes

The effect of perturbing the interfacial water structure in liposomes on the glycoprotein arrangement in the bilayer was investigated. This perturbation was achieved by a series of reagents called structure makers and breakers. The glycoprotein arrangement in the liposomes was determined by fluorescence measurement with 1-anilino-2-naphtalene sulphonate (ANS). A dependence of (n) (number of binding sites for ANS on the glycoprotein molecule) with concentration of structure maker and breaker reagents was observed. The results have been interpreted as a possible new arrangement of membrane-bound glycoprotein, due to the effect of perturbing the interfacial water structure in the liposomes.     

The role of glycine and prolines in connective tissue fiber staining with hydrogen bonding dyes

Extensive hydrogen bonding of dyes to connective tissue fibers is made possible by the high content of the amino acids proline and glycine in elastin and collagens. Proline confers an extended polypeptide structure and glycine is the only amino acid whose specific side group, -H, is so small that it forms no obstacle to hydrogen bonding between the peptide group and external molecules. Thus, a high proportion of the peptide groups in fibrous proteins are directly accessible to hydrogen bonding groups dye molecules.     

Number of water molecules coupled to the transport of sodium, potassium and hydrogen ions via gramicidin, nonactin or valinomycin.

The number of water molecules (n) coupled to the transport of cations across lipid membranes was determined in two different wats: directly from the electro-osmotic volume flux per ion, and by the use of Onsager's relation, from the open circuit streaming potential produced by an osmotic pressure difference. The results of the two approaches were in general agreement. Monoolein membranes were formed on the ends of polyethylene or Teflon tubing connected to a microliter syringe and the volume change necessary to keep the membrane at a fixed position was measured. It was necessary to make corrections for unstirred layer effects. The results for gramicidin were: n approximately 12 for 0.15 M KCl and NaCl, n approximately 6 for 3.0 M KCl and NaCl, and n approximately 0 for 0.01 M HCl. For nonactin, n approximately 4 for both 0.15 and 3.0 M KCl and NaCl. Valinomycin (for 0.15 M KCl) behaved like nonactin. It is shown that for a channel mechanism, in general, n is less than or equal to the number of water molecules in a channel that does not contain any cations. Thus, the n of 12 for the 0.15 M salts implies that the gramicidin channel can hold at least 12 water molecules. This places an important constraint on models of the channel structure. The n of 0 for HCl is consistent with a process in which protons jump along a continuous row of water molecules. The decrease of n with the 3.0 M salts may indicate that the channel becomes multiply occupied at high salt concentrations. The n of 4 for nonactin and valinomycin means that at least four water molecules are associated with the carrier . cation complex, probably in the interstices between the complex and the disordered lipid.     

Role of hydrogen bonding in the interaction between a xylan binding module and xylan

NMR studies of the internal family 2b carbohydrate binding module (CBM2b-1) of Cellulomonas fimi xylanase 11A have identified six polar residues and two aromatic residues that interact with its target ligand, xylan. To investigate the importance of the various interactions, free energy and enthalpy changes have been measured for the binding of xylan to native and mutant forms of CBM2b-1. The data show that the two aromatic residues, Trp 259 and Trp 291, play a critical role in the binding, and similarly that mutants N264A and T316A have no affinity for the xylose polymer. Interestingly, mutations E257A, Q288A, N292A, E257A/Q288A, E257A/N292A, and E257A/N292A/Q288A do not significantly diminish the affinity of CBM2b-1 for the xylose polymers, but do influence the thermodynamics driving the protein-carbohydrate interactions. These thermodynamic parameters have been interpreted in light of a fresh understanding of enthalpy-entropy compensation and show the following. (1) For proteins whose ligands are bound on an exposed surface, hydrogen bonding confers little specificity or affinity. It also displays little cooperativity. Most specificity and affinity derive from binding between the face of sugar rings and aromatic rings. (2) Loss of hydrogen bonding interactions leads to a redistribution of the remaining bonding interactions such that the entropic mobility of the ligand is maximized, at the expense (if necessary) of enthalpically favorable bonds. (3) Changes in entropy and enthalpy in the binding between polysaccharide and a range of mutants can be interpreted by considering changes in binding and flexibility, without any need to consider solvent reorganization.     

Role of hydrogen bonding in red cell aggregation.

The role of hydrogen bonding in red cell aggregation induced by dextran was studied with the use of urea, an inhibitor for hydrogen bonding. In order to avoid hemolysis of red cells by the high concentration of urea, the studies were performed on human red cells hardened in glutaraldehyde. The degree of red cell aggregation at Hct = 45% was estimated by the use of a coaxial cylinder viscometer. The viscometric aggregation index (VAI) was calculated from viscosity values at shear rates of 52 sec-1 (eta H) and 0.05 sec-1 (eta L); VAI = (eta L - eta H)/eta H. Red cells with surface charge intact and with charge removal by neuraminidase treatment were studied. Urea at high concentrations, e.g., 6 M, significantly inhibited red cell aggregation induced by dextran. These findings indicate that hydrogen bonding plays an important role in dextran-induced red cell aggregation. An understanding of the nature of the forces involved in red cell aggregation serves to establish the physicochemical principles of cell-to-cell interactions induced by macromolecules.     

The critical role of substrate-protein hydrogen bonding in the control of regioselective hydroxylation in p450cin

Cytochrome P450cin (CYP176A1) is a bacterial P450 isolated from Citrobacter braakii that catalyzes the hydroxylation of cineole to (S)-6beta-hydroxycineole. This initiates the biodegradation of cineole, enabling C. braakii to live on cineole as its sole source of carbon and energy. P450cin lacks the almost universally conserved threonine residue believed to be involved in dioxygen activation and instead contains an asparagine at this position (Asn-242). To investigate the role of Asn-242 in P450cin catalysis, it was converted to alanine, and the resultant mutant was characterized. The characteristic CO-bound spectrum and spectrally determined K(D) for substrate binding were unchanged in the mutant. The x-ray crystal structures of the substrate-free and -bound N242A mutant were determined and show that the only significant change is in a reorientation of the substrate such that (R)-6alpha-hydroxycineole should be a major product. Molecular dynamics simulations of both wild type and mutant are consistent with the change in regio- and stereoselectivity predicted from the crystal structure. The mutation has only a modest effect on enzyme activity and on the diversion of the NADPH-reducing equivalent toward unproductive peroxide formation. Product profile analysis shows that (R)-6alpha-hydroxycineole is the main product, which is consistent with the crystal structure. These results demonstrate that Asn-242 is not a functional replacement for the conserved threonine in other P450s but, rather, is critical in controlling regioselective substrate oxidation.