Fluorescence polarization studies of lysozyme and lysozyme-saccharide complexes

The fluorescence polarization properties of hen egg white lysozyme and of an iodine oxidized derivative of lysozyme in which tryptophan-108 was selectively modified, were investigated. Using the addition law of anisotropy of mixed systems, the contribution of tryptophan-108 to the anisotropy spectrum of lysozyme and lysozyme-chitotetraose complex was separated. The rate of fluorescence polarization was studied as a function of pH. The major contribution to this rate is shown to arise from internal rotations of the indole side-chain of tryptophan-108 as well as from structural changes around tryptophan-62 and 63. From the dependence of the fluorescence polarization of lysozyme and IL with saccharide concentration, the existence of the simultaneous binding of two saccharide molecules to the enzyme cleft was inferred. At low chitotetraose concentration, the subsites A, B and C are occupied with an association constant of 8 × 10⁴m^?1 whereas at high saccharide concentration, both subsites A–B–C and E–F are occupied. The association constants of a series of saccharides to subsites E–F were measured and all found to be around 2 × 10²m^?1. The dependence of the rate of depolarization with saccharide concentration was determined and showed that, upon binding of the first saccharide molecule to subsites A, B and C, the rate of internal rotation of tryptophan-108 and tryptophan-62 and 63 was much reduced whereas upon further binding of a saccharide molecule in subsites E–F the rates are enhanced. This behaviour was interpreted as an indication that the binding of saccharide in subsites E–F induces changes in conformation of the enzyme which affect the entire active site architecture.     

Conformational rearrangements of bacteriophage T4 lysozyme during its binding to the inhibitor]

The structural changes of bacteriophage T4 lysozyme during its binding to the inhibitor, i. e. disaccharide-tetrapeptide N-acetylglucosaminyl-N-acetylmuraminyl - L - alanyl-gamma-D-glutaminyl - mesodiaminopimelyl-D-alanine) isolated from Escherichia coli cell wall have been studied. During the inhibitor binding to the protein the degree of helicity decreases by approximately 14% as was shown using the circular dichroism technique. The changes in optical properties of tryptophane, tyrosine and phenylalanine residues detected by UV difference and fluorescence spectroscopy have been observed. Based on the experimental data and a comparison of spatial organization of phage T4 lysozyme and chicken egg-white lysozyme made it possible to develop a structural model of phage T4 lysozyme functioning. This model may account for the differences in specificity of action of bacteriophage T4 and chicken egg-white lysozymes and allows to establish the role of the "extra" part of phage lysozyme. According to the model, at the first stage of binding the peptide part of the substrate comes in contact with the "upper" (with respect to the cleft) part of the protein molecule (residues 106--116 and 135--140). This results in rearrangement of the molecule, with opening of the cleft at the second stage. This makes possible the access of the polysaccharide part of the substrate of the active site and a subsequent hydrolysis of the beta (1 leads to 4) glycoside bond.     

Left-sided substrate binding of lysozyme: evidence for the involvement of asparagine-46 in the initial binding of substrate to chicken lysozyme.

The "right-sided" and "left-sided" substrate binding modes at the lower saccharide binding subsites (D-F sites) of chicken lysozyme were investigated by utilizing mutant lysozymes secreted from yeast. We constructed the following mutant lysozymes; "left-sided" substitution of Asn46 to Asp, deletion of Thr47, and insertion of Gly between Thr47 and Asp48 and "right-sided" substitution of Asn37 to Gly. Analyses of their activities and substrate binding abilities showed that Asn46 and Thr47 are involved in the initial enzyme-substrate complex and Asn37 is involved in the transition state. These results support an earlier proposal that interactions between substrate and residues at the left side of lysozyme stabilize a catalytically inactive enzyme-substrate complex, while interactions between substrate and residues at the right side stabilize the catalytically active complex [Pincus, M. R., & Scheraga, H. A. (1979) Macromolecules 12, 633-644]. These results are also consistent with the proposed kinetic mechanism for lysozyme reaction that the rearrangement of an initial enzyme-substrate complex (beta-complex) to another complex (gamma-complex) is required for catalytic hydrolysis [Banerjee S. K., Holler, E., Hess, G. P., & Rupley, J. A. (1975) J. Biol. Chem. 250, 4355-4367].     

Tryptophan fluorescence lifetimes in lysozyme.

Tryptophan fluorescence lifetimes at pH 2 and pH 8 have been obtained for lysozyme and for lysozyme derivatives in which tryptophan-62 or tryptophan-108 or both are nonfluorescent. The lifetimes range from about 0.5 ns to 2.8 ns for the various emitting tryptophans. The tryptophan lifetimes appear to increase with exposure of tryptophan to solvent, but intramolecular contacts, probably with cystine residues, can considerably shorten the lifetime. Intertryptophanyl interactions can also affect fluorescence lifetimes. The trytophan-108 lifetime in lysozyme is shorter than in the derivative in which tryptophan-62 is oxidized; this is ascribed to energy transfer from tryptophan-108 to tryptophan-62. From the lifetime results the relative intensities emitted by specific tryptophans can be estimated, and these values also support the existence of intertryptophanyl energy transfer. The emission intensity from tryptophan-62 is greater in the presence of tryptophan-108, and the emission intensity of tryptophan-108 appears to be greater in the absence of tryptophan-62. Conformational effects accompanying chemical modification of tryptophan cannot be completely ruled out, however. The tryptophan-62 lifetime at pH 8 in lysozyme is shorter than in the derivatives, which might indicate a subtle conformational effect. Studies with tri-(N-acetyl-glucosamine)-protein complexes indicate that both the tryptophan lifetimes and the number of emitting tryptophans may be changing upon complexation. The results illustrate the usefulness and the limitations of lifetime measurements in understanding protein fluorescence.     

An NMR and MD study of complexes of bacteriophage lambda lysozyme with tetra- and hexa-N-acetylchitohexaose

The X-ray structure of lysozyme from bacteriophage lambda (λ lysozyme) in complex with the inhibitor hexa-N-acetylchitohexaose (NAG6) (PDB: 3D3D) has been reported previously showing sugar units from two molecules of NAG6 bound in the active site. One NAG6 is bound with four sugar units in the ABCD sites and the other with two sugar units in the E′F′ sites potentially representing the cleavage reaction products; each NAG6 cross links two neighboring λ lysozyme molecules. Here we use NMR and MD simulations to study the interaction of λ lysozyme with the inhibitors NAG4 and NAG6 in solution. This allows us to study the interactions within the complex prior to cleavage of the polysaccharide. ¹H^N and ¹⁵N chemical shifts of λ lysozyme resonances were followed during NAG4/NAG6 titrations. The chemical shift changes were similar in the two titrations, consistent with sugars binding to the cleft between the upper and lower domains; the NMR data show no evidence for simultaneous binding of a NAG6 to two λ lysozyme molecules. Six 150 ns MD simulations of λ lysozyme in complex with NAG4 or NAG6 were performed starting from different conformations. The simulations with both NAG4 and NAG6 show stable binding of sugars across the D/E active site providing low energy models for the enzyme-inhibitor complexes. The MD simulations identify different binding subsites for the 5th and 6th sugars consistent with the NMR data. The structural information gained from the NMR experiments and MD simulations have been used to model the enzyme-peptidoglycan complex.     

Modelling of lysozyme binding to a cation exchange surface at atomic detail: the role of flexibility

Different approaches were made to predict the adsorbed orientation based on rigid, flexible, or a mixture of both models. To determine the role of flexibility during adsorption, the orientation of lysozyme adsorbed to a negatively charged ligand surface was predicted by a rigid and a flexible model based on two differing protein structures at atomic resolution. For the rigid model, the protein structures were placed at different distances from the ligand surface and the electrostatic interaction energy was calculated for all possible orientations. The results were compared to a flexible model where the binding to the ligand surface was modeled by multiple molecular dynamics simulations starting with 14 initial orientations. Different aspects of the adsorption process were not covered by the rigid model and only detectable by the flexible model. Whereas the results of the rigid model depended sensitively on the protein-surface distance and the protein structure, the preferred orientation obtained by the flexible model was closer to a previous experimental determined orientation, robust toward the initial orientation and independent of the initial protein structure. Additionally, it was possible to obtain insights into the preferred binding process of lysozyme on a negatively charged surface by the flexible model.     

Crystal structures of egg-white lysozyme of hen in acetate-free medium and of lysozyme complexes with N-acetylglucosamine and beta-methyl N-acetylglucosaminide.

The binding of beta-methyl N-acetylglucosaminide (betaMeGlcNAc) to egg-white lysozyme of hen in the tetragonal crystal form was studied by X-ray diffraction techniques to a resolution of 0.25 nm. The binding of the beta-methyl glycoside is almost identical with the binding of beta-N-acetylglucosamine (betaGlcNAc). Real-space refinement of the lysozyme-alpha/beta GlcNAc and lysozyme-betaMeGlcNAc complexes allowed preliminary analysis of the conformational changes observed on binding monosaccharide inhibitors, specially in the region involving tryptophan-62 and residues 70--76. Tetagonal lysozyme crystals, grown in the absence of acetate ions, were examined by X-ray diffraction to 0.25nm resolution. The resulting difference Fourier synthesis shows no firm evidence for bound acetate ions and indicates only minor conformational changes in the side-chain positions of aspartic acid-101 and asparagine-103. The close similarity of the lysozyme structures in the presence and absence of acetate is contrary to expectations from previous n.m.r. studies.     

Raman spectroscopic characterization of tryptophan side chains in lysozyme bound to inhibitors: role of the hydrophobic box in the enzymatic function.

The state of H-bonding and the hydrophobic interaction of six tryptophan side chains in lysozyme bound to substrate-analogous inhibitors were investigated by combining H----D exchange labeling and Raman difference spectroscopy. The frequency of the W17 band due to Trp-63 shifts downward upon inhibitor binding, indicating a specific and strong H-bond formation between the N1 site of the side chain and the inhibitor molecule. On the other hand, the H-bonding state of Trp-62 in the complex is as weak as that in inhibitor-free lysozyme, suggesting no contribution of this residue to the inhibitor binding. Intensity increases of W17 and W18 bands observed upon inhibitor binding are, respectively, ascribed to an increase at Trp-28 and a decrease at Trp-111 in hydrophobic interactions with the environment. The environmental changes are explained consistently by a movement of the Met-105 side chain sandwiched by two indole rings of Trp-28 and 111 in the direction from Trp-111 to Trp-28. The sandwich structure in a core domain, hydrophobic box, and its rearrangement are considered to play an important role in the enzymatic function of lysozyme.     

Crystal structure of a phage library-derived single-chain Fv fragment complexed with turkey egg-white lysozyme at 2.0 A resolution

The three-dimensional structure of the single-chain Fv fragment 1F9 in complex with turkey egg-white lysozyme (TEL) has been determined to a nominal resolution of 2.0 A by X-ray diffraction. The scFv fragment 1F9 was derived from phage-display libraries in two steps and binds both hen and turkey egg-white lysozyme, although the level of binding affinity is two orders of magnitude greater for the turkey lysozyme. The comparison of the crystal structure with a model of the single-chain Fv fragment 1F9 in complex with hen egg-white lysozyme (HEL) reveals that in the latter a clash between Asp101 in lysozyme and Trp98 of the complementarity determining region H3 of the heavy chain variable domain occurs. This is the only explanation apparent from the crystal structure for the better binding of TEL compared to HEL.The binding site topology on the paratope is not simply a planar surface as is usually found in antibody-protein interfaces, but includes a cleft between the light chain variable domain and heavy chain variable domain large enough to accommodate a loop from the lysozyme. The scFv fragment 1F9 recognizes an epitope on TEL that differs from the three antigenic determinants recognized in other known crystal structures of monoclonal antibodies in complex with lysozyme.     

Protein structural change upon ligand binding correlates with enzymatic reaction mechanism

Overall structural changes of enzymes in response to ligand binding were investigated by database analysis of 62 non-redundant enzymes whose ligand-unbound and ligand-bound forms were available in the Protein Data Bank. The results of analysis indicate that transferases often undergo large rigid-body domain motions upon ligand binding, while other enzymes, most typically, hydrolases, change their structures to a small extent. It was also found that the solvent accessibility of the substrate molecule was low in transferases but high in hydrolases. These differences are explained by the enzymatic reaction mechanisms. The transferase reaction requires the catalytic groups to be insulated from the water environment, and thus transferases bury the ligand molecule inside the protein by closing the cleft. On the other hand, the hydrolase reaction involves the surrounding water molecules and occurs at the protein surface, requiring only a small structural change.     

Agonist-induced isomerization in a glutamate receptor ligand-binding domain. A kinetic and mutagenetic analysis

Agonist binding to glutamate receptor ion channels occurs within an extracellular domain (S1S2) that retains ligand affinity when expressed separately. S1S2 is homologous to periplasmic binding proteins, and it has been proposed that a Venus flytrap-style cleft closure triggers opening of glutamate receptor ion channels. Here we compare the kinetics of S1S2-agonist binding to those of the periplasmic binding proteins and show that the reaction involves an initial rapid association, followed by slower conformational changes that stabilize the complex: "docking" followed by "locking." The motion detected here reflects the mechanism by which the energy of glutamate binding is converted into protein conformational changes within S1S2 alone. In the intact channel, these load-free conformational changes are harnessed and possibly modified as the agonist binding reaction is used to drive channel opening and subsequent desensitization. Using mutagenesis, key residues in each step were identified, and their roles were interpreted in light of a published S1S2 crystal structure. In contrast to the Venus flytrap proposal, which focuses on motion between the two lobes as the readout for agonist binding, we argue that smaller, localized conformational rearrangements allow agonists to bridge the cleft, consistent with published hydrodynamic measurements.     

The oxidative refolding of hen lysozyme and its catalysis by protein disulfide isomerase.

The oxidative refolding of hen lysozyme has been studied by a variety of time-resolved biophysical methods in conjunction with analysis of folding intermediates using reverse-phase HPLC. In order to achieve this, refolding conditions were designed to reduce aggregation during the early stages of the folding reaction. A complex ensemble of relatively unstructured intermediates with on average two disulfide bonds is formed rapidly from the fully reduced protein after initiation of folding. Following structural collapse, the majority of molecules slowly form the four-disulfide-containing fully native protein via rearrangement of a highly native-like, kinetically trapped intermediate, des-[76-94], although a significant population (approximately 30%) appears to fold more quickly via other three-disulfide intermediates. The folding catalyst PDI increases dramatically both yields and rates of lysozyme refolding, largely by facilitating the conversion of des-[76-94] to the native state. This suggests that acceleration of the folding rate may be an important factor in avoiding aggregation in the intracellular environment.     

1H-NMR study of the intramolecular interaction of a substrate analogue covalently attached to aspartic acid-101 in lysozyme

We prepared the lysozyme derivative in which the beta-carboxyl group of Asp101 was modified with alpha-O-methyl N-glycylglucosaminide as an amide by means of the carbodimide reaction (alpha-MGG lysozyme). Since Asp101 residue is located at the edge of the active site cleft, a 1H-NMR study was carried out for this derivative in order to investigate the interaction between the introduced substituent and the active site cleft. It was confirmed that the alpha-MGG moiety sat in the active site cleft in alpha-MGG lysozyme from the reduction of line broadening of the NH-proton of Trp63 located in the active site cleft, the remarkable chemical shift change of the methyl group of the alpha-MGG moiety upon adding a trimer of N-acetyl-D-glucosamine [(NAG)3], and the NOE between the C6-proton resonance of Trp63 and the methyl resonance of the alpha-MGG moiety. Furthermore, alpha-MGG lysozyme had increased thermal stability compared with native lysozyme. Therefore, it was concluded that the alpha-MGG moiety covalently attached to Asp101 interacted with the active site cleft to increase the thermal stability of lysozyme.     

The C-terminus of glutathione S-transferase A1-1 is required for entropically-driven ligand binding

Binding of a hydrophobic glutathione product conjugate to rGST A1-1 proceeds via a two-step mechanism, including rapid ligand docking, followed by a slow isomerization to the final [GST.ligand] complex, which involves the localization of the flexible C-terminal helix. These kinetically resolved steps have been observed previously by stopped-flow fluorescence with the wild-type rGST A1-1, which contains a native Trp-21 approximately 20 A from the ligand binding site at the intrasubunit domain-domain interface. To confirm this binding mechanism, as well as elucidate the effects of truncation of the C-terminus, we have further characterized the binding and dissociation of the glutathione-ethacrynic acid product conjugate (GS-EA) to wild-type, F222W:W21F, and Delta209-222 rGST A1-1 and wild-type hGST A1-1. Although modest kinetic differences were observed between the hGST A1-1 and rGST A1-1, stopped-flow binding studies with GS-EA verified that the two-step mechanism of ligand binding is not unique to the GST A1-1 isoform from rat. An F222W:W21F rGST A1-1 double mutant provides a direct fluorescence probe of changes in the environment of the C-terminal residue. The observation of two relaxation times during ligand binding and dissociation to F222W:W21F suggests that the C-terminus has an intermediate conformation following ligand docking, which is distinct from its conformation in the apoenzyme or localized helical state. For the wild-type, Delta209-222, and F222W:W21F proteins, variable-temperature stopped-flow experiments were performed and activation parameters calculated for the individual steps of the binding reaction. Activation parameters for the binding reaction coordinate illustrate that the C-terminus provides a significant entropic contribution to ligand binding, which is completely realized within the initial docking step of the binding mechanism. In contrast, the slow isomerization step is enthalpically driven. The partitioning of entropic and enthalpic components of binding energy was confirmed by isothermal titration calorimetry with wild-type and Delta209-222 rGST A1-1.     

Placing single-molecule T4 lysozyme enzymes on a bacterial cell surface: toward probing single-molecule enzymatic reaction in living cells

The T4 lysozyme enzymatic hydrolyzation reaction of bacterial cell walls is an important biological process, and single-molecule enzymatic reaction dynamics have been studied under physiological condition using purified Escherichia coli cell walls as substrates. Here, we report progress toward characterizing the T4 lysozyme enzymatic reaction on a living bacterial cell wall using a combined single-molecule placement and spectroscopy. Placing a dye-labeled single T4 lysozyme molecule on a targeted bacterial cell wall by using a hydrodynamic microinjection approach, we monitored single-molecule rotational motions during binding, attachment to, and dissociation from the cell wall by tracing single-molecule fluorescence intensity time trajectories and polarization. The single-molecule attachment duration of the T4 lysozyme to the cell wall during enzymatic reactions was typically shorter than the photobleaching time under physiological conditions. Applying single-molecule fluorescence polarization measurements to characterize the binding and motions of the T4 lysozyme molecules, we observed that the motions of wild-type and mutant T4 lysozyme proteins are essentially the same whether under an enzymatic reaction or not. The changing of the fluorescence polarization suggests that the motions of the T4 lysozyme are associated with orientational rotations. This observation also suggests that the T4 lysozyme binding-unbinding motions on cell walls involve a complex mechanism beyond a single-step first-order rate process.     

Electron donation in Photosystem II

The electron transfer resulting from illumination and dark storage of PS II has been studied using EPR signals from several electron carriers. The recombination of D⁺ (Signal II) and Q⁻_A formed by illumination occurred during dark storage at 77 K and was used to deplete reaction centres of D⁺. The donor D was then shown to be oxidized in the dark by the S₂ state of the oxygen-evolving complex. A slow change which occurred during dark storage of PS II samples was detected using the power saturation characteristics of D. We interpret this effect on D to be an indirect result of a rearrangement of the manganese complex during long-term dark adaptation. A role for D in the stability, protection and perhaps initial manganese binding of the oxygen-evolving complex is suggested.     

Structural investigation of inhibitor designs targeting 3-dehydroquinate dehydratase from the shikimate pathway of Mycobacterium tuberculosis

The shikimate pathway is essential in Mycobacterium tuberculosis and its absence from humans makes the enzymes of this pathway potential drug targets. In the present paper, we provide structural insights into ligand and inhibitor binding to 3-dehydroquinate dehydratase (dehydroquinase) from M. tuberculosis (MtDHQase), the third enzyme of the shikimate pathway. The enzyme has been crystallized in complex with its reaction product, 3-dehydroshikimate, and with six different competitive inhibitors. The inhibitor 2,3-anhydroquinate mimics the flattened enol/enolate reaction intermediate and serves as an anchor molecule for four of the inhibitors investigated. MtDHQase also forms a complex with citrazinic acid, a planar analogue of the reaction product. The structure of MtDHQase in complex with a 2,3-anhydroquinate moiety attached to a biaryl group shows that this group extends to an active-site subpocket inducing significant structural rearrangement. The flexible extensions of inhibitors designed to form π-stacking interactions with the catalytic Tyr24 have been investigated. The high-resolution crystal structures of the MtDHQase complexes provide structural evidence for the role of the loop residues 19-24 in MtDHQase ligand binding and catalytic mechanism and provide a rationale for the design and efficacy of inhibitors.     

A salt-bridge motif involved in ligand binding and large-scale domain motions of the maltose-binding protein

The uptake of nutrients is essential for the survival of bacterial cells. Many specialized systems have evolved, such as the maltose-dependent ABC transport system that transfers oligosaccharides through the cytoplasmic membrane. The maltose/maltodextrin-binding protein (MBP) serves as an initial high-affinity binding component in the periplasm that delivers the bound sugar into the cognate ABC transporter MalFGK(2). We have investigated the domain motions induced by the binding of the ligand maltotriose into the binding cleft using molecular dynamics simulations. We find that MBP is predominantly in the open state without ligand and in the closed state with ligand bound. Oligosaccharide binding induces a closure motion (30.0 degrees rotation), whereas ligand removal leads to domain opening (32.6 degrees rotation) around a well-defined hinge affecting key areas relevant for chemotaxis and transport. Our simulations suggest that a "hook-and-eye" motif is involved in the binding. A salt bridge between Glu-111 and Lys-15 forms that effectively locks the protein-ligand complex in a semiclosed conformation inhibiting any further opening and promoting complete closure. This previously unrecognized feature seems to secure the ligand in the binding site and keeps MBP in the closed conformation and suggests a role in the initial steps of substrate transport.     

Mapping the CGRP receptor ligand binding domain: Tryptophan-84 of RAMP1 is critical for agonist and antagonist binding

The calcitonin receptor-like receptor (CLR) associates with the accessory protein RAMP1 to form a receptor for the neuropeptide calcitonin gene-related peptide (CGRP). Multiple lines of evidence have implicated CGRP in the pathophysiology of migraine headache making the CGRP receptor an attractive target for development of small-molecule antagonists as a novel treatment for this debilitating condition. The CGRP receptor antagonists telcagepant and olcegepant (BIBN4096BS) have demonstrated clinical efficacy in the treatment of migraine and there is now a need to better understand how these molecules interact with the receptor. Previous work has shown the extracellular portion of RAMP1 to be important for binding of these antagonists, with tryptophan-74 being a key interaction site. The crystal structure of the extracellular portion of human RAMP1 placed tryptophan-74 in a hydrophobic patch hypothesized to interact with CGRP receptor ligands and also identified nearby residues that may be important for ligand binding. In this study we explored the role played by these residues of RAMP1 using an alanine replacement strategy. We confirmed a role for tryptophan-74 in antagonist binding and also identified arginine-67 as being important for binding of telcagepant but not compound 3, a close analog of BIBN4096BS. We also identified tryptophan-84 as being critical for both high-affinity binding of the non-peptide antagonists as well as the peptides CGRP and CGRP(8-37). These data for the first time pinpoint a specific RAMP1 residue important for both antagonist and agonist potency and are consistent with the N-terminal domain of RAMP1 forming the binding pocket interface with CLR.     

X-ray characterisation of an additional binding site in lysozyme

Bromophenol red (BPR) binds to lysozyme and inhibits its activity against bacterial cell walls, but not against the polysaccharide component of peptidoglycan. The binding site of BPR in the enzyme has been characterised by X-ray analysis of the complex at 5.5A resolution. The new binding site, which is outside the cleft close to subsite F, is presumably involved in interactions with the peptide component of peptidoglycan, in the action of lysozyme against bacterial cell walls.