Refinement of an enzyme complex with inhibitor bound at partial occupancy. Hen egg-white lysozyme and tri-N-acetylchitotriose at 1.75 A resolution.

The structure of the tri-N-acetylchitotriose inhibitor complex of hen egg-white lysozyme has been refined at 1.75 A resolution, using data collected from a complex crystal with ligand bound at less than full occupancy. To determine the exact value of the inhibitor occupancy, a model comprising unliganded and sugar-bound protein molecules was generated and refined against the 1.75 A data, using a modified version of the Hendrickson & Konnert least-squares procedure. The crystallographic R-factor for the model was found to fall to a minimum at 55% bound sugar. Conventional refinement assuming unit occupancy was found to yield incorrect thermal and positional parameters. Application of the same refinement procedures to an earlier 2.0 A data set, collected independently on different complex crystals by Blake et al. gave less consistent results than the 1.75 A refinement. From an analysis of the high resolution structure a detailed picture of the protein-carbohydrate interactions in the non-productive complex has emerged, together with the conformation and mobility changes that accompany ligand binding. The specificity of interaction between the protein and inhibitor, bound in subsites A to C of the active site, is seen to be generated primarily by an extensive network of hydrogen bonds, both to the protein itself and to bound solvent molecules. The latter also play an important role in maintaining the structural integrity of the active site cleft in the apo-protein.     

Lysozyme revisited: crystallographic evidence for distortion of an N-acetylmuramic acid residue bound in site D.

A structure of the trisaccharide 2-acetamido-2-deoxy-D-muramic acid-beta (1----4)-2-acetamido-2-deoxy-D-glucose-beta (1----4)-2-acetamido-2-deoxy-D-muramic acid (NAM-NAG-NAM), bound to subsites B, C and D in the active-site cleft of hen egg-white lysozyme has been determined and refined at 1.5 A resolution. The resulting atomic co-ordinates indicate that the NAM residue in site D is distorted from the full 4C1 chair conformation to one in which the ring atoms C-1, C-2, O-5 and C-5 are approximately coplanar, and the hydroxymethyl group is positioned axially (a conformation best described as a sofa). This finding supports the original proposals that suggested the ground-state conformation of the sugar bound in site D is strained to one that more closely resembles the geometry required for the oxocarbonium-ion transition state, the next step along the reaction pathway. Additionally, detailed analysis at 1.5 A resolution of the environments of the catalytic residues Glu35 and Asp52 provides new information on the properties that may allow lysozyme to promote the stabilization of an unusually long-lived oxocarbonium-ion transition state. Intermolecular interactions between the N-acetylmuramic acid residue in site D and the lysozyme molecule that contribute to the saccharide ring distortion include: close packing of the O-3' lactyl group with a hydrogen-bonded "platform" of enzyme residues (Asp52, Asn46, Asn59, Ser50 and Asp48), a close contact between the hydroxymethyl group of ring D and the 2'-acetamido group of ring C and a strong hydrogen-bonded interaction between the NH group of Val109 and O-6 of ring D that stabilizes the observed quasi-axial orientation of the -CH2OH group. Additionally, the structure of this complex shows a strong hydrogen bond between the carboxyl group of Glu35 and the beta-anomeric hydroxyl group of the NAM residue in site D. The hydrogen-bonded environment of Asp52 in the native enzyme and in the complex coupled with the very unfavorable direction of approach of the potential carboxylate nucleophile makes it most unlikely that there is a covalent glycosylenzyme intermediate on the hydrolysis pathway of hen egg-white lysozyme.     

Crystal structure of thiamin phosphate synthase from Bacillus subtilis at 1.25 A resolution.

The crystal structure of Bacillus subtilis thiamin phosphate synthase complexed with the reaction products thiamin phosphate and pyrophosphate has been determined by multiwavelength anomalous diffraction phasing techniques and refined to 1.25 A resolution. Thiamin phosphate synthase is an alpha/beta protein with a triosephosphate isomerase fold. The active site is in a pocket formed primarily by the loop regions, residues 59-67 (A loop, joining alpha3 and beta2), residues 109-114 (B loop, joining alpha5 and beta4), and residues 151-168 (C loop, joining alpha7 and beta6). The high-resolution structure of thiamin phosphate synthase complexed with its reaction products described here provides a detailed picture of the catalytically important interactions between the enzyme and the substrates. The structure and other mechanistic studies are consistent with a reaction mechanism involving the ionization of 4-amino-2-methyl-5-hydroxymethylpyrimidine pyrophosphate at the active site to give the pyrimidine carbocation. Trapping of the carbocation by the thiazole followed by product dissociation completes the reaction. The ionization step is catalyzed by orienting the C-O bond perpendicular to the plane of the pyrimidine, by hydrogen bonding between the C4' amino group and one of the terminal oxygen atoms of the pyrophosphate, and by extensive hydrogen bonding and electrostatic interactions between the pyrophosphate and the enzyme.     

Probing residue-level unfolding during lysozyme precipitation

We have employed nuclear magnetic resonance (NMR) measurements of hydrogen exchange to identify residue-level conformational changes in hen egg white lysozyme (HEWL) as induced by salt precipitation. Deuterated HEWL was dissolved into a phosphate (H2O) buffer and precipitated at pH 2.1 upon addition of solid KSCN or (ND4)2SO4, allowing isotope labeling of unfolded regions. After 1 h, each precipitate was then dissolved at pH 3.8 to initiate refolding and preserve labeling and subsequently purified for NMR analysis. HEWL precipitated by 1.0 M KSCN exhibited increased hydrogen exchange at 14 residues out of 42 normally well-protected in the native state. Of the affected residues, 9 were situated in the beta-sheet/loop domain. A similar, though less extensive, effect was observed at 0.2 M KSCN. Precipitation by 1.2 M (ND4)2SO4 resulted in none of the changes detected with KSCN. The popularity of ammonium sulfate as a precipitant is thus supported by this observed preservation of structural integrity. KSCN, in comparison, produced partial unfolding of specific regions in HEWL due most likely to known preferential interactions between -SCN and proteins. The severity of unfolding increased with KSCN concentration such that, at 1.0 M KSCN, almost the entire beta-sheet/loop domain of HEWL was disrupted. Even so, a portion of the HEWL core encompassed by three alpha-helices remained intact, possibly facilitating precipitate dissolution.     

Structural characterization of protein-denaturant interactions: crystal structures of hen egg-white lysozyme in complex with DMSO and guanidinium chloride

A variety of physico-chemical methods employ chemical denaturants to unfold proteins, and study different biophysical processes involved therein. Chemical denaturants are believed to induce unfolding by stabilizing the unfolded state of proteins over the folded state, either macroscopically or through specific interactions. In order to characterize the nature of specific interactions between proteins and denaturants, we have solved crystal structures of hen egg-white lysozyme complexed with denaturants, and report here dimethyl sulfoxide and guanidinium chloride complexes. The dimethyl sulfoxide molecules and guanidinium ions were seen to bind the protein at specific sites and were involved in characteristic interactions. They share a major binding site between them, the C site in the sugar binding cleft of the enzyme. Although the overall conformations of the complexes were very similar to the native structure, spectacular conformational changes were seen to occur locally. Temperature factors were also seen to drop dramatically in the local regions close to the denaturant binding sites. An interesting observation of the present study was the generation of a sodium ion binding site in hen egg-white lysozyme in the presence of denaturants, which was hitherto unknown in any of the other lysozyme structures solved so far. Loss of some of the crucial side chain-main chain interactions may form the initial events in lysozyme unfolding.     

Ester-linked hen egg white lysozyme shows a compact fold in a molecular dynamics simulation - possible causes and sensitivity of experimentally observable quantities to structural changes maintaining this compact fold

Prediction and understanding of the folding and stability of the 3D structure of proteins is still a challenge. The different atomic interactions, such as non polar contacts and hydrogen bonding, are known but their exact relative weights and roles when contributing to protein folding and stability are not identified. Initiated by a previous molecular dynamics simulation of fully ester-linked hen egg white lysozyme (HEWL), which showed a more compact fold of the ester-linked molecule compared to the native one, three variants of this protein are analyzed in the present study. These are 129-residue native HEWL, partly ester-linked HEWL, in which only 34 peptide linkages that are not involved in the helical or β-strand parts of native HEWL were replaced by ester linkages, and fully (126 residues) ester-linked HEWL. Native and partly ester-linked HEWL showed comparable behaviour, whereas fully ester-linked HEWL could not maintain the native secondary structure of HEWL in the simulation and adopted a more compact fold. The conformational changes were analyzed by comparing simulation averaged values of quantities that can be measured by NMR, such as (1)H-(15)N backbone order parameters, residual dipolar couplings, proton-proton NOE distances and (3)J-couplings with the corresponding values derived from experimental NMR data for native HEWL. The information content of the latter appeared to be insufficient to detect the local conformational rearrangements upon esterification of the loop regions of the protein. For fully ester-linked HEWL, a significantly reduced agreement was observed. Upon esterification, the backbone-side chain and side chain-side chain hydrogen-bonding pattern of HEWL changes to maintain its compactness and thus the structural stability of the ester-linked lysozymes.     

A highly amyloidogenic region of hen lysozyme

Amyloid fibrils obtained after incubating hen egg-white lysozyme (HEWL) at pH 2.0 and 65 degrees C for extended periods of time have been found to consist predominantly of fragments of the protein corresponding to residues 49-100, 49-101, 53-100 and 53-101, derived largely from the partial acid hydrolysis of Asp-X peptide bonds. These internal fragments of HEWL encompass part of the beta-domain and all the residues forming the C-helix in the native protein, and contain two internal disulfide bridges Cys64-Cys80 and Cys76-Cys94. The complementary protein fragments, including helices A, B and D of the native protein, are not significantly incorporated into the network of fibrils, but remain largely soluble, in agreement with their predicted lower propensities to aggregate. Further analysis of the properties of different regions of HEWL to form amyloid fibrils was carried out by studying fragments produced by limited proteolysis of the protein by pepsin. Here, we show that only fragment 57-107, but not fragment 1-38/108-129, is able to generate well-defined amyloid fibrils under the conditions used. This finding is of particular importance, as the beta-domain and C-helix of the highly homologous human lysozyme have been shown to unfold locally in the amyloidogenic variant D67H, which is associated with the familial cases of systemic amyloidosis linked to lysozyme deposition. The identification of the highly amyloidogenic character of this region of the polypeptide chain provides strong support for the involvement of partially unfolded species in the initiation of the aggregation events that lead to amyloid deposition in clinical disease.     

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.     

Molecular dynamics simulation of thionated hen egg white lysozyme

Understanding of the driving forces of protein folding is a complex challenge because different types of interactions play a varying role. To investigate the role of hydrogen bonding involving the backbone, the effect of thio substitutions in a protein, hen egg white lysozyme (HEWL), was investigated through molecular dynamics simulations of native as well as partly (only residues in loops) and fully thionated HEWL using the GROMOS 54A7 force field. The results of the three simulations show that the structural properties of fully thionated HEWL clearly differ from those of the native protein, while for partly thionated HEWL they only changed slightly compared with native HEWL. The analysis of the torsional-angle distributions and hydrogen bonds in the backbone suggests that the α-helical segments of native HEWL tend to show a propensity to convert to 3(10)-helical geometry in fully thionated HEWL. A comparison of the simulated quantities with experimental NMR data such as nuclear overhauser effect (NOE) atom-atom distance bounds and (3)J((H)(N)(H)(α))-couplings measured for native HEWL illustrates that the information content of these quantities with respect to the structural changes induced by thionation of the protein backbone is rather limited.     

1H-NMR study on the structure of lysozyme from guinea hen egg white.

The structure of lysozyme from guinea hen egg white (GEWL), which differs from hen egg white lysozyme (HEWL) by ten amino acid substitutions, was investigated by nuclear magnetic resonance (NMR) spectroscopy. GEWL and HEWL were very similar to each other in their tertiary structure as judged from the profile of 1H-NMR spectra, pH titration, and an N-acetylglucosamine trisaccharide [(GlcNAc)3 binding experiment. However, we have noticed several characteristics which distinguish GEWL from HEWL. The signal of Trp 108 indole N1H of GEWL was shifted upfield by about 0.3 ppm when compared with that of HEWL, and its hydrogen exchange was faster than that of HEWL. The pKa values of Glu 35 estimated from the pH titration curve of Trp 108 indole N1H were different between GEWL and HEWL. From a careful examination of spectral changes caused by (GlcNAc)3 binding, the changes in the chemical shift values of Trp 28 C5H and Asn 59 alpha CH of GEWL were found to be slightly larger than those of HEWL. Ile 55 of HEWL is replaced by valine in GEWL. Such a replacement may affect the neighboring hydrogen bonding between the main chain C = O of Leu 56 and Trp 108 indole N1H, resulting in a change in the microenvironment of the substrate-binding site near Trp 108.     

Comparison of goose-type, chicken-type, and phage-type lysozymes illustrates the changes that occur in both amino acid sequence and three-dimensional structure during evolution

The three-dimensional structure of goose-type lysozyme (GEWL), determined by x-ray crystallography and refined at high resolution, has similarities to the structures of hen (chicken) egg-white lysozyme (HEWL) and bacteriophage T4 lysozyme (T4L). The nature of the structural correspondence suggests that all three classes of lysozyme diverged from a common evolutionary precursor, even though their amino acid sequences appear to be unrelated (Grütter et al. 1983). In this paper we make detailed comparisons of goose-type, chicken-type, and phage-type lysozymes. The lysozymes have undergone conformational changes at both the global and the local level. As in the globins, there are corresponding alpha-helices that have rigid-body displacements relative to each other, but in some cases corresponding helices have increased or decreased in length, and in other cases there are helices in one structure that have no counterpart in another. Independent of the overall structural correspondence among the three lysozyme backbones is another, distinct correspondence between a set of three consecutive alpha-helices in GEWL and three consecutive alpha-helices in T4L. This structural correspondence could be due, in part, to a common energetically favorable contact between the first and the third helices. There are similarities in the active sites of the three lysozymes, but also one striking difference. Glu 73 (GEWL) spatially corresponds to Glu 35 (HEWL) and to Glu 11 (T4L). On the other hand, there are two aspartates in the GEWL active site, Asp 86 and Asp 97, neither of which corresponds exactly to Asp 52 (HEWL) or Asp 20 (T4L). (The discrepancy in the location of the carboxyl groups is about 10 A for Asp 86 and 4 A for Asp 97.) This lack of structural correspondence may reflect some differences in the mechanisms of action of the three lysozymes. When the amino acid sequences of the three lysozyme types are aligned according to their structural correspondence, there is still no apparent relationship between the sequences except for possible weak matching in the vicinity of the active sites.     

Effect of 1-methyl-3-octyleimmidazolium chloride on the stability and activity of lysozyme: a spectroscopic and molecular dynamics studies

Herein, the binding of 1-methyl-3-octylimidazolium chloride [OMIM][Cl] ionic liquid with hen egg white lysozyme (HEWL) has been studied using fluorescence, time resolved fluorescence, UV–visible and circular dichroism (CD) spectroscopy, in combination with computational study. The fluorescence results revealed that [OMIM][Cl] quenches the fluorophore of HEWL through static quenching mechanism. The calculated thermodynamic parameters show that [OMIM][Cl] bind with HEWL through hydrophobic interactions. In addition, the negative value of Gibbs energy change (?G) indicates that the binding process was spontaneous. Furthermore, UV–vis and CD results indicate that [OMIM][Cl] induce the conformational change in HEWL and increase its enzymatic activity. Additionally, molecular docking results showed that [OMIM][Cl] binds at the active site of HEWL where both the fluorophore residues (Trp108 and Trp62) and the catalytic residues (Glu35 and Asp52) reside. Molecular dynamic simulation results show the reduction of intra-molecular hydrogen bond of HEWL when it binds with [OMIM][Cl].     

A truncated glucoamylase gene fusion for heterologous protein secretion from Aspergillus niger

Abstract The secreted yield of hen egg-white lysozyme (HEWL) from the filamentous fungus Aspergillus niger was increased 10–20-fold by constructing a novel gene fusion. The cDNA sequence encoding mature HEWL was fused in frame to part of the native A. niger gene encoding glucoamylase ( gla A), separated by a proteolytic cleavage site for in vivo processing. Using this construct, peak secreted HEWL yields of 1 g/l were obtained in A. niger shake flask cultures compared to about 50 mg/l when using an expression cassette lacking any gla A coding sequence. The portion of gla A used in the gene fusion encoded the first 498 amino acids of glucoamylase (G498) and comprised its secretion signal, the catalytic domain and most of the O-glycosylated linker region which, in the entire glucoamylase molecule, spatially separates and links the catalytic and starch-binding domains.     

Mechanistic inferences from the binding of ligands to LpxC, a metal-dependent deacetylase

The metal-dependent deacetylase LpxC catalyzes the first committed step of lipid A biosynthesis in Gram-negative bacteria. Accordingly, LpxC is an attractive target for the development of inhibitors that may serve as potential new antibiotics for the treatment of Gram-negative bacterial infections. Here, we report the 2.7 A resolution X-ray crystal structure of LpxC complexed with the substrate analogue inhibitor TU-514 and the 2.0 A resolution structure of LpxC complexed with imidazole. The X-ray crystal structure of LpxC complexed with TU-514 allows for a detailed examination of the coordination geometry of the catalytic zinc ion and other enzyme-inhibitor interactions in the active site. The hydroxamate group of TU-514 forms a bidentate chelate complex with the zinc ion and makes hydrogen bond interactions with conserved active site residues E78, H265, and T191. The inhibitor C-4 hydroxyl group makes direct hydrogen bond interactions with E197 and H58. Finally, the C-3 myristate moiety of the inhibitor binds in the hydrophobic tunnel of the active site. These intermolecular interactions provide a foundation for understanding structural aspects of enzyme-substrate and enzyme-inhibitor affinity. Comparison of the TU-514 complex with cacodylate and imidazole complexes suggests a possible substrate diphosphate binding site and highlights residues that may stabilize the tetrahedral intermediate and its flanking transition states in catalysis. Evidence of a catalytic zinc ion in the native zinc enzyme coordinated by H79, H238, D242, and two water molecules with square pyramidal geometry is also presented. These results suggest that the native state of this metallohydrolase may contain a pentacoordinate zinc ion, which contrasts with the native states of archetypical zinc hydrolases such as thermolysin and carboxypeptidase A.     

Crystal structure of Turkey egg-white lysozyme: Results of the molecular replacement method at 5 Å resolution

Turkey egg-white lysozyme differs from hen egg-white lysozyme in its primary structure in 7 of the 129 residues. We have determined the rotational and translational parameters relating the known co-ordinates of hen egg-white lysozyme molecule to the turkey lysozyme. The rotational parameters were determined using the rotation function, the translational parameters were determined by placing the properly rotated molecule systematically at all positions within the unit cell and searching for those positions producing few intermolecular contacts between the α-carbon atoms of one molecule and all its neighbors. These parameters were refined by minimizing the conventional R factor between observed and calculated structure amplitudes. The final rotational and translational parameters give an R value of 46.7% for reflections with d spacings between 6 Å and 12 Å and have 7 intermolecular contacts closer than 5 Å between the a carbon atoms of one molecule and all its neighbors. An electron density map has been calculated at 5 Å resolution; the packing of the molecules in this form appears to present the entire length of the active cleft in the vicinity of the crystallographic 6-fold axis and does not appear to be blocked by neighboring molecules.     

A molecular dynamics analysis of protein structural elements

The relation between protein secondary structure and internal motions was examined by using molecular dynamics to calculate positional fluctuations of individual helix, beta-sheet, and loop structural elements in free and substrate-bound hen egg-white lysozyme. The time development of the fluctuations revealed a general correspondence between structure and dynamics; the fluctuations of the helices and beta-sheets converged within the 101 psec period of the simulation and were lower than average in magnitude, while the fluctuations of the loop regions were not converged and were mostly larger than average in magnitude. Notable exceptions to this pattern occurred in the substrate-bound simulation. A loop region (residues 101-107) of the active site cleft had significantly reduced motion due to interactions with the substrate. Moreover, part of a loop and a 3(10) helix (residues of 67-88) not in contact with the substrate showed a marked increase in fluctuations. That these differences in dynamics of free and substrate-bound lysozyme did not result simply from sampling errors was established by an analysis of the variations in the fluctuations of the two halves of the 101 psec simulation of free lysozyme. Concerted transitions of four to five mainchain phi and psi angles between dihedral wells were shown to be responsible for large coordinate shifts in the loops. These transitions displaced six or fewer residues and took place either abruptly, in 1 psec or less, or with a diffusive character over 5-10 psec. Displacements of rigid secondary structures involved longer timescale motions in bound lysozyme; a 0.5 A rms change in the position of a helix occurred over the 55 psec simulation period. This helix reorientation within the protein appears to be a response to substrate binding. There was little correlation between the solvent accessible surface area and the dynamics of the different structural elements.     

Structure of the pigeon lysozyme and its relationship with other type c lysozymes

1. The secondary structure of the pigeon egg-white lysozyme shows important differences when compared to other type c lysozymes. These differences are mainly located at the region comprising residues 77-84. This segment contains one alpha-helix in the lysozymes c studied by means of an X-ray analysis, while the residues at such positions in pigeon lysozyme would form two beta-bends. 2. Analysis of the tertiary structure of the pigeon lysozyme by means of hydropathy profiles reveals that the above segment seems to be more hydrophilic in the pigeon enzyme than in other type c lysozymes. 3. Though a certain similarity to the calcium-binding loop of alpha-lactalbumins is detected in pigeon lysozyme, the circular dichroism spectra of the protein at neutral pH do not change in the presence of Ca2+ ions. 4. The presented structural analysis is discussed in terms of function-structure and antigenicity relationships between the type c lysozymes.     

Crystallization of the Fab fragments of anti-peptide monoclonal antibodies and a complex with peptide.

The antigen-binding fragments of four monoclonal antibodies that cross-react with both the "loop" peptide of hen egg-white lysozyme (residues 57 to 84) against which they were raised, and with the native protein (HEL) have been crystallized. One of these fragments also crystallizes as a complex with the peptide antigen.     

Crystal structure of Anaerobiospirillum succiniciproducens PEP carboxykinase reveals an important active site loop

The 2.2 Angstroms resolution crystal structure of the enzyme phosphoenolpyruvate carboxykinase (PCK) from the bacterium Anaerobiospirillum succiniciproducens complexed with ATP, Mg(2+), Mn(2+) and the transition state analogue oxalate has been solved. The 2.4 Angstroms resolution native structure of A. succiniciproducens PCK has also been determined. It has been found that upon binding of substrate, PCK undergoes a conformational change. Two domains of the molecule fold towards each other, with the substrates and metal ions held in a cleft formed between the two domains. This domain movement is believed to accelerate the reaction PCK catalyzes by forcing bulk solvent molecules out of the active site. Although the crystal structure of A. succiniciproducens PCK with bound substrate and metal ions is related to the structures of PCK from Escherichia coli and Trypanosoma cruzi, it is the first crystal structure from this class of enzymes that clearly shows an important surface loop (residues 383-397) from the C-terminal domain, hydrogen bonding with the peptide backbone of the active site residue Arg60. The interaction between the surface loop and the active site backbone, which is a parallel beta-sheet, seems to be a feature unique of A. succiniciproducens PCK. The association between the loop and the active site is the third type of interaction found in PCK that is thought to play a part in the domain closure. This loop also appears to help accelerate catalysis by functioning as a 'lid' that shields water molecules from the active site.     

A structural basis for the interaction of urea with lysozyme.

The effect of urea on the crystal structure of hen egg-white lysozyme has been investigated using X-ray crystallography. High resolution structures have been determined from crystals grown in the presence of 0, 0.7, 2, 3, 4, and 5 M urea and from crystals soaked in 9 M urea. All the forms are essentially isomorphous with the native type II crystals, and the derived structures exhibit excellent geometry and RMS differences from ideality in bond distances and angles. Comparison of the urea complex structures with the native enzyme (type II form, at 1.5 A resolution) indicates that the effect of urea is minimal over the concentration range studied. The mean difference in backbone conformation between the native enzyme and its urea complexes varies from 0.18 to 0.49 A. Conformational changes are limited to flexible surface loops (Thr 69-Asn 74, Ser 100-Asn 103), the active site loop (Asn 59-Cys 80), and the C-terminus (Cys 127-Leu 129). Urea molecules are bound to distinct sites on the surface of the protein. One molecule is bound to the active site cleft's C subsite, at all concentrations, in a fashion analogous to that of the N-acetyl substituent of substrate and inhibitor sugars normally bound to this site. Occupation of this subsite by urea alone does not appear to induce the conformational changes associated with inhibitor binding.