Crystal structure of a papain-E-64 complex

E-64 [1-[N-[(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl] amino]-4-guanidinobutane] is an irreversible inhibitor of many cysteine proteases. A papain-E-64 complex was crystallized at pH 6.3 by using the hanging drop method. Three different crystal forms grew in 3-7 days; the form chosen for structure analysis has space group P212121, with a = 42.91(4) A, b = 102.02(6) A, c = 49.73(2) A, and Z = 4. Diffraction data were measured to 2.4-A resolution, giving 9367 unique reflections. The papain structure was solved by use of the molecular replacement method, and then the inhibitor was located from a difference electron density map and fitted with the aid of a PS330 computer graphics system. The structure of the complex was refined to R = 23.3%. Our analysis shows that a covalent link is formed between the sulfur of the active-site cysteine 25 and the C-2 atom of the inhibitor. Contrary to earlier predictions, the E-64 inhibitor clearly interacts with the S subsites on the enzyme rather than the S' subsites, and papain's histidine 159 imidazole group plays a binding rather than a catalytic role in the inactivation process.     

Refined x-ray structure of papain.E-64-c complex at 2.1-A resolution.

E-64-c, a synthetic cysteine protease inhibitor designed from E-64, binds to papain through a thioether covalent bond. The x-ray diffraction data for 2.1-A resolution were used to determine the three-dimensional structure of this complex and refined it to R = 0.159. 0.159. In the complex structure, the configurational conversion from S to R took place on the epoxy carbon of E-64-c, implying that the nucleophilic attack of the Cys-25 thiol group occurs at the opposite side of the epoxy oxygen atom. The leucyl and isoamylamide groups of E-64-c were strongly fixed to papain S subsites by specific interactions, including hydrogen bonding to the Gly-66 residue. The carboxyl-terminal anion of E-64-c formed an electrostatic interaction with the protonated His-159 imidazole ring (O-...HN+ = 3.76 A) and consequently prevented the participation of this residue in the hydrolytic charge-relay system. No significant distortion caused by the binding of E-64-c was shown in the secondary structure of papain. It is important to note that inhibitor and substrate have opposite binding modes for the peptide groups. The possible relationship between the binding mode and inhibitory activity is discussed on the basis of the crystal structure of this complex.     

A vibrating flexible chain in a molecular cage: crystal structure of the complex cyclomaltoheptaose (beta-cyclodextrin)-1,4-butanediol.6.25H2O.

A single crystal X-ray diffraction study of the title complex carried out at room temperature revealed space group P2(1), a = 21.199(12), b = 9.973(3), c = 15.271(8) A, beta = 110.87(3) degrees, V = 3017(3) A3, 4681 unique reflections with Fo greater than 1 sigma (Fo). The structure was refined to R = 0.069, resolution lambda/2sin theta max = 0.89 A. The crystal packing is of the cage type and is isomorphous to that of beta-cyclodextrin (beta CD) dodecahydrate. One 1,4-butanediol and approximately 1.25 water molecules are enclosed in each beta CD cavity. The hydroxyl groups of the 1,4-butanediol molecule are located at each end of the cavity and form hydrogen bonds with neighboring water and beta CD molecules. The flexible (CH2)4 moiety vibrates extensively in the central part of the cavity. Water molecules and hydroxyl groups are chelated between O-6 and O-5 of at least five glucose residues.     

A trigonal form of the idarubicin:d(CGATCG) complex; crystal and molecular structure at 2.0 A resolution.

The X-ray crystal structure of the complex between the anthracycline idarubicin and d(CGATCG) has been solved by molecular replacement and refined to a resolution of 2.0 A. The final R-factor is 0.19 for 3768 reflections with Fo > or = 2 sigma (Fo). The complex crystallizes in the trigonal space group P31 with unit cell parameters a = b = 52.996(4), c = 33.065(2) A, alpha = beta = 90 degree, gamma = 120 degree. The asymmetric unit consists of two duplexes, each one being complexed with two idarubicin drugs intercalated at the CpG steps, one spermine and 160 water molecules. The molecular packing underlines major groove-major groove interactions between neighbouring helices, and an unusually low value of the occupied fraction of the unit cell due to a large solvent channel of approximately 30 A diameter. This is the first trigonal crystal form of a DNA-anthracycline complex. The structure is compared with the previously reported structure of the same complex crystallizing in a tetragonal form. The geometry of both the double helices and the intercalation site are conserved as are the intramolecular interactions despite the different crystal forms.     

Structure of variant-3 scorpion neurotoxin from Centruroides sculpturatus Ewing, refined at 1.8 A resolution

The three-dimensional structure of the variant-3 protein neurotoxin from the scorpion Centruroides sculpturatus Ewing has been determined by X-ray diffraction data. The initial model for the 65-residue protein was obtained at 3 A resolution by multiple-isomorphous-replacement methods. The structure was refined at 1.8 A resolution by restrained difference-Fourier methods, and by free-atom, block-diagonal least-squares. Considering the 4900 reflections for which d = 1.8-7 A and Fo greater than 2.5 sigma (Fo), the final R-index is 0.16 for the restrained model, and 0.14 for the free-atom model. Average estimated errors in atomic co-ordinates are about 0.1 A. The refined structure includes 492 protein atoms; one molecule of 2-methyl-2,4-pentanediol, which is tightly bound in a hydrophobic pocket on the surface of the protein; and 72 additional solvent sites. The major secondary structural features are two and a half turns of alpha-helix and a three-strand stretch of antiparallel beta-sheet. The helix is connected to the middle strand of the beta-sheet by two disulfide bridges, and a third disulfide bridge is located nearby. Several loops extend out of this dense core of secondary structure. The protein displays several reverse turns and a highly contorted proline-rich, COOH-terminal segment. One of the proline residues (Pro59) assumes a cis-conformation. The structure involves 44 intramolecular hydrogen bonds. The crystallographic results suggest two major corrections in the published primary structure; one of these has been confirmed by new chemical sequence data. The protein displays a large flattened surface that contains a high concentration of hydrophobic residues, along with most of the conserved amino acids that are found in the scorpion neurotoxins.     

A comparison between the binding modes of a substrate and inhibitor to papain as observed in complex crystal structures

On the basis of the crystal structures of papain complexed with the substrate analogue benzyloxycarbonyl-L-phenylalanyl-L-alanine chloromethyl-ketone (Drenth, J., Kalk, K.H., and Swen, H.M. (1976) Biochemistry 15, 3731-3738) and with the inhibitor E-64-c, the binding modes were compared at the atomic level to clarify the functional difference between the substrate and inhibitor. Irrespective of the reverse chemical bonding in the peptide bonds, both the molecules are located at the S subsites of papain with similar interactions. However, the inhibitory activity of E-64-c is characterized by the stereochemical function of a carboxyoxirane ring and the tight binding of the isopentylaminoleucyl side chain to the S subsites.     

Complex formation by bovine trypsin and a tetrapeptide (Leu-Arg-Pro-Gly-NH2): X-ray structure analysis of the complex in the orthorhombic crystal form with low molecular packing density

The title tetrapeptide, Leu-Arg-Pro-Gly-NH₂, forms a complex with trypsin in a novel orthorhombic crystal form with low molecular packing density. The complex formation was directly evidenced by X-ray crystallography. The crystal structure at 1.8 Å resolution was refined to anR-factor of 20.5% for 13,923 reflection data, which were measured with synchrotron radiation. The tetrapeptide is bound to trypsin at the active site, and the binding mode is very similar to that of a bovine pancreatic trypsin inhibitor (BPTI):trypsin complex. The tetrapeptide:trypsin complex is the first observation that a peptide forms a stable complex with trypsin.     

Crystallographic analysis of a complex between human immunodeficiency virus type 1 protease and acetyl-pepstatin at 2.0-A resolution

The mode of binding of acetyl-pepstatin to the protease from the human immunodeficiency virus type 1 (HIV-1) has been determined by x-ray diffraction analysis. Crystals of an acetyl-pepstatin-HIV-1 protease complex were obtained in space group P2(1)2(1)2 (unit cell dimensions a = 58.39 A, b = 86.70 A, c = 46.27 A) by precipitation with sodium chloride. The structure was phased by molecular replacement methods, and a model for the structure was refined using diffraction data to 2.0 A resolution (R = 0.176 for 12901 reflections with I greater than sigma (I); deviation of bond distances from ideal values = 0.018 A; 172 solvent molecules included). The structure of the protein in the complex has been compared with the structure of the enzyme without the ligand. A core of 44 amino acids in each monomer, including residues in the active site and residues at the dimer interface, remains unchanged on binding of the inhibitor (root mean square deviation of alpha carbon positions = 0.39 A). The remaining 55 residues in each monomer undergo substantial rearrangement, with the most dramatic changes occurring at residues 44-57 (these residues comprise the so-called flaps of the enzyme). The flaps interact with one another and with the inhibitor so as to largely preserve the 2-fold symmetry of the protein. The inhibitor is bound in two approximately symmetric orientations. In both orientations the peptidyl backbone of the inhibitor is extended; a network of hydrogen bonds is formed between the inhibitor and the main body of the protein as well as between the inhibitor and the flaps. Hydrophobic side chains of residues in the body of the protein form partial binding sites for the side chains of the inhibitor; hydrophobic side chains of residues in the flaps complete these binding sites.     

Structure of the complex formed by bovine trypsin and bovine pancreatic trypsin inhibitor. II. Crystallographic refinement at 1.9 A resolution

The crystal structure of the complex formed by bovine trypsin and bovine pancreatic trypsin inhibitor has been refined with data to 1.9 Å resolution, using a procedure described by Deisenhofer &; Steigemann (1974) in their refinement of the crystal structure of the free inhibitor. This procedure involves cycles consisting of phase calculation using the current atomic model, Fourier synthesis using these phases and the observed structure factor amplitudes and Diamond's real-space refinement (Diamond, 1971,1974). At various stages, difference Fourier syntheses are calculated to detect and correct gross errors in the model and to localize solvent molecules.The refinement progressed smoothly, starting with the model obtained from the isomorphous Fourier map at 2.6 Å resolution. The R-factor is 0.23 for 20,500 significantly measured reflections to 1.9 Å resolution, using an over-all temperature factor of 20 Ų. The estimated standard deviation of atomic positions is 0.09 Å.An objective assessment of the upper limit of the error in the atomic coordinates of the final model is possible by comparing the inhibitor component in the model of the complex with the refined structure of the free inhibitor (Deisenhofer &; Steigemann, 1974). The mean deviation of main-chain atoms of the two molecular models in internal segments is 0.25 Å, of main-chain dihedral angles 5.1 ° and side-chain dihedral angles 6.5 °.A comparison of the trypsin component with α-chymotrypsin (Birktoft &; Blow, 1972) showed a mean deviation of main-chain atoms of 0.75 Å. The structures are closely similar and the various deletions and insertions cause local structural differences only.     

Crystal structure of a complex formed between proteolytically-generated lactoferrin fragment and proteinase K

Lactoferrin is an iron binding glycoprotein with a molecular weight of 80 kDa. The molecule is divided into two lobes representing the N-terminal and C-terminal halves of the polypeptide chain, each containing an iron binding site. The serine proteinases such as trypsin, chymotrypsin, and pepsin hydrolyze lactoferrin into two unequal halves while proteinase K divides this protein into two equal halves. In the first step of hydrolysis by proteinase K, the C- and N-lobes, each having a molecular weight of approximately 40 kDa, are generated. In the next step, the lobes are further hydrolyzed into small molecular weight peptides. The proteinase K isolated from the hydrolyzed product does not show enzymatic activity suggesting that the enzyme is inhibited. Furthermore, the hydrolysis experiments on N-lobe and C-lobe showed that the inhibitory fragment came from the C-lobe. The purified lactoferrin fragment was found to be a decapeptide with an amino acid sequence of H₂N-Val-Ala-Gln-Gly-Ala-Ala-Gly-Leu-Ala-COOH. The complex formed between proteinase K and lactoferrin fragment was crystallized by microdialysis. The crystals belonged to the monoclinic space group P2₁with cell dimensions a = 44.4 Å, b = 38.6 Å, c = 79.2 Å, β = 105.8^o and Z = 2. The crystal structure has been determined at 2.4 Å resolution. It has been refined to an R factor of 0.163 for 9044 reflections. The Lf-fragment forms several intermolecular interactions with proteinase K. The Ser-224 Oγ and His-57 Nϵ2 move away to a distance of 3.68 Å in the complex. In the crystal structure, Gln-3I (I indicates inhibitor i.e., lactoferrin fragment) is involved in a direct intermolecular interaction with a symmetry related proteinase K molecule through a strong hydrogen bond with Asp-254. The mode of intermolecular interactions in the complex conformational features of the enzyme and placement of the fragment with respect to the enzyme resemble with the molecular complex of proteinase K with its natural inhibitor PKI3 from wheat. Proteins 33:30–38, 1998. © 1998 Wiley-Liss, Inc.     

His92Ala mutation in ribonuclease T1 induces segmental flexibility. An X-ray study.

In the genetically mutated ribonuclease T1 His92Ala (RNase T1 His92Ala), deletion of the active site His92 imidazole leads to an inactive enzyme. Attempts to crystallize RNase T1 His92Ala under conditions used for wild-type enzyme failed, and a modified protocol produced two crystal forms, one obtained with polyethylene glycol (PEG), and the other with phosphate as precipitants. Space groups are identical to wild-type RNase T1, P2(1)2(1)2(1), but unit cell dimensions differ significantly, associated with different molecular packings in the crystals; they are a = 31.04 A, b = 62.31 A, c = 43.70 A for PEG-derived crystals and a = 32.76 A, b = 55.13 A, c = 43.29 A for phosphate-derived crystals, compared to a = 48.73 A, b = 46.39 A, c = 41.10 A for uncomplexed wild-type RNase T1. The crystal structures were solved by molecular replacement and refined by stereochemically restrained least-squares methods based on Fo greater than or equal to sigma (Fo) of 3712 reflections in the resolution range 10 to 2.2 A (R = 15.8%) for the PEG-derived crystal and based on Fo greater than or equal to sigma (Fo) of 6258 reflections in the resolution range 10 to 1.8 A (R = 14.8%) for the phosphate-derived crystal. The His92Ala mutation deletes the hydrogen bond His92N epsilon H ... O Asn99 of wild-type RNase T1, thereby inducing structural flexibility and conformational changes in the loop 91 to 101 which is located at the periphery of the globular enzyme. This loop is stabilized in the wild-type protein by two beta-turns of which only one is retained in the crystals obtained with PEG. In the crystals grown with phosphate as precipitant, both beta-turns are deleted and the segment Gly94-Ala95-Ser96-Gly97 is so disordered that it is not seen at all. In addition, the geometry of the guanine binding site in both mutant studies is different from "empty" wild-type RNase T1 but similar to that found in complexes with guanosine derivatives: the Glu46 side-chain carboxylate hydrogen bonds to Tyr42 O eta; water molecules that are present in the guanine binding site of "empty" wild-type RNase T1 are displaced; the Asn43-Asn44 peptide is flipped such that phi/psi-angles of Asn44 are in alpha L-conformation (that is observed in wild-type enzyme when guanine is bound).(ABSTRACT TRUNCATED AT 400 WORDS)     

Complex formation by bovine trypsin and a tetrapeptide (Leu-Arg-Pro-Gly-NH2): X-ray structure analysis of the complex in the orthorhombic crystal form with low molecular packing density

The title tetrapeptide, Leu-Arg-Pro-Gly-NH₂, forms a complex with trypsin in a novel orthorhombic crystal form with low molecular packing density. The complex formation was directly evidenced by X-ray crystallography. The crystal structure at 1.8 Å resolution was refined to anR-factor of 20.5% for 13,923 reflection data, which were measured with synchrotron radiation. The tetrapeptide is bound to trypsin at the active site, and the binding mode is very similar to that of a bovine pancreatic trypsin inhibitor (BPTI):trypsin complex. The tetrapeptide:trypsin complex is the first observation that a peptide forms a stable complex with trypsin.     

Pyruvate site of pyruvate phosphate dikinase: crystal structure of the enzyme-phosphonopyruvate complex, and mutant analysis.

Crystals of pyruvate phosphate dikinase in complex with a substrate analogue inhibitor, phosphonopyruvate (K(i) = 3 microM), have been obtained in the presence of Mg(2+). The structure has been determined and refined at 2.2 A resolution, revealing that the Mg(2+)-bound phosphonopyruvate binds in the alpha/beta-barrel's central channel, at the C-termini of the beta-strands. The mode of binding resembles closely the previously proposed PEP substrate binding mode, inferred by the homology of the structure (but not sequence homology) to pyruvate kinase. Kinetic analysis of site-directed mutants, probing residues involved in inhibitor binding, showed that all mutations resulted in inactivation, confirming the key role that these residues play in catalysis. Comparison between the structure of the PPDK-phosphonopyruvate complex and the structures of two complexes of pyruvate kinase, one with Mg(2+)-bound phospholactate and the other with Mg(2+)-oxalate and ATP, revealed that the two enzymes share some key features that facilitate common modes of substrate binding. There are also important structural differences; most notably, the machinery for acid/base catalysis is different.     

Nonintercalative binding of proflavin to Z-DNA: structure of a complex between d(5BrC-G-5BrC-G) and proflavin

The crystal structure of a disordered 1:1 complex between the tetradeoxyoligomer d(5BrC-G-5BrC-G) and proflavin has been determined and refined to an R factor of 26.9% for 474 reflections initially in space group P6(5) and to an R factor of 22.2% for 475 reflections in space group P2(1), both at 2-A resolution with Fobsd greater than or equal to 4.0. The unit cell constants are a = b = 17.9 A, c = 44.5 A, and gamma = 120 degrees. The final models are essentially the same in the two space groups with greater disorder in space group P6(5). In space group P2(1), the asymmetric unit is a tetranucleotide duplex, two sandwiched proflavin molecules, and four "outside-bound" proflavins. The tetranucleotide duplex is in the Z conformation and is located at the origin of the unit cell with a pair of proflavins sandwiched between the tetranucleotides. Thus, the tetranucleotides and proflavin dimers stack alternatively forming a quasi-continuous helix with the helix axis coincident with the c axis. The structure analysis revealed the presence of outside-bound proflavins as well. It is interesting that one type of outside-bound proflavins occupies a similar environment as the cobalt hexaammines in their complex with the decadeoxyoligomer d(CGTACGTACG) [Brennan, R. G., Westhof, E., & Sundaralingam, M. (1986) J. Biomol. Struct. Dyn. 3, 649]. Crystals of the latter are isomorphous to the present complex. The outside-bound proflavins penetrate the deep minor groove, thereby closing it off, and provide a visualization of a quasi-internal mode of binding of proflavin to a nucleic acid.     

Crystal structure and molecular conformation of achatin-I (H-Gly-D-Phe-Ala-Asp-OH), an endogenous neuropeptide containing a D-amino acid residue.

In order to investigate the active conformation of achatin-I (H-Gly-D-Phe-Ala-Asp-OH), an endogenous neuropeptide from the Achatina fulica ganglia, its crystal structure and molecular conformation were analysed by the X-ray diffraction method. Crystals from methanol/dioxane are monoclinic, space group P2(1) with a = 5.083(1), b = 9.125(1), c = 20.939(3) A, beta = 94.73(1) degrees. The structure was solved by direct methods and refined to R = 0.051 for 1714 independent reflections with /Fo/ greater than sigma (Fo). The molecule exists as a zwitterion with the Gly N-terminal end protonated and Asp beta-carboxyl deprotonated; the C-terminal of Asp is in a neutral state. The molecule takes a kind of beta turn structure with the D-Phe-Ala residues at the corner of the bend. This turn conformation is primarily formed by the strong intramolecular hydrogen bonds of NH(Gly)...O delta 1 (Asp) and NH(Asp)...O delta 1 (Asp) pairs, thus forming a 15-membered ring structure. Judging from the published data concerning the structure-activity relationship, this turn conformation may reflect an important feature related to the neuroexcitatory activity of achatin-I.     

Complex of rat transthyretin with tetraiodothyroacetic acid refined at 2.1 and 1.8 A resolution

The crystal structure of rat transthyretin (rTTR) complex with 3,5,3',5'-tetraiodothyroacetic acid (T4Ac) was determined at 1.8 A resolution with low temperature synchrotron data collected at CHESS. The structure was refined to R = 0.207 and Rfree = 0.24 with the use of 8-1.8 A data. The additional 8000 reflections from the incomplete 2.1-1.8 data shell, included in the refinement, reduced the Rfree index by 1.3%. Structure comparison with the model refined against the complete 8-2.1 A data revealed no differences in the ligand orientation and the conformation of the polypeptide chain in the core regions. However, the high-resolution data included in the refinement improved the model in the flexible regions poorly defined with the lower resolution data. Also additional sixteen water molecules were found in the difference map calculated with the extended data. The structure revealed both forward and reverse binding of tetraiodothyroacetic acid in one binding site and two modes of forward ligand binding in the second site, with the phenolic iodine atoms occupying different sets of the halogen binding pockets.     

Three-dimensional structure of catalase from Micrococcus lysodeikticus at 1.5 A resolution.

The three-dimensional crystal structure of catalase from Micrococcus lysodeikticus has been solved by multiple isomorphous replacement and refined at 1.5 A resolution. The subunit of the tetrameric molecule of 222 symmetry consists of a single polypeptide chain of about 500 amino acid residues and one haem group. The crystals belong to space group P4(2)2(1)2 with unit cell parameters a = b = 106.7 A, c = 106.3 A, and there is one subunit of the tetramer per asymmetric unit. The amino acid sequence has been tentatively determined by computer graphics model building and comparison with the known three-dimensional structure of beef liver catalase and sequences of several other catalases. The atomic model has been refined by Hendrickson and Konnert's least-squares minimisation against 94,315 reflections between 8 A and 1.5 A. The final model consists of 3,977 non-hydrogen atoms of the protein and haem group, 426 water molecules and one sulphate ion. The secondary and tertiary structures of the bacterial catalase have been analyzed and a comparison with the structure of beef liver catalase has been made.     

Binding diversity of a noncovalent-type low-molecular-weight serine protease inhibitor and function of a catalytic water molecule: X-ray crystal structure of PKSI-527--inhibited trypsin

PKSI-527 is a noncovalent-type low-molecular-weight inhibitor. The X-ray crystal structure of the trypsin-PKSI-527 complex revealed a binding mode (Form II) different from the previously reported one (Form I) [Nakamura, M. et al. (1995) Biochem. Biophys. Res. Commun. 213, 583--587]. In contrast to the previous case, the electron density of the inhibitor revealed the whole structure clearly. Each structural part of the inhibitor in Forms I and II was differently located at the active site, although the modes of binding of the terminal amino group to the Asp189 carboxyl group were similar. This binding diversity, which is a characteristic of the noncovalent-type low-molecular-weight inhibitor, provides a suitable example for estimating the possible mechanism toward the enzymatic inhibition, together with the structural basis necessary for a specific inhibitor. The mode of binding in Form II reflects the inhibitor-specific situation and is in contrast with the substrate-mimetic binding mode for Form I. Based on the generally accepted catalytic mechanism for serine protease, we propose that a water molecule located at the catalytic site plays an important role in blocking the catalytic function of the reactive Ser193 OH group.     

Crystal structure of chartreusin derivative A132

The crystal structure of chartreusin derivative A132 (benzilidene chartreusin) has been determined by single-crystal X-ray diffraction. The space group is C2 with unit cell dimensions, a=18.482(4), b=8.749(3), c=43.906(2) A, beta=94.87(2) degrees, and the structure was refined to R-factors of 0.2365 (6585 all unique reflections) and 0.087 (2914 reflections with F(o)>4 sigma(F(o))) by a full-matrix least-squares method. There are two molecules in an asymmetric unit. Both molecules have similar structures, which are favorable to bind with DNA in the minor groove. A modeling study of the A132-DNA complex based on the X-ray structures suggests that the sugar moiety of A132 may play an important role in recognizing the sequence of DNA base pairs.