Refined 1.7 A X-ray crystallographic structure of P-30 protein, an amphibian ribonuclease with anti-tumor activity.

The X-ray crystallographic structure of P-30 protein (Onconase) has been solved by multiple isomorphous replacement and the structure has been refined at 1.7 A resolution to a conventional R-factor of 0.178. The molecular model comprises all 826 non-hydrogen protein atoms, 96 solvent molecules and a sulfate anion that is bound at the active site. The molecular structure is similar to that of ribonuclease A. The active site cleft is located at the junction of two three-stranded beta-sheets and the N-terminal helix. A sulfate anion is non-covalently bound by Lys9, His10, His97, Phe98 and an intermolecular contact involving Lys55' from a neighboring molecule. The N-terminal pyroglutamyl (Pyr) residue is part of the active site and its O epsilon 1 atom forms a hydrogen bond with the Lys9 N zeta. The previously constructed comparative molecular model of P-30 based on ribonuclease A correctly predicted the overall fold of P-30 and the conformation of its active site residues. The model failed to predict the conformation of Pyr1 and the conformation of the two loops following helix alpha 3 and strand beta 3.

X-ray crystallographic structure of recombinant eosinophil-derived neurotoxin at 1.83 A resolution.

The X-ray crystallographic structure of recombinant eosinophil-derived neurotoxin (rEDN) has been determined by molecular replacement methods and refined at 1.83 A resolution to a conventional R-factor ( = sigma magnitute of (magnitute of F(zero)-magnitude of Fc)/ sigma magnitude of F(zero) of 0.152 with excellent stereochemistry. The molecular model of rEDN contains all 1081 non-hydrogen protein atoms, two non-covalently bound sulfate anions and 121 ordered solvent molecules. The polypeptide fold of rEDN is related to those observed in the homologous structures of RNase A, Onconase and angiogenin. rEDN is one of the largest members of the pyrimidine-specific ribonuclease superfamily of vertebrates and has small insertions in four of its seven loop structures and a large insertion from Asp115 to Tyr123. The non-covalently bound SO4(A) and SO4(B) anions occupy phosphate-binding subsites of rEDN. The active site SO4(A) anion makes contacts in rEDN that are similar to those in RNase A and involve the side-chain atoms of Gln14, His15 and His129, and the NH group of Leu130. The SO4(B) anion makes contacts with the side-chain atoms of Arg36 and Asn39 and the main-chain atoms of Asn39 and Gln40. The equivalent residues of RNase A cannot make contacts similar to those observed in rEDN. The SO4(B) binding site of rEDN likely corresponds to the P-1 subsite and may be representative of how other homologous RNases bind the P-1 phosphate.

Refined X-ray crystallographic structure of the poliovirus 3C gene product.

The X-ray crystallographic structure of the recombinant poliovirus 3C gene product (Mahoney strain) has been determined by single isomorphous replacement and non-crystallographic symmetry averaging and refined at 2.1 A resolution. Poliovirus 3C is comprised of two six-stranded antiparallel beta-barrel domains and is structurally similar to the chymotrypsin-like serine proteinases. The shallow active site cleft is located at the junction of the two beta-barrel domains and contains a His40, Glu71, Cys147 catalytic triad. The polypeptide loop preceding Cys147 is flexible and likely undergoes a conformational change upon substrate binding. The specificity pockets for poliovirus 3C are well-defined and modeling studies account for the known substrate specificity of this proteinase. Poliovirus 3C also participates in the formation of the viral replicative initiation complex where it specifically recognizes and binds the RNA stem-loop structure in the 5' non-translated region of its own genome. The RNA recognition site of 3C is located on the opposite side of the molecule in relation to its proteolytic active site and is centered about the conserved KFRDIR sequence of the domain linker. The recognition site is well-defined and also includes residues from the amino and carboxy-terminal helices. The two molecules in the asymmetric unit are related by an approximate 2-fold, non-crystallographic symmetry and form an intermolecular antiparallel beta-sheet at their interface.

Crystal structure of human pepsin and its complex with pepstatin.

The three-dimensional crystal structure of human pepsin and that of its complex with pepstatin have been solved by X-ray crystallographic methods. The native pepsin structure has been refined with data collected to 2.2 A resolution to an R-factor of 19.7%. The pepsin:pepstatin structure has been refined with data to 2.0 A resolution to an R-factor of 18.5%. The hydrogen bonding interactions and the conformation adopted by pepstatin are very similar to those found in complexes of pepstatin with other aspartic proteinases. The enzyme undergoes a conformational change upon inhibitor binding to enclose the inhibitor more tightly. The analysis of the binding sites indicates that they form an extended tube without distinct binding pockets. By comparing the residues on the binding surface with those of the other human aspartic proteinases, it has been possible to rationalize some of the experimental data concerning the different specificities. At the S1 site, valine at position 120 in renin instead of isoleucine, as in the other enzymes, allows for binding of larger hydrophobic residues. The possibility of multiple conformations for the P2 residue makes the analysis of the S2 site difficult. However, it is possible to see that the specific interactions that renin makes with histidine at P2 would not be possible in the case of the other enzymes. At the S3 site, the smaller volume that is accessible in pepsin compared to the other enzymes is consistent with its preference for smaller residues at the P3 position.

Structure of a sialic acid-activating synthetase, CMP-acylneuraminate synthetase in the presence and absence of CDP.

The x-ray crystallographic structure of selenomethionyl cytosine-5'-monophosphate-acylneuraminate synthetase (CMP-NeuAc synthetase) from Neisseria meningitidis has been determined at 2.0-A resolution using multiple-wavelength anomalous dispersion phasing, and a second structure, in the presence of the substrate analogue CDP, has been determined at 2.2-A resolution by molecular replacement. This work identifies the active site residues for this class of enzyme for the first time. The detailed interactions between the enzyme and CDP within the mononucleotide-binding pocket are directly observed, and the acylneuraminate-binding pocket has also been identified. A model of acylneuraminate bound to CMP-NeuAc synthetase has been constructed and provides a structural basis for understanding the mechanism of production of "activated" sialic acids. Sialic acids are key saccharide components on the surface of mammalian cells and can be virulence factors in a variety of bacterial species (e.g. Neisseria, Haemophilus, group B streptococci, etc.). As such, the identification of the bacterial CMP-NeuAc synthetase active site can serve as a starting point for rational drug design strategies.

The structure of UDP-N-acetylglucosamine 2-epimerase reveals homology to phosphoglycosyl transferases.

Bacterial UDP-N-acetylglucosamine 2-epimerase catalyzes the reversible epimerization at C-2 of UDP-N-acetylglucosamine (UDP-GlcNAc) and thereby provides bacteria with UDP-N-acetylmannosamine (UDP-ManNAc), the activated donor of ManNAc residues. ManNAc is critical for several processes in bacteria, including formation of the antiphagocytic capsular polysaccharide of pathogens such as Streptococcus pneumoniae types 19F and 19A. We have determined the X-ray structure (2.5 A) of UDP-GlcNAc 2-epimerase with bound UDP and identified a previously unsuspected structural homology with the enzymes glycogen phosphorylase and T4 phage beta-glucosyltransferase. The relationship to these phosphoglycosyl transferases is very intriguing in terms of possible similarities in the catalytic mechanisms. Specifically, this observation is consistent with the proposal that the UDP-GlcNAc 2-epimerase-catalyzed elimination and re-addition of UDP to the glycal intermediate may proceed through a transition state with significant oxocarbenium ion-like character. The homodimeric epimerase is composed of two similar alpha/beta/alpha sandwich domains with the active site located in the deep cleft at the domain interface. Comparison of the multiple copies in the asymmetric unit has revealed that the epimerase can undergo a 10 degrees interdomain rotation that is implicated in the regulatory mechanism. A structure-based sequence alignment has identified several basic residues in the active site that may be involved in the proton transfer at C-2 or stabilization of the proposed oxocarbenium ion-like transition state. This insight into the structure of the bacterial epimerase is applicable to the homologous N-terminal domain of the bifunctional mammalian UDP-GlcNAc "hydrolyzing" 2-epimerase/ManNAc kinase that catalyzes the rate-determining step in the sialic acid biosynthetic pathway.

The first structure of UDP-glucose dehydrogenase reveals the catalytic residues necessary for the two-fold oxidation.

Bacterial UDP-glucose dehydrogenase (UDPGlcDH) is essential for formation of the antiphagocytic capsule that protects many virulent bacteria such as Streptococcus pyogenes andStreptococcus pneumoniae type 3 from the host's immune system. We have determined the X-ray structures of both native and Cys260Ser UDPGlcDH from S. pyogenes (74% similarity to S. pneumoniae) in ternary complexes with UDP-xylose/NAD(+) and UDP-glucuronic acid/NAD(H), respectively. The 402 residue homodimeric UDPGlcDH is composed of an N-terminal NAD(+) dinucleotide binding domain and a C-terminal UDP-sugar binding domain connected by a long (48 A) central alpha-helix. The first 290 residues of UDPGlcDH share structural homology with 6-phosphogluconate dehydrogenase, including conservation of an active site lysine and asparagine that are implicated in the enzyme mechanism. Also proposed to participate in the catalytic mechanism are a threonine and a glutamate that hydrogen bond to a conserved active site water molecule suitably positioned for general acid/base catalysis.

Comparative molecular modeling and crystallization of P-30 protein: a novel antitumor protein of Rana pipiens oocytes and early embryos.

The P-30 protein (Onconase) of Rana pipiens oocytes and early embryos is homologous to members of the pancreatic ribonuclease superfamily and exhibits an antitumor activity in vitro and in vivo. It appears that the ribonucleolytic activity of P-30 protein may be required for its antitumor effects. A comparative molecular model of P-30 protein has been constructed based upon the known three-dimensional structure of bovine pancreatic RNase A in order to provide structural information. Functionally, these enzymes hydrolyze oligoribonucleotides to pyrimidine-3'-phosphate monoesters and 5'-OH ribonucleotides. In the modeling procedure, automated sequence alignments were revised based upon the inspection of the RNase A structure before the amino acids of the P-30 protein were assigned the coordinates of the RNase A template. The inevitable intermolecular steric clashes that result were relieved on an interactive graphics device through the adjustment of side chain torsion angles. This process was followed by energy minimization of the model, which served to optimize stereochemical geometry and to relieve any remaining unacceptably close contacts. The resulting model retains the essential features of RNase A as sequence insertions and deletions are almost exclusively found in exposed surface loops. The all atom superposition of active site residues of the P-30 protein model and an identically minimized RNase A structure has a root mean square deviation of 0.52 A. Though tentative, the model is consistent with a pyrimidine specificity.(ABSTRACT TRUNCATED AT 250 WORDS)

The structure of L-ribulose-5-phosphate 4-epimerase: an aldolase-like platform for epimerization.

The structure of L-ribulose-5-phosphate 4-epimerase from E. coli has been solved to 2.4 A resolution using X-ray diffraction data. The structure is homo-tetrameric and displays C(4) symmetry. Each subunit has a single domain comprised of a central beta-sheet flanked on either side by layers of alpha-helices. The active site is identified by the position of the catalytic zinc residue and is located at the interface between two adjacent subunits. A remarkable feature of the structure is that it shows a very close resemblance to that of L-fuculose-1-phosphate aldolase. This is consistent with the notion that both enzymes belong to a superfamily of epimerases/aldolases that catalyze carbon-carbon bond cleavage reactions via a metal-stabilized enolate intermediate. Detailed inspection of the epimerase structure, however, indicates that despite the close overall structural similarity to class II aldolases, the enzyme has evolved distinct active site features that promote its particular chemistry.

Crystal structure of the class D beta-lactamase OXA-10.

We report the crystal structure of a class D beta-lactamase, the broad spectrum enzyme OXA-10 from Pseudomonas aeruginosa at 2.0 A resolution. There are significant differences between the overall fold observed in this structure and those of the evolutionarily related class A and class C beta-lactamases. Furthermore, the structure suggests the unique, cation mediated formation of a homodimer. Kinetic and hydrodynamic data shows that the dimer is a relevant species in solution and is the more active form of the enzyme. Comparison of the molecular details of the active sites of the class A and class C enzymes with the OXA-10 structure reveals that there is no counterpart in OXA-10 to the residues proposed to act as general bases in either of these enzymes (Glu 166 and Tyr 150, respectively). Our structures of the native and chloride inhibited forms of OXA-10 suggest that the class D enzymes have evolved a distinct catalytic mechanism for beta-lactam hydrolysis. Clinical variants of OXA-10 are also discussed in light of the structure.

The refined crystal structure of the 3C gene product from hepatitis A virus: specific proteinase activity and RNA recognition.

The virally encoded 3C proteinases of picornaviruses process the polyprotein produced by the translation of polycistronic viral mRNA. The X-ray crystallographic structure of a catalytically active mutant of the hepatitis A virus (HAV) 3C proteinase (C24S) has been determined. Crystals of this mutant of HAV 3C are triclinic with unit cell dimensions a = 53.6 A, b = 53.5 A, c = 53.2 A, alpha = 99.1 degrees, beta = 129.0 degrees, and gamma = 103.3 degrees. There are two molecules of HAV 3C in the unit cell of this crystal form. The structure has been refined to an R factor of 0.211 (Rfree = 0.265) at 2.0-A resolution. Both molecules fold into the characteristic two-domain structure of the chymotrypsin-like serine proteinases. The active-site and substrate-binding regions are located in a surface groove between the two beta-barrel domains. The catalytic Cys 172 S(gamma) and His 44 N(epsilon2) are separated by 3.9 A; the oxyanion hole adopts the same conformation as that seen in the serine proteinases. The side chain of Asp 84, the residue expected to form the third member of the catalytic triad, is pointed away from the side chain of His 44 and is locked in an ion pair interaction with the epsilon-amino group of Lys 202. A water molecule is hydrogen bonded to His 44 N(delta1). The side-chain phenolic hydroxyl group of Tyr 143 is close to this water and to His 44 N(delta1) and may be negatively charged. The glutamine specificity for P1 residues of substrate cleavage sites is attributed to the presence of a highly conserved His 191 in the S1 pocket. A very unusual environment of two water molecules and a buried glutamate contribute to the imidazole tautomer believed to be important in the P1 specificity. HAV 3C proteinase has the conserved RNA recognition sequence KFRDI located in the interdomain connection loop on the side of the molecule diametrically opposite the proteolytic site. This segment of polypeptide is located between the N- and C-terminal helices, and its conformation results in the formation of a well-defined surface with a strongly charged electrostatic potential. Presumably, this surface of HAV 3C participates in the recognition of the 5' and 3' nontranslated regions of the RNA genome during viral replication.