Jan Drenth

Structure of papain refined at 1.65 A resolution

Papain is a sulfhydryl protease from the latex of the papaya fruit. Its molecules consist of one polypeptide chain with 212 amino acid residues. The chain is folded into two domains with the active site in a groove between the domains. We have refined the crystal structure of papain, in which the sulfhydryl group was oxidized, by a restrained least-squares procedure at 1.65 A to an R-factor of 16.1%. The estimated accuracy in the atomic co-ordinates is 0.1 A, except for disordered atoms. All phi/psi angles for non-glycine residues are found within the outer limit boundary of a Ramachandran plot and this provides another check on the quality of the model. In the alpha-helical parts of the structure, the C = O bonds are directed more away from the helix axis than in a classical alpha-helix, leading to somewhat longer hydrogen bonds, 2.98 A, compared to 2.89 A. The hydrogen-bonding parameters and conformational angles in the anti-parallel beta-sheet structure show a large diversity. Hydrogen bonds in the core of the sheet are generally shorter than those at the more twisted ends. The average value is 2.91 A. The hydrogen bond distance Ni+3-Oi in turns is relatively long and the geometry is far from linear. Hydrogen bond formation, therefore, is perhaps not an essential prerequisite for turn formation. Although the crystallization medium is 62% (w/w) methanol in water, only 29 out of 224 solvent molecules can be regarded with any certainty as methanol molecules. The water molecules play an important role in maintaining structural stability. This is specially true for internal water. Twenty-one water molecules are located in contact areas between adjacent papain molecules. It seems as if the enzyme is trapped in a grid of water molecules with only a limited number of direct interactions between the protein molecules. The residues in the active site cleft belong to the most static parts of the structure. In general, disorder in atomic positions increases when going from the interior of the protein molecule to its surface. This behavior was quantified and it was found that the point of minimum disorder is near the molecular centroid.

Structure of bovine pancreatic phospholipase A2 at 1.7A resolution.

Abstract The crystal structure of bovine pancreatic phospholipase A2 has been refined to 1.7 A resolution. The starting model for this refinement was the previously published structure at a resolution of 2.4 A (Dijkstra et al., 1978). This model was adjusted to the multiple isomorphous replacement map with Diamonds real space refinement program (Diamond, 1971,1974) and subsequently refined using Agarwals least-squares method (Agarwal, 1978). The final crystallographic R-factor is 17.1% and the estimated root-mean-square error in the positional parameters is 0.12 A. The refined model allowed a detailed survey of the hydrogen-bonding pattern in the molecule. The essential calcium ion is located in the active site and is stabilized by one carboxyl group as well as by a peptide loop with many residues unvaried in all known phospholipase A2 sequences. Five of the oxygen ligands octahedrally surround the ion. The sixth octahedral position is shared between one of the carboxylate oxygens of Asp49 and a water molecule. The entrance to the active site is surrounded by residues involved in the binding of micelle substrates. The N-terminal region plays an important role here. Its α-NH+3 group is buried and interacts with Gln4, the carbonyl oxygen of Asn71 and a fully enclosed water molecule, which provides a link between the N terminus and several active site residues. A total of 106 water molecules was located in the final structure, most of them in a two-layer shell around the protein molecule. The mobility in the structure was derived from the individual atomic temperature factors. Minimum mobility is found for the main chain atoms in the central part of the two long α-helices. The active site is rather rigid.

Thiol proteases - comparative studies based on the high-resolution structures of papain and actinidin, and on amino acid sequence information for cathepsins B and H, and stem bromelain.

An accurate three-dimensional structure is known for papain (1.65 A resolution) and actinidin (1.7 A). A detailed comparison of these two structures was performed to determine the effect of amino acid changes on the conformation. It appeared that, despite only 48% identity in their amino acid sequence, different crystallization conditions and different X-ray data collection techniques, their structures are surprisingly similar with a root-mean-square difference of 0.40 A between 76% of the main-chain atoms (differences less than 3 sigma). Insertions and deletions cause larger differences but they alter the conformation over a very limited range of two to three residues only. Conformations of identical side-chains are generally retained to the same extent as the main-chain conformation. If they do change, this is due to a modified local environment. Several examples are described. Spatial positions of hydrogen bonds are conserved to a greater extent than are the specific groups involved. The greatest structural similarity is found for the active site residues of papain and actinidin, for the internal water molecules and for the main-chain conformation of residues in alpha-helices and anti-parallel beta-sheet structure. This was reflected also in the similarity of the temperature factors. It suggests that the secondary structural elements form the skeleton of the molecule and that their interaction is the main factor in directing the fold of the polypeptide chain. Therefore, substitution of residues in the skeleton will, in general, have the most drastic effect on the conformation of the protein molecule. In papain and actinidin, some main-chain-side-chain hydrogen bonds are also strongly conserved and these may determine the folding of non-repetitive parts of the structure. Furthermore, we included primary structure information for three homologous thiol proteases: stem bromelain, and the cathepsins B and H. By combining the three-dimensional structural information for papain and actinidin with sequence homologies and identities, we conclude that the overall folding pattern of the polypeptide chain is grossly the same in all five proteases, and that they utilize the same catalytic mechanism.

The structure of papain.

Publisher Summary The fruits of the tropical papaya tree have latex that contains several enzymes. This chapter focuses on two different proteolytic enzymes: chymopapain and papain. Both enzymes belong to the group of proteolytic plant enzymes that require a sulfhydryl group for activity. The chapter discusses the enzymatic properties of papain. Papain can catalyze a number of reaction—namely, acyl-enzyme intermediate, hydrolysis, transferase action, and specificity. X-ray diffraction can provide a wealth of information on a protein structure. The papain molecule consists of one folded polypeptide chain of 212 residues. The sequence determination of these residues is an example of the mutual interaction between purely chemical and X-ray methods. Because of the many difficulties that papain presented to the chemists, only a tentative sequence could be published in 1964. Some of the overlaps were not found to be as conclusive as desired, and at least one gap existed in the sequence. The available information, however, permitted the interpretation of the electron density map. Moreover, the difficulties in the early chemical sequence studies on papain because of the insolubility of the denatured protein and many of its peptides is circumvented by the use of completely carboxymethylated papain that has been fully maleylated.

Structure of porcine pancreatic phospholipase A2 at 2.6 A resolution and comparison with bovine phospholipase A2.

The previously published three-dimensional structure of porcine pancreatic prophospholipase A2 at 3 A resolution was found to be incompatible with the structures of bovine phospholipase A2 and bovine prophospholipase A2. This was unexpected because of the very homologous amino acid sequences of these enzymes. Therefore, the crystal structure of the porcine enzyme was redetermined using molecular replacement methods with bovine phospholipase as the parent model. The structure was crystallographically refined at 2.6 A resolution by fast Fourier transform and restrained least-squares procedures to an R-factor of 0.241. The crystals appeared to contain phospholipase A2 and not prophospholipase A2. Apparently the protein is slowly converted under the crystallization conditions employed. Our investigation shows that, in contrast to the previous report, the three-dimensional structure of porcine phospholipase A2 is very similar to that of bovine phospholipase A2, including the active site. Smaller differences were observed in some residues involved in the binding of aggregated substrates. However, an appreciable conformational difference is in the loop 59 to 70, where a single substitution at position 63 (bovine Val leads to porcine Phe) causes a complete rearrangement of the peptide chain. In addition to the calcium ion in the active site, a second calcium ion is present in the crystals; this is located on a crystallographic 2-fold axis and stabilizes the interaction between two neighbouring molecules.

Comparison of the three-dimensional protein and nucleotide structure of the FAD-binding domain of p-hydroxybenzoate hydroxylase with the FAD- as well as NADPH-binding domains of glutathione reductase.

The chain fold of the FAD-binding domain of p-hydroxybenzoate hydroxylase resembles the chain folds of the two nucleotide-binding domains of glutathione reductase. This fold consists of a four-stranded parallel beta-sheet sandwiched between a three-stranded antiparallel beta-sheet and alpha-helices. The nucleotides bind in similar positions relative to this chain fold. The best superposition of the folds has been established and geometrically quantified, giving rise to an equivalencing scheme for 110 residue positions, of which only four residues are identical in all three domains. It is discussed whether this chain fold is also present in a number of other FAD-binding proteins with known sequence. After the second strand of the parallel beta-sheet both FAD-binding domains contain long chain excursions, which make intimate contacts to rather distant parts of the respective molecules. In the environment of the isoalloxazine rings we observe interesting similarities. In both enzymes the si-face of this ring is covered by polypeptide, and only the re-face is accessible for the cofactor NADPH. Furthermore, there is a long alpha-helix in each enzyme, which points with its N-terminal start to the O-2 alpha region of isoalloxazine. These helices are spatially in the same position with respect to the isoalloxazine ring but are at quite different positions along the polypeptide chain. Since they can stabilize a negative charge around O-2 alpha, they may be important for the catalytic processes.

Crystal structure of the p-hydroxybenzoate hydroxylase-substrate complex refined at 1.9 Å resolution: analysis of the enzyme-substrate and enzyme-product complexes

Using synchrotron radiation, the X-ray diffraction intensities of crystals of p-hydroxy-benzoate hydroxylase, complexed with the substrate p-hydroxybenzoate, were measured to a resolution of 1.9 A. Restrained least-squares refinement alternated with rebuilding in electron density maps yielded an atom model of the enzyme-substrate complex with a crystallographic R-factor of 15.6% for 31,148 reflections between 6.0 and 1.9 A. A total of 330 solvent molecules was located. In the final model, only three residues have deviating phi-psi angle combinations. One of them, the active site residue Arg44, has a well-defined electron density and may be strained to adopt this conformation for efficient catalysis. The mode of binding of FAD is distinctly different for the different components of the coenzyme. The adenine ring is engaged in three water-mediated hydrogen bonds with the protein, while making only one direct hydrogen bond with the enzyme. The pyrophosphate moiety makes five water-mediated versus three direct hydrogen bonds. The ribityl and ribose moieties make only direct hydrogen bonds, in all cases, except one, with side-chain atoms. The isoalloxazine ring also makes only direct hydrogen bonds, but virtually only with main-chain atoms. The conformation of FAD in p-hydroxybenzoate hydroxylase is strikingly similar to that in glutathione reductase, while the riboflavin-binding parts of these two enzymes have no structural similarity at all. The refined 1.9 A structure of the p-hydroxybenzoate hydroxylase-substrate complex was the basis of further refinement of the 2.3 A structure of the enzyme-product complex. The result was a final R-factor of 16.7% for 14,339 reflections between 6.0 and 2.3 A and an improved geometry. Comparison between the complexes indicated only small differences in the active site region, where the product molecule is rotated by 14 degrees compared with the substrate in the enzyme-substrate complex. During the refinements of the enzyme-substrate and enzyme-product complexes, the flavin ring was allowed to bend or twist by imposing planarity restraints on the benzene and pyrimidine ring, but not on the flavin ring as a whole. The observed angle between the benzene ring and the pyrimidine ring was 10 degrees for the enzyme-substrate complex and 19 degrees for the enzyme-product complex. Because of the high temperature factors of the flavin ring in the enzyme-product complex, the latter value should be treated with caution. Six out of eight peptide residues near the flavin ring are oriented with their nitrogen atom pointing towards the ring.(ABSTRACT TRUNCATED AT 400 WORDS)

Three-dimensional Structure and Disulfide Bond Connections in Bovine Pancreatic Phospholipase A2

Bovine pancreatic phospholipase A2 (Mr = 14,000) has been crystallized and its three-dimensional structure determined by X-ray diffraction analysis to a resolution of 2.4 A. Three heavy-atom derivatives were used in the phase calculations with inclusion of the anomalous dispersion differences. The resulting electron density map allowed an easy and unambiguous tracing of the peptide chain. Two of the seven disulfide connections appeared to be different from what was suggested by the earlier chemical and structural work. The bovine phospholipase A2 structure contains about 50% α-helix and 10% β-structure. The bovine enzyme structure was found to deviate substantially from the previously published porcine prophospholipase structure.

Structure of quinoprotein methylamine dehydrogenase at 2.25 A resolution.

The three‐dimensional structure of quinoprotein methylamine dehydrogenase from Thiobacillus versutus has been determined at 2.25 A resolution by a combination of multiple isomorphous replacement, phase extension by solvent flattening and partial structure phasing using molecular dynamics refinement. In the resulting map, the polypeptide chain for both subunits could be followed and an X‐ray sequence was established. The tetrameric enzyme, made up of two heavy (H) and two light (L) subunits, is a flat parallellepiped with overall dimensions of approximately 76 x 61 x 45 A. The H subunit, comprising 370 residues, is made up of two distinct segments: the first 31 residues form an extension which embraces one of the L subunits; the remaining residues are found in a disc‐shaped domain. This domain is formed by a circular arrangement of seven topologically identical four‐stranded antiparallel beta‐sheets, with approximately 7‐fold symmetry. In spite of distinct differences, this arrangement is reminiscent of the structure found in influenza virus neuraminidase. The L subunit consists of 121 residues, out of which 53 form a beta‐sheet scaffold of a central three‐stranded antiparallel sheet flanked by two shorter two‐stranded antiparallel sheets. The remaining residues are found in segments of irregular structure. This subunit is stabilized by six disulphide bridges, plus two covalent bridges involving the quinone co‐factor and residues 57 and 107 of this subunit. The active site is located in a channel at the interface region between the H and L subunits, and the electron density in this part of the molecule suggests that the co‐factor of this enzyme is not pyrrolo quinoline quinone (PQQ) itself, but might be instead a precursor of PQQ.

Crystal structure of p-hydroxybenzoate hydroxylase complexed with its reaction product 3,4-dihydroxybenzoate

Crystals of the flavin-containing enzyme p-hydroxybenzoate hydroxylase (PHBHase) complexed with its reaction product were investigated in order to obtain insight into the catalytic cycle of this enzyme involving two substrates and two cofactors. PHBHase was crystallized initially with its substrate, p-hydroxybenzoate and the substrate was then converted into the product 3,4-dihydroxybenzoate by allowing the catalytic reaction to proceed in the crystals. In addition, crystals were soaked in mother liquor containing a high concentration of this product. Data up to 2.3 A (1 A = 0.1 nm) were collected by the oscillation method and the structure of the enzyme product complex was refined by alternate restrained least-squares procedures and model building by computer graphics techniques. A total of 273 solvent molecules could be located, four of them being presumably sulfate ions. The R-factor for 14,339 reflections between 6.0 A and 2.3 A is 19.3%. The 3-hydroxyl group of the product introduced by the enzyme is clearly visible in the electron density, showing unambiguously which carbon atom of the substrate is hydroxylated. A clear picture of the hydroxylation site is obtained. The plane of the product is rotated 21 degrees with respect to the plane of the substrate in the current model of enzyme-substrate complex. The 4-hydroxyl group of the product is hydrogen bonded to the hydroxyl group of Tyr201, its carboxyl group is interacting with the side-chains of Tyr222, Arg214 and Ser212, while the newly introduced 3-hydroxyl group makes a hydrogen bond with the backbone carbonyl oxygen of Pro293.