Masao Fujinaga

Crystal and molecular structures of the complex of α-chymotrypsin with its inhibitor Turkey ovomucoid third domain at 1.8 Å resolution

The molecular structure of the complex between bovine pancreatic α-chymotrypsin (EC 3.4.4.5) and the third domain of the Kazal-type ovomucoid from Turkey (OMTKY3) has been determined crystallographically by the molecular replacement method. Restrained-parameter least-squares refinement of the molecular model of the complex has led to a conventional agreement factor R of 0.168 for the 19,466 reflections in the 1.8 A (1 A = 0.1 nm) resolution shell [I ≥ σ(I)]. The reactive site loop of OMTKY3, from Lys131 to Arg211 (I indicates inhibitor), is highly complementary to the surface of α-chymotrypsin in the complex. A total of 13 residues on the inhibitor make 113 contacts of less than 4.0 A with 21 residues of the enzyme. A short contact (2.95 A) from Oγ of Ser195 to the carbonylcarbon atom of the scissile bond between Leu181 and Glu191 is present; in spite of it, this peptide remains planar and undistorted. Analysis of the interactions of the inhibitor with chymotrypsin explains the enhanced specificity that chymotrypsin has for P′3 arginine residues. There is a water-mediated ion pair between the guanidinium group on this residue and the carboxylate of Asp64. Comparison of the structure of the α-chymotrypsin portion of this complex with the several structures of α and γ-chymotrypsin in the uncomplexed form shows a high degree of structural equivalence (root-mean-square deviation of the 234 common α-carbon atoms averages 0.38 A). Significant differences occur mainly in two regions Lys36 to Phe39 and Ser75 to Lys79. Among the 21 residues that are in contact with the ovomucoid domain, only Phe39 and Tyr146 change their conformations significantly as a result of forming the complex. Comparison of the structure of the OMTKY3 domain in this complex to that of the same inhibitor bound to a serine proteinase from Streptomyces griseus (SGPB) shows a central core of 44 amino acids (the central α-helix and flanking small 3-stranded β-sheet) that have α-carbon atoms fitting to within 1.0 A (root-mean-square deviation of 0.45 A) whereas the residues of the reactive-site loop differ in position by up to 1.9 A (Cα of Leu181). The ovomucoid domain has a built-in conformational flexibility that allows it to adapt to the active sites of different enzymes. A comparison of the SGPB and α-chymotrypsin molecules is made and the water molecules bound at the inhibitor-enzyme interface in both complexes are analysed for similarities and differences.

Experiences with a new translation-function program

The development of a new translation-function program is reported. It is one that uses a linear correlation coefficient to determine the correct position of an oriented molecule in the crystal cell. The method has been implemented in a computer program called BRUTE. The program can also refine the orientation of the model and accept a set of atoms with fixed positions. Comparison of the correlation coefficient with other translation functions indicates that it is comparable to or slightly better than the rest. The most important feature of the program is its ability to adjust the orientation of the model. This allows for errors in the orientation obtained from the rotation function to be corrected.

Refined structure of α-lytic protease at 1.7 Å resolution analysis of hydrogen bonding and solvent structure

The structure of alpha-lytic protease, a serine protease produced by the bacterium Lysobacter enzymogenes, has been refined at 1.7 A resolution. The conventional R-factor is 0.131 for the 14,996 reflections between 8 and 1.7 A resolution with I greater than or equal to 2 sigma (I). The model consists of 1391 protein atoms, two sulfate ions and 156 water molecules. The overall root-meansquare error is estimated to be about 0.14 A. The refined structure was compared with homologous enzymes alpha-chymotrypsin and Streptomyces griseus protease A and B. A new sequence numbering was derived based on the alignment of these structures. The comparison showed that the greatest structural homology is around the active site residues Asp102, His57 and Ser195, and that basic folding pathways are maintained despite chemical changes in the hydrophobic cores. The hydrogen bonds in the structure were tabulated and the distances and angles of interaction are similar to those found in small molecules. The analysis also revealed the presence of close intraresidue interactions. There are only a few direct intermolecular hydrogen bonds. Most intermolecular interactions involve bridging solvent molecules. The structural importance of hydrogen bonds involving the side-chain of Asx residues is discussed. All the negatively charged groups have a counterion nearby, while the excess positively charged groups are exposed to the solvent. One of the sulfate ions is located near the active site, whereas the other is close to the N terminus. Of the 156 water molecules, only seven are not involved in a hydrogen bond. Six of these have polar groups nearby, while the remaining one is in very weak density. There are nine internal water molecules, consisting of two monomers, two dimers and one trimer. No significant second shell of solvent is observed.

Rat submaxillary gland serine protease, tonin. Structure solution and refinement at 1.8 A resolution.

Tonin is a mammalian serine protease that is capable of generating the vasoconstrictive agent, angiotensin II, directly from its precursor protein, angiotensinogen, a process that normally requires two enzymes, renin and angiotensin-converting enzyme. The X-ray crystallographic structure determination and refinement of tonin at 1.8 A resolution and the analysis of the resulting model are reported. The initial phases were obtained by the method of molecular replacement using as the search model the structure of bovine trypsin. The refined model of tonin consists of 227 amino acid residues out of the 235 in the complete molecule, 149 water molecules, and one zinc ion. The R-factor (R = sigma Fo - Fc/sigma Fo) is 0.196 for the 14,997 measured data between 8 and 1.8 A resolution with I greater than or equal to sigma (I). It is estimated that the overall root-mean-square error in the coordinates is about 0.3 A. The structure of tonin that has been determined is not in its active conformation, but one that has been perturbed by the binding of Zn2+ in the active site. Zn2+ was included in the buffer to aid the crystallization. Nevertheless, the structure of tonin that is described is for the most part similar to its native form as indicated by the close tertiary structural homology with kallikrein. The differences in the structures of the two enzymes are concentrated in several loop regions; these structural differences are probably responsible for the differences in their reactivities and specificities.

Refined structure of porcine pepsinogen at 1.8 A resolution.

The molecular structure of porcine pepsinogen at 1.8 A resolution has been determined by a combination of molecular replacement and multiple isomorphous phasing techniques. The resulting structure was refined by restrained-parameter least-squares methods. The final R factor [formula: see text] is 0.164 for 32,264 reflections with I greater than or equal to sigma (I) in the resolution range of 8.0 to 1.8 A. The model consists of 2785 protein atoms in 370 residues, a phosphoryl group on Ser68 and 238 ordered water molecules. The resulting molecular stereochemistry is consistent with a well-refined crystal structure with co-ordinate accuracy in the range of 0.10 to 0.15 A for the well-ordered regions of the molecule (B less than 15 A2). For the enzyme portion of the zymogen, the root-mean-square difference in C alpha atom co-ordinates with the refined porcine pepsin structure is 0.90 A (284 common atoms) and with the C alpha atoms of penicillopepsin it is 1.63 A (275 common atoms). The additional 44 N-terminal amino acids of the prosegment (Leu1p to Leu44p, using the letter p after the residue number to distinguish the residues of the prosegment) adopt a relatively compact structure consisting of a long beta-strand followed by two approximately orthogonal alpha-helices and a short 3(10)-helix. Intimate contacts, both electrostatic and hydrophobic interactions, are made with residues in the pepsin active site. The N-terminal beta-strand, Leu1p to Leu6p, forms part of the six-stranded beta-sheet common to the aspartic proteinases. In the zymogen the first 13 residues of pepsin, Ile1 to Glu13, adopt a completely different conformation from that of the mature enzyme. The C alpha atom of Ile1 must move approximately 44 A in going from its position in the inactive zymogen to its observed position in active pepsin. Electrostatic interactions of Lys36pN and hydrogen-bonding interactions of Tyr37pOH, and Tyr90H with the two catalytic aspartate groups, Asp32 and Asp215, prevent substrate access to the active site of the zymogen. We have made a detailed comparison of the mammalian pepsinogen fold with the fungal aspartic proteinase fold of penicillopepsin, used for the molecular replacement solution. A structurally derived alignment of the two sequences is presented.