Anita R. Sielecki

Structure and refinement of penicillopepsin at 1.8 A resolution.

Abstract Penicillopepsin, the aspartyl protease from the mould Penicillium janthinellum , has had its molecular structure refined by a restrained-parameter least-squares procedure at 1.8 A resolution to a conventional R -factor of 0.136. The estimated co-ordinate accuracy for the majority of the 2363 atoms of the enzyme is better than 0.12 A. The average atomic thermal vibration parameter, B , for the atoms of the enzyme is 14.5 A 2 . One determining factor of this low average B value is the large central hydrophobic core, in which there are two prominent clusters of aromatic residues, one of nine, the other of seven residues. The N and C-terminal domains of penicillopepsin display an approximate 2-fold symmetry: 70 residue pairs are topologically equivalent, related by a rotation of 177 ° and a translation of 1.2 A. The analysis of the secondary structural features of the molecule reveals non-linear hydrogen bonding. In penicillopepsin, there is no difference in the mean hydrogen-bond parameters for the elements of α-helix, parallel or antiparallel β-pleated sheet. The mean values for these structural elements are: NO, 2.90 A; NHO, 1.95 A; NĤO, 160 °. The average hydrogen-bond parameters of the reverse β-turns and the 3 10 helices are distinctly different from the above values. The analysis of sidechain conformational angles χ 1 and χ 2 penicillopepsin and other enzyme structures refined in this laboratory shows much narrower distributions as compared with those compiled from unrefined protein structures. The close proximity of the carboxyl groups of Asp33 and Asp213 suggests that they share a proton in a tight hydrogen-bonded environment (Asp33OD2 to Asp213OD1 is 2.87 A). There are several solvent molecules in the active site region and, in particular, O39 forms hydrogen-bonded interactions with both aspartate residues. The disposition of the two carboxyl groups suggests that neither is likely to be involved in a direct nucleophilic attack on the scissile bond of a substrate. The average atomic B -factors of the residues in this region of the molecule are between 5 and 8 A 2 , confirming the proposal that conformational mobility of the active site residues has no role in the enzymatic mechanism. However, conformational mobility of neighbouring regions of the molecule e.g. the “flap” containing Tyr75, is verified by the high B -factors for those residues. The positions of 319 solvent sites per asymmetric unit have been selected from difference electron density maps and refined. Thirteen have been classified as internal, and several of these may have key roles during catalysis. The positively charged N ζ atom of Lys304 forms hydrogen bonds to the carboxylate of Asp14 (internal ion pair) and to two internal water molecules O5 and O25. The protonated side-chain of Asp300 forms a hydrogen bond to Thr214O, 2.78 A, and is the recipient of a hydrogen bond from a surface pocket water molecule O46. There is no possibility for direct interaction between Asp300 and Lys304 without large conformational changes of their environment. The intermolecular packing involves many protein-protein contacts (66 residues) with a large number of solvent molecules involved in bridging between polar residues at the contact surface. The penicillopepsin molecules resemble an approximate hexagonal close-packing of spheres with each molecule having 12 “nearest” neighbours.

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.

Structures of product and inhibitor complexes of Streptomyces griseus protease A at 1.8 A resolution. A model for serine protease catalysis.

Abstract This paper describes the 1.8 A resolution structure of the microbial enzyme Streptomyces griseus protease A in its native conformation, and in complexes with Ac-Pro-Ala-Pro-Phe-OH (I), Ac-Pro-Ala-Pro-Tyr-OH (II) and Ac-Pro-Ala-ProPhe-H (IV), all at pH 4.1. Each of these structures has been extensively refined by restrained parameter least-squares. The agreement factors ( R = Σ∥F o ¦—¦F c ∥/Σ¦F 0 ¦ ) are 0.130, 0.133, 0.122 and 0.142, respectively. The resultant electron density maps show that the peptide aldehyde (IV) forms a covalent hemiacetal bond with Ser195, the refined distance from the carbonyl carbon of the aldehyde to Oγ of Ser195 being 1.73 A. The corresponding distances in the protease-peptide I and II complexes are 2.58 A and 2.66 A, respectively, but there is no continuous electron density from Oγ to these carbonyl carbon atoms. Only three regions of the protease undergo conformational changes > 0.15 A upon binding of the peptides; namely, those segments comprising binding sites S2 to S4. Comparison of the overall binding modes of the three tetrapeptides in the complexes indicates that the peptide aldehyde (IV) moves in a concerted manner towards His57 and Ser195, due to the formation of the covalent hemiacetal bond; the overall root-mean-square co-ordinate difference in the 33 atoms common to I and IV is 0.34 A. A very large conformational movement of the imidazole ring of His57 (χ1 changing by 101 ° and χ2 by 7 °) is observed in the aldehyde complex. Of 200 water molecules located within the first contact shell of the enzyme, only four are internally bound, two of which are structurally equivalent to internal water molecules in the pancreatic serine proteases. There is a chain of water molecules ordered approximately parallel to the polypeptide chain forming the S1 to S3 binding sites. Sixteen water molecules which occupy the active site vicinity (Sielecki et al., 1979) are displaced by the product and inhibitor molecules when they are bound to the enzyme. The results from this study provide evidence for some important modifications to the reaction pathway of serine proteases, from formation of the initial E · S complex to the final dissociation of the E · P complex. We consider that the main motive forces for conversion of the enzyme-substrate complex to the covalent tetrahedral intermediate are the electrostatic interactions of the peptide dipole moments of the oxyanion binding site. We propose that the acyl enzyme is a high energy ester with a pyramidal carbonyl carbon atom, the carbonyl oxygen atom remaining in the strongly polarizing electrostatic field of the oxyanion site. The electronic strain accumulated in the acyl enzyme is released in the formation of the tetrahedral product intermediate. The most stable intermediate in this reaction sequence at pH 4.1 is the enzyme-product complex, which we have isolated in two cases (I and II). The close contact from Oγ of Ser195 to the carbonyl carbon atom of the product indicates that the energy barrier between the tetrahedral product and the planar carboxylate product intermediates must be relatively small, and is consistent with a partial bond between these two atoms.

Protein structure refinement: Streptomyces griseus serine protease A at 1.8 A resolution.

Abstract The crystal structure of the bacterial serine protease from Streptomyces griseus (SGPA) has been refined at 1.8 A resolution by a restrained parameter least-squares procedure ( Konnert, 1976 ) to a conventional R factor of 0.139 for 12662 statistically significant reflections [ I > 3 σ ( I )]. The number of variable parameters in the final model was 5912 which included positional and individual thermal parameters of the enzyme, and positions, B factors and occupancies of 175 solvent molecules. The algorithm used for this refinement allows for the simultaneous restraint on bond distances and distances related to interbond angles, the coplanarity of atoms in planar groups, the conservation of chirality of asymmetric centres, non-bonded contact distances, conformational torsional angles and individual isotropic temperature factors. The refined structure of SGPA differs from ideal bond lengths by an overall root-mean-square deviation of 0.02 A; the corresponding value for angle distances is 0.038 A. Comparison of the phase angles for the shell of data, 8.0 to 2.8 A, between the multiple isomorphous replacement phases (Brayer et al. , 1978 a ) and the refined phases, indicates an overall difference (r.m.s.) of 56.6 °. The average conformational angle of the peptide bond (ω) is 179.7 ° (root-mean-square deviation ± 2.5 °) for the 180 peptide bonds of SGPA. Of the 175 solvent molecules included during the course of the refinement, 22 with occupancies ranging from 1.00 to 0.38 are located in the active site and the substrate binding region. It was not until these water molecules were included in the refinement process that the active Ser195 adopted its final conformation ( χ 1 = −77 °). The resulting distance from O γ of Ser195 to N e 2 of His57 is 3.1 A, which, when taken with the observed distortion from linearity (50 °), indicates a rather weak interaction.

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.

Crystal structure studies and inhibition kinetics of tripeptide chloromethyl ketone inhibitors with Streptomyces griseus protease B.

Abstract The bacterial serine protease, SGPB, was inhibited by two specific tripeptide chloromethyl ketones, N-t-butyloxycarbonyl- l -alanylglycyl- l -phenylalanine chloromethyl ketone (BocAGFCK) and N-t-butyloxycarbonyl-glycyl- l -leucyl- l -phenylalanine chloromethyl ketone (BocGLFCK). Crystals of the inhibited complexes were grown and examined by X-ray crystallographic methods. The peptide backbone of each inhibitor is bound by three hydrogen bonds to the main chain of residues Ser214 to Gly216. There are two well-characterized hydrophobic pockets, S1 and S2, on the surface of SGPB which accommodate the P1 and P2 side-chains of the BocGLFCK inhibitor. A conformational change of Tyr171 is induced by the binding of this inhibitor. Both inhibitors make two covalent bonds to the SGPB enzyme. The imidazole ring of His57 is alkylated at the Nϵ2 atom and Oγ of Ser195 forms a hemiketal bond with the carbonyl-carbon atom of the inhibitor. Comparison of the binding modes of the two tripeptides in conjunction with the differences in their inhibition constants (KI) allows one to estimate the binding energy of the leucyl side-chain as −2.6 kcal mol−1. The importance of an electrophilic component in the serine protease mechanism, which involves the polarization of the susceptible carbonyl bond of a substrate or inhibitor by the peptide NH groups of Gly193 and Ser195 is discussed.

Refined Crystal Structure of Rat Parvalbumin, a Mammalian α-lineage Parvalbumin, at 2·0 Å Resolution

We present here the X-ray crystal structure of the rat α-parvalbumin from fast twitch muscle. This protein (Mr 11·8kDa) crystallizes in space group P212121 with unit cell dimensions of a=34·3 A, b=55·0 A, c=156·1 A and three molecules in the asymmetric unit. The protein structure was solved by the molecular replacement method and has been refined to a crystallographic R-factor (R=Σ∥Fo|-|Fc∥/Σ|Fo|)) of 0·181 for all reflections with I/σ(I)(l) ≥ 2 (I=intensity) between 8·0 and 2·0 A resolution. The molecules located most easily in the molecular replacement rotation function had lower overall thermal motion parameters and higher numbers of intermolecular crystal packing contacts. The overall fold of the polypeptide chain for the rat α-parvalbumin is similar to other known parvalbumin structures (root-mean-square deviations in α-carbon atom positions range from 0·60 to 0·87 A). There are two Ca2+-binding sites in parvalbumins, and them is some evidence for a third ion-binding site, adjacent to the CD site, in the rat species. The level of structural variability among the best-ordered regions of the three independent rat, α-parvalbumin molecules in the crystallographic asymmetric unit, is two to three times higher than the mean coordinate error (0·10 A), indicating flexibility in the molecule. Sequence differences between α and β-lineage parvalbumins result in repacking of the hydrophobic core and some shifts in the protein backbone. The shifts are localized, however, and entire helices do not shift as rigid units.

The Molecular Structure of Human Progastricsin and its Comparison with that of Porcine Pepsinogen

Mammalian aspartic proteinases are synthesized as inactive precursors or zymogens. Stomach zymogens undergo a conversion to the active enzyme form autocatalytically at pH < 5.0 (1). The human gastric juice has two major groups of aspartic proteinases, the pepsins (EC3.4.23.1) and the gastricsins (EC3.4.23.3). Progastricsin or pepsinogen C (PGC) is converted to gastricsin by removal of the 43 amino-terminal residues of the prosegment. The resulting mature gastricsin has 329 amino acid residues. The sequence of human PGC has been determined independently in two laboratories by nucleotide sequencing of the gene (2) and of cDNA clones (3).