Michael N. G. James

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.

Refined crystal structure of troponin C from turkey skeletal muscle at 2.0 A resolution.

The crystal structure of troponin C from turkey skeletal muscle has been refined at 2.0 A resolution (1 A = 0.1 nm). The resulting crystallographic R factor (R = sigma[[Fo[-[Fc[[/sigma[Fo[, where [Fo[ and [Fc[ are the observed and calculated structure factor amplitudes) is 0.155 for the 8054 reflections with intensities I greater than or equal to 2 sigma(I) within the 10 A to 2.0 A resolution range. With 66% of the residues in helical conformation, troponin C provides a good sample for helix analysis. The mean alpha-helix dihedral angles (phi, psi = -62 degrees, -42 degrees) agree with values observed for helical regions in other proteins. The helices are all curved and/or kinked. In particular, the 31 amino acid long inter-domain helix is smoothly curved, with a rather large radius of curvature of 137 A. Helix packing is different in the Ca2+-free domain (N-terminal) and the Ca2+-bound domain (C-terminal). The inter-helix angles for the two helix-loop-helix motifs in the regulatory domain are 133 degrees and 151 degrees, whereas the value for the two motifs in the C-terminal domain is 110 degrees, as observed in the EF-hands of parvalbumin. These differences affect the packing of the respective hydrophobic cores of each domain, in particular the disposition of aromatic rings. Pairwise arrangement of Ca2+-binding loops is common to both states, but the conformation is markedly different. Conversion of one to the other can be achieved by small cumulative changes of main-chain dihedral angles. The integrity of loop structure is maintained by numerous electrostatic interactions. Both salt bridges and carboxyl-carboxylate interactions are observed in TnC. There are more intramolecular (9) than intermolecular (1) salt bridges. Carboxyl-carboxylate interactions occur because the pH of the crystals is 5.0 and there is a multitude of aspartate and glutamate residues. One is intramolecular and four are intermolecular. Polar side-chain interactions occur more commonly with main-chain carbonyls and amides than with other polar side-chains. These interactions are mostly short range, and are similar to those observed in other proteins with one exception: negatively charged side-chains interact more frequently with main-chain carbonyl oxygen atoms. However, out of 19 such interactions, 10 involve oxygen atoms of the Ca2+ ligands. These unfavorable interactions are compensated by the favorable interactions with the Ca2+ ions and with main-chain amides. They are a trivial consequence of the tight fold of the Ca2+-binding loops.

Molecular structure of an aspartic proteinase zymogen, porcine pepsinogen, at 1.8 A resolution.

The only well-understood mechanism of zymogen activation is that of the serine proteinases, in which proteolytic cleavage leads to conformational changes resulting in a functional active site. A different mechanism is now unveiled by the crystal structure of pepsinogen. Salt bridges that stabilize the positioning of the N-terminal proenzyme segment across the active site of pepsin are disrupted at low pH, releasing the amino-terminal segment and thereby exposing the catalytic apparatus and the substrate-binding sites.

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.

Calcium-binding sites in proteins : a structural perspective

Publisher Summary This chapter describes and compares the calcium (Ca 2+ )-binding sites in proteins, attempting to extract common features from them. It determines the functional and structural parameters of a regular protein Ca 2+ -binding site by helix–loop–helix proteins, serine proteinases, and other Ca 2+ -binding proteins. Three broad classes of functions are: (1) Ca 2+ modulation of protein action, (2) Ca 2+ stabilization of protein structure, and (3) involvement of the Ca 2+ ion in enzymatic catalysis. The Ca 2+ -modulated proteins have altered interactions with other proteins on binding of Ca 2+ . Binding Ca 2+ stabilizes some proteins against thermal or chaotropic denaturation, or proteolytic degradation. The chapter also discusses on what are the structural correlates of stronger or weaker Ca 2+ binding and how Ca 2+ - binding sites distinguish between Ca 2+ and the very similar Mg 2+ or other ions. Ca 2+ may be the ion of choice in so many biological roles because of its relative flexibility in preferred coordination number and ligand geometry. The relationships between the protein structure at a Ca 2+ -binding site and the function of the ion within the protein are both subtle and complex.

Crystal structure of Human beta-hexosaminidase B: Understanding the molecular basis of Sandhoff and Tay-Sachs disease

In humans, two major beta-hexosaminidase isoenzymes exist: Hex A and Hex B. Hex A is a heterodimer of subunits alpha and beta (60% identity), whereas Hex B is a homodimer of beta-subunits. Interest in human beta-hexosaminidase stems from its association with Tay-Sachs and Sandhoff disease; these are prototypical lysosomal storage disorders resulting from the abnormal accumulation of G(M2)-ganglioside (G(M2)). Hex A degrades G(M2) by removing a terminal N-acetyl-D-galactosamine (beta-GalNAc) residue, and this activity requires the G(M2)-activator, a protein which solubilizes the ganglioside for presentation to Hex A. We present here the crystal structure of human Hex B, alone (2.4A) and in complex with the mechanistic inhibitors GalNAc-isofagomine (2.2A) or NAG-thiazoline (2.5A). From these, and the known X-ray structure of the G(M2)-activator, we have modeled Hex A in complex with the activator and ganglioside. Together, our crystallographic and modeling data demonstrate how alpha and beta-subunits dimerize to form either Hex A or Hex B, how these isoenzymes hydrolyze diverse substrates, and how many documented point mutations cause Sandhoff disease (beta-subunit mutations) and Tay-Sachs disease (alpha-subunit mutations).

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.

The crystal structure of the rhomboid peptidase from Haemophilus influenzae provides insight into intramembrane proteolysis.

Rhomboid peptidases are members of a family of regulated intramembrane peptidases that cleave the transmembrane segments of integral membrane proteins. Rhomboid peptidases have been shown to play a major role in developmental processes in Drosophila and in mitochondrial maintenance in yeast. Most recently, the function of rhomboid peptidases has been directly linked to apoptosis. We have solved the structure of the rhomboid peptidase from Haemophilus influenzae (hiGlpG) to 2.2-Å resolution. The phasing for the crystals of hiGlpG was provided mainly by molecular replacement, by using the coordinates of the Escherichia coli rhomboid (ecGlpG). The structural results on these rhomboid peptidases have allowed us to speculate on the catalytic mechanism of substrate cleavage in a membranous environment. We have identified the relative disposition of the nucleophilic serine to the general base/acid function of the conserved histidine. Modeling a tetrapeptide substrate in the context of the rhomboid structure reveals an oxyanion hole comprising the side chain of a second conserved histidine and the main-chain NH of the nucleophilic serine residue. In both hiGlpG and ecGlpG structures, a water molecule occupies this oxyanion hole.

A potent new mode of beta-lactamase inhibition revealed by the 1.7 A X-ray crystallographic structure of the TEM-1-BLIP complex.

The structure of TEM-1 β-lactamase complexed with the inhibitor BLIP has been determined at 1.7 Å resolution. The two tandemly repeated domains of BLIP form a polar, concave surface that docks onto a predominantly polar, convex protrusion on the enzyme. The ability of BLIP to adapt to a variety of class A β-lactamases is most likely due to an observed flexibility between the two domains of the inhibitor and to an extensive layer of water molecules entrapped between the enzyme and inhibitor. A β-hairpin loop from domain 1 of BLIP is inserted into the active site of the β-lactamase. The carboxylate of Asp 49 forms hydrogen bonds to four conserved, catalytic residues in the β-lactamase, thereby mimicking the position of the penicillin G carboxylate observed in the acyl–enzyme complex of TEM-1 with substrate. This β-hairpin may serve as a template with which to create a new family of peptide-analogue β-lactamase inhibitors.