An analysis of subdomain orientation, conformational change and disorder in relation to crystal packing of aspartic proteinases.

The analysis reported here describes detailed structural studies of endothiapepsin (the aspartic proteinase from Endothia parasitica), with and without bound inhibitors, and human pepsin 3b. Comparison of multiple crystal structures of members of the aspartic proteinase family has revealed small but significant differences in domain orientation in different crystal forms. In this paper, it is shown that these differences in domain orientation do not necessarily correlate with the presence or absence of bound inhibitors, but appear to stem at least partly from crystal contacts mediated by sulfate ions. However, since the same inherent flexibility of the structure is observed for other enzymes in this family such as human pepsin, the native structure of which is also reported here, the observed domain movements may well have implications for the mechanism of catalysis.

Structure determination of the head-tail connector of bacteriophage phi29.

The head-tail connector of bacteriophage phi29 is composed of 12 36 kDa subunits with 12-fold symmetry. It is the central component of a rotary motor that packages the genomic dsDNA into preformed proheads. This motor consists of the head-tail connector, surrounded by a phi29-encoded, 174-base, RNA and a viral ATPase protein, both of which have fivefold symmetry in three-dimensional cryo-electron microscopy reconstructions. DNA is translocated into the prohead through a 36 A diameter pore in the center of the connector, where the DNA takes the role of a motor spindle. The helical nature of the DNA allows the rotational action of the connector to be transformed into a linear translation of the DNA. The crystal structure determination of connector crystals in space group C2 was initiated by molecular replacement, using an approximately 20 A resolution model derived from cryo-electron microscopy. The model phases were extended to 3.5 A resolution using 12-fold non-crystallographic symmetry averaging and solvent flattening. Although this electron density was not interpretable, the phases were adequate to locate the position of 24 mercury sites of a thimerosal heavy-atom derivative. The resultant 3.2 A single isomorphous replacement phases were improved using density modification, producing an interpretable electron-density map. The crystallographically refined structure was used as a molecular-replacement model to solve the structures of two other crystal forms of the connector molecule. One of these was in the same space group and almost isomorphous, whereas the other was in space group P2(1)2(1)2. The structural differences between the oligomeric connector molecules in the three crystal forms and between different monomers within each crystal show that the structure is relatively flexible, particularly in the protruding domain at the wide end of the connector. This domain probably acts as a bearing, allowing the connector to rotate within the pentagonal portal of the prohead during DNA packaging.

Structure of the bacteriophage phi29 DNA packaging motor.

Motors generating mechanical force, powered by the hydrolysis of ATP, translocate double-stranded DNA into preformed capsids (proheads) of bacterial viruses and certain animal viruses. Here we describe the motor that packages the double-stranded DNA of the Bacillus subtilis bacteriophage phi29 into a precursor capsid. We determined the structure of the head-tail connector--the central component of the phi29 DNA packaging motor--to 3.2 A resolution by means of X-ray crystallography. We then fitted the connector into the electron densities of the prohead and of the partially packaged prohead as determined using cryo-electron microscopy and image reconstruction analysis. Our results suggest that the prohead plus dodecameric connector, prohead RNA, viral ATPase and DNA comprise a rotary motor with the head-prohead RNA-ATPase complex acting as a stator, the DNA acting as a spindle, and the connector as a ball-race. The helical nature of the DNA converts the rotary action of the connector into translation of the DNA.

X-ray structures of five renin inhibitors bound to saccharopepsin: exploration of active-site specificity.

Saccharopepsin is a vacuolar aspartic proteinase involved in activation of a number of hydrolases. The enzyme has great structural homology to mammalian aspartic proteinases including human renin and we have used it as a model system to study the binding of renin inhibitors by X-ray crystallography. Five medium-to-high resolution structures of saccharopepsin complexed with transition-state analogue renin inhibitors were determined. The structure of a cyclic peptide inhibitor (PD-129,541) complexed with the proteinase was solved to 2.5 A resolution. This inhibitor has low affinity for human renin yet binds very tightly to the yeast proteinase (K(i)=4 nM). The high affinity of this inhibitor can be attributed to its bulky cyclic moiety spanning P(2)-P(3)' and other residues that appear to optimally fit the binding sub-sites of the enzyme. Superposition of the saccharopepsin structure on that of renin showed that a movement of the loop 286-301 relative to renin facilitates tighter binding of this inhibitor to saccharopepsin. Our 2.8 A resolution structure of the complex with CP-108,420 shows that its benzimidazole P(3 )replacement retains one of the standard hydrogen bonds that normally involve the inhibitor's main-chain. This suggests a non-peptide lead in overcoming the problem of susceptible peptide bonds in the design of aspartic proteinase inhibitors. CP-72,647 which possesses a basic histidine residue at P(2), has a high affinity for renin (K(i)=5 nM) but proves to be a poor inhibitor for saccharopepsin (K(i)=3.7 microM). This may stem from the fact that the histidine residue would not bind favourably with the predominantly hydrophobic S(2) sub-site of saccharopepsin.

Purification, crystallization and initial X-ray analysis of the head-tail connector of bacteriophage phi29.

The head-tail connector of bacteriophage phi29, an oligomer of gene product 10 (gp10), was crystallized into various forms. The most useful of these were an orthorhombic P22(1)2(1) form (unit-cell parameters a = 143.0, b = 157.0, c = 245.2 A), a monoclinic C2 form (a = 160.7, b = 143.6, c = 221.0 A, beta = 97.8 degrees ) and another monoclinic C2 form (a = 177.0, b = 169.1, c = 185.2 A, beta = 114.1 degrees ). Frozen crystals diffracted to about 3.2 A resolution. There is one connector per crystallographic asymmetric unit in each case. Rotation functions show the connector to be a dodecamer. Translation functions readily determined the position of the 12-fold axis in each unit cell. The structure is being determined by 12-fold electron-density averaging within each crystal and by averaging between the various crystal forms.

Purification, co-crystallization and preliminary X--ray analysis of the natural aspartic proteinase inhibitor IA3 complexed with saccharopepsin from Saccharomyces cerevisiae.

The vacuolar aspartic proteinase from baker's yeast, saccharopepsin, has been co-crystallized with its natural inhibitor I(A)3, found in the cytosol. The I(A)3-saccharopepsin complex crystals belong to the space group P6(2)22, with unit-cell parameters a = b = 192.1, c = 59. 80 A and one molecule per asymmetric unit. The initial X-ray analysis of the complex indicates that the crystals diffract to 5.0 A, similar to native saccharopepsin crystals. This is probably a consequence in part of glycosylation of the native saccharopepsin. Full structural analysis of the complex crystal is in progress.

A 2.3 A resolution structure of chymosin complexed with a reduced bond inhibitor shows that the active site beta-hairpin flap is rearranged when compared with the native crystal structure.

In the crystal structure of uncomplexed native chymosin, the beta-hairpin at the active site, known as 'the flap', adopts a different conformation from that of other aspartic proteinases. This conformation would prevent the mode of binding of substrates/inhibitors generally found in other aspartic proteinase complexes. We now report the X-ray analysis of chymosin complexed with a reduced bond inhibitor CP-113972 ¿(2R,3S)-isopropyl 3-[(L-prolyl-p-iodo-L-phenylalanyl-S-methyl-cysteinyl)amino-4]-cyclohexy l-2-hydroxybutanoate¿ at 2.3 A resolution in a novel crystal form of spacegroup R32. The structure has been refined by restrained least-squares methods to a final R-factor of 0.19 for a total of 11 988 independent reflections in the resolution range 10 to 2.3 A. The extended beta-strand conformation of the inhibitor allows hydrogen bonds within the active site, while its sidechains make both electrostatic and hydrophobic interactions with residues lining the specificity pockets S4-->S1. The flap closes over the active site cleft in a way that closely resembles that of other previously determined aspartic proteinase inhibitor complexes. We conclude that the usual position and conformation of the flap found in other aspartic proteinases is available to native chymosin. The conformation observed in the native crystal form may result from intermolecular interactions between symmetry-related molecules in the crystal lattice.

Engineering a change in metal-ion specificity of the iron-dependent superoxide dismutase from Mycobacterium tuberculosis-- X-ray structure analysis of site-directed mutants.

We have refined the X-ray structures of two site-directed mutants of the iron-dependent superoxide dismutase (SOD) from Mycobacterium tuberculosis. These mutations which affect residue 145 in the enzyme (H145Q and H145E) were designed to alter its metal-ion specificity. This residue is either Gln or His in homologous SOD enzymes and has previously been shown to play a role in active-site interactions since its side-chain helps to coordinate the metal ion via a solvent molecule which is thought to be a hydroxide ion. The mutations were based on the observation that in the closely homologous manganese dependent SOD from Mycobacterium leprae, the only significant difference from the M. tuberculosis SOD within 10 A of the metal-binding site is the substitution of Gln for His at position 145. Hence an H145Q mutant of the M. tuberculosis (TB) SOD was engineered to investigate this residue's role in metal ion dependence and an isosteric H145E mutant was also expressed. The X-ray structures of the H145Q and H145E mutants have been solved at resolutions of 4.0 A and 2.5 A, respectively, confirming that neither mutation has any gross effects on the conformation of the enzyme or the structure of the active site. The residue substitutions are accommodated in the enzyme's three-dimensional structure by small local conformational changes. Peroxide inhibition experiments and atomic absorption spectroscopy establish surprisingly the H145E mutant SOD has manganese bound to it whereas the H145Q mutant SOD retains iron as the active-site metal. This alteration in metal specificity may reflect on the preference of manganese ions for anionic ligands.

The three-dimensional structure at 2.4 A resolution of glycosylated proteinase A from the lysosome-like vacuole of Saccharomyces cerevisiae.

The crystal structures of glycosylated native proteinase A, an aspartic proteinase found in the vacuole of Saccharomyces cerevisiae, and its complex with a difluorostatone-containing tripeptide have been determined by molecular replacement to 3.5 A and 2.4 A resolutions, respectively. Superposition of the bound and native forms gave an r.m.s. difference of 0.6 A largely reflecting the poor resolution of the native crystal structure. The secondary and tertiary structures are highly similar to those found in porcine pepsin and lysosomal cathepsin D; superposition of the structure of proteinase A bound to the difluorostatone inhibitor on those of pepsin and cathepsin D gave pairwise r.m.s. differences for C(alpha) atoms of 1.36 A and 0.88 A. Most differences occur in loop regions. Comparison of the structure of the proteinase A-difluorostatone complex with that of endothiapepsin bound with the same inhibitor shows that the conformation and hydrogen bond interactions of the inhibitor in the active site are very similar, even though the enzymes have only 27% sequence identity. Electron density for the crystal structure of the proteinase A complex reveals five residues of the oligosaccharide structure attached to Asn67: Man-(1 --> 2)-alpha-Man-(1 --> 3)-beta-Man-(1 --> 4)-beta-GlcNAc-(1 --> 4)-beta-GlcNAc-Asn-67. The first three residues of the oligosaccharide cover the same region of the protein surface as those of the oligosaccharide attached to the equivalent position in cathepsin D. The second carbohydrate attachment site is disordered beyond the first carbohydrate residue in both enzymes.

X-ray structure analysis of an engineered Fe-superoxide dismutase Gly-Ala mutant with significantly reduced stability to denaturant.

We have refined the X-ray structure of a site-directed G152A mutant of the iron-dependent superoxide dismutase from Mycobacterium tuberculosis at 2.9 angstroms resolution. The mutation which replaces a glycine residue in a surface loop with alanine was designed to alter the conformation of this loop region which has previously been shown to play a crucial structural role in quaternary interactions within the SOD tetramer. Gly-152 was targeted as it has dihedral angles (phi = 83.1 degrees, psi = -0.3 degrees) close to the left-handed alpha-helical conformation which is rarely adopted by other amino acids except asparagine. Gly-152 was replaced by alanine as it has similar size and polarity, yet has a very low tendency to adopt similar conformations. X-ray data collection on crystals of this mutant at 2.9 angstroms resolution and subsequent least-squares refinement to an R-value of 0.169 clearly establish that the loop conformation is unaffected. Fluorescence studies of guanidine hydrochloride denaturation establish that the mutant is 4 kcal/mol less stable than the wild-type enzyme. Our results indicate that strict conformational constraints imposed upon a region of polypeptide, due for example to interactions with a neighbouring subunit, may force an alanine residue to adopt this sterically hindered conformation with a consequent reduction in stability of the folded conformation.

X-ray structure analysis of the iron-dependent superoxide dismutase from Mycobacterium tuberculosis at 2.0 Angstroms resolution reveals novel dimer-dimer interactions.

The X-ray structure of the tetrameric iron-dependent superoxide dismutase from Mycobacterium tuberculosis has been refined to an R-factor of 0.167 and a correlation coefficient of 0.954 at 2.0 A resolution. The crystals are monoclinic P2(1) and have four subunits related by strong non-crystallographic 222 (or D2) symmetry in the asymmetric unit. 198 of the 207 amino acids of each subunit are defined by the electron density which shows that they adopt the conserved fold of other iron- or manganese-dependent SODs. The structure can be divided into two domains, the N-terminal domain involving an extended region followed by two projecting antiparallel alpha-helices, and the C-terminal domain containing four more helical segments with a three-stranded antiparallel beta-sheet inserted sequentially between the fourth and fifth helices. The catalytic iron is co-ordinated by five ligands: three histidines (residues 28, 76 and 164), one aspartate (160) and a solvent molecule. The inferred positions of protons at the active site are consistent with the solvent ligand being a hydroxide ion. This ligand interacts with His145 in the Mycobacterium tuberculosis SOD. In the highly homologous Mycobacterium leprae Mn-SOD, the histidine is replaced by glutamine, this being the only significant residue difference within 10 A of the Fe3+. The nature of the amino acid at this position may influence the metal ion specificity of these enzymes. The subunits of the Mycobacterium tuberculosis SOD associate by polar contacts to form dimers, which closely resemble those of other dimeric or tetrameric Fe- or Mn-SODs. However, the dimer-dimer interactions within the tetramer are novel, being dominated by dimerisation of the 144 to 152 loop regions which connect the outer two beta-strands of the three-membered beta-sheet. This contrasts strongly with the other tetrameric Fe- or Mn-SODs where the dimer-dimer association is dominated by the projecting alpha alpha-turn in the N-terminal domain.

Comparisons of the three-dimensional structures, specificities and glycosylation of renins, yeast proteinase A and cathepsin D.

The crystal structures of complexes of the aspartic proteinases, human and mouse renins, yeast proteinase A and cathepsin D, with peptide analogue inhibitors are compared. Differences occur in the relative positions of the domain comprising residues 190-302 (pepsin numbering) compared to the remaining structure and in the nature and position of the irregular regions joining the beta-strands and alpha-helices. The first three of the five residues of the oligosaccharide structures attached to Asn 67 of yeast proteinase and cathepsin D cover the same region of the protein surface. All enzymes have an unusual, proline-rich region (292-297) which acts as a second flap (in addition to that involving residues 72-81). This covers the active site cleft, but can be very close to the substrate/inhibitor at P3' and P4' only in the renins.

X-ray analysis at 2.0 A resolution of mouse submaxillary renin complexed with a decapeptide inhibitor CH-66, based on the 4-16 fragment of rat angiotensinogen.

The structure of mouse submaxillary renin complexed with a decapeptide inhibitor, CH-66 (Piv-His-Pro-Phe-His-Leu-OH-Leu-Tyr-Tyr-Ser-NH2), where Piv denotes a pivaloyl blocking group, and -OH- denotes a hydroxyethylene (-(S)CHOH-CH2-) transition state isostere as a scissile bond surrogate, has been refined to an agreement factor of 0.18 at 2.0 A resolution. The positions of 10,038 protein atoms and 364 inhibitor atoms (4 independent protein inhibitor complexes), as well as of 613 solvent atoms, have been determined with an estimated root-mean-square (r.m.s.) error of 0.21 A. The r.m.s. deviation from ideality for bond distances is 0.026 A, and for angle distances is 0.0543 A. We have compared the three-dimensional structure of mouse renin with other aspartic proteinases, using rigid-body analysis with respect to shifts involving the domain comprising residues 190 to 302. In terms of the relative orientation of domains, mouse submaxillary renin is closest to human renin with only a 1.7 degrees difference in domain orientation. Porcine pepsin (the molecular replacement model) differs structurally from mouse renin by a 6.9 degrees domain rotation, whereas endothiapepsin, a fungal aspartic proteinase, differs by 18.8 degrees. The triple proline loop (residues 292 to 294), which is structurally opposite the active-site "flap" (residues 72 to 83), gives renin a superficial resemblance to the fold of the retroviral proteinases. The inhibitor is bound in an extended conformation along the active-site cleft, and the hydroxyethylene moiety forms hydrogen bonds with both catalytic aspartate carboxylates. The complex is stabilized by hydrogen bonds between the main chain of the inhibitor and the enzyme. All side-chains of the inhibitor are in van der Waals contact with groups in the enzyme and define ten specificity sub-sites. This study shows how renin has compact sub-sites due to the positioning of secondary structure elements, to complementary substitutions and to the residue composition of its loops close to the active site, leading to extreme specificity towards its prohormone substrate, angiotensinogen. We have analysed the micro-environment of each of the buried charged groups in order to predict their ionization states.

Analyses of ligand binding in five endothiapepsin crystal complexes and their use in the design and evaluation of novel renin inhibitors.

Five renin inhibitors were cocrystallized with endothiapepsin, a fungal enzyme homologous to renin. Crystal structures of inhibitor-bound complexes have provided invaluable insight regarding the three-dimensional structure of the aspartic proteinase family of enzymes, as well as the steric and polar interactions that occur between the proteins and the bound ligands. Beyond this, subtleties of binding have been revealed, including multiple subsite binding modes and subsite interdependencies. This information has been applied in the design of novel potent renin inhibitors and in the understanding of structure-activity relationships and enzyme selectivities.

Crystallization and preliminary crystallographic characterization of aspartic proteinase-A from baker's yeast and its complexes with inhibitors.

The aspartic proteinase from yeast vacuoles, proteinase-A, has been crystallized with and without non-hydrolysable transition-state analogue inhibitors. The native enzyme crystals belong to the space group I2(1)2(1)2(1), with two molecules per asymmetric unit. The inhibitor complex crystals are trigonal with space group P3(2)21 and with one molecule in the asymmetric unit. Preliminary X-ray analysis of both native enzyme and its complexes indicate that the complexes diffract to higher resolution than the native crystals. This is probably due to reduced flexibility in the enzyme-inhibitor complex.

X-ray analyses of aspartic proteinases. V. Structure and refinement at 2.0 A resolution of the aspartic proteinase from Mucor pusillus.

The structure of mucor pusillus pepsin (EC 3.4.23.6), the aspartic proteinase from Mucor pusillus, has been refined to a crystallographic R-factor of 16.2% at 2.0 A resolution. The positions of 2638 protein atoms, 221 solvent atoms and a sulphate ion have been determined with an estimated root-mean-square (r.m.s.) error of 0.15 to 0.20 A. In the final model, the r.m.s. deviation from ideality for bond distances is 0.022 A, and for angle distances it is 0.050 A. Comparison of the overall three-dimensional structure with other aspartic proteinases shows that mucor pusillus pepsin is as distant from the other fungal enzymes as it is from those of mammalian origin. Analysis of a rigid body shift of residues 190 to 302 shows that mucor pusillus pepsin displays one of the largest shifts relative to other aspartic proteinases (14.4 degrees relative to endothiapepsin) and that changes have occurred at the interface between the two rigid bodies to accommodate this large shift. A new sequence alignment has been obtained on the basis of the three-dimensional structure, enabling the positions of large insertions to be identified. Analysis of secondary structure shows the beta-sheet to be well conserved whereas alpha-helical elements are more variable. A new alpha-helix hN4 is formed by a six-residue insertion between positions 131 and 132. Most insertions occur in loop regions: -5 to 1 (five residues relative to porcine pepsin): 115 to 116 (six residues); 186 to 187 (four residues); 263 to 264 (seven residues); 278 to 279 (four residues); and 326 to 332 (six residues). The active site residues are highly conserved in mucor pusillus pepsin; r.m.s. difference with rhizopuspepsin is 0.37 A for 25 C alpha atom pairs. However, residue 303, which is generally conserved as an aspartate, is changed to an asparagine in mucor pusillus pepsin, possibly influencing pH optimum. Substantial changes have occurred in the substrate binding cleft in the region of S1 and S3 due to the insertion between 115 and 116 and the rearrangement of loop 9-13. Residue Asn219 necessitates a shift in position of substrate main-chain atoms to maintain hydrogen bonding pattern. Invariant residues Asp11 and Tyr14 have undergone a major change in conformation apparently due to localized changes in molecular structure. Both these residues have been implicated in zymogen stability and activation.

X-ray analyses of peptide-inhibitor complexes define the structural basis of specificity for human and mouse renins.

X-ray analyses have defined the three-dimensional structures of crystals of mouse and human renins complexed with peptide inhibitors at resolutions of 1.9 and 2.8 A, respectively. The exquisite specificity of renin arises partly from ordered loop regions at the periphery of the binding cleft. Although the pattern of main-chain hydrogen bonding in other aspartic proteinase inhibitor complexes is conserved in renins, differences in the positions of secondary structure elements (particularly helices) also lead to improved specificity in renins for angiotensinogen substrates.

Crystallization and preliminary X-ray analysis of complexes of peptide inhibitors with human recombinant and mouse submandibular renins.

Inhibitor-complexed crystals of mouse and human renins suitable for X-ray analysis have been prepared. The mouse renin is complexed with a non-hydrolysable decapeptide analogue of rat angiotensinogen containing a hydroxyethylene isostere in place of the scissile bond. The crystals are monoclinic, space group P2(1) with cell dimensions a = 78.3 A, b = 117.8 A, c = 85.9 A, beta = 101.18 degrees containing four molecules per asymmetric unit. The human renin is fully glycosylated and complexed with a tetrapeptide containing norstatine. The complex crystallises in the cubic space group P2(1)3 with a = 143.1 A and has two molecules in the asymmetric unit. The rotation function of the mouse renin complex indicates pseudo 222 symmetry while that of human renin indicates a pseudo 2-fold axis. Full structural analyses of the two complexes are underway.