Crystal structure analysis of deamino-oxytocin: conformational flexibility and receptor binding.

Two crystal structures of deamino-oxytocin have been determined at better than 1.1A resolution from isomorphous replacement and anomalous scattering x-ray measurements. In each of two crystal forms there are two closely related conformers with disulfide bridges of different chirality, which may be important in receptor recognition and activation.

X-ray analyses of aspartic proteinases. II. Three-dimensional structure of the hexagonal crystal form of porcine pepsin at 2.3 A resolution.

The molecular structure of the hexagonal crystal form of porcine pepsin (EC 3.4.23.1), an aspartic proteinase from the gastric mucosa, has been determined by molecular replacement using the fungal enzyme, penicillopepsin (EC 3.4.23.6), as the search model. This defined the space group as P6522 and refinement led to an R-factor of 0.190 at 2.3 A resolution. The positions of 2425 non-hydrogen protein atoms in 326 residues have been determined and the model contains 371 water molecules. The structure is bilobal, consisting of two predominantly beta-sheet lobes which, as in other aspartic proteinases, are related by a pseudo 2-fold axis. The strands of the mixed beta-sheets (1N and 1C) of each lobe are related by an intra-lobe topological 2-fold symmetry. Two further beta-sheets, 2N and 2C, are each composed of two topologically related beta-hairpins folded below the 1N and 1C sheets. A further six-stranded sheet (3) spans the two lobes and forms a structure resembling an arch upon which the four other sheets reside. The interface between sheets 1N and 1C forms the catalytic centre consisting of absolutely conserved aspartate residues 32 and 215, which are shielded from solvent by a beta-hairpin loop (75 to 78). The crystal structure of a mammalian aspartic proteinase indicates that interactions with substrate may be more extensive on the prime side of the active site cleft than in the fungal enzymes and involve Tyr189 and the loop 290 to 295, perhaps contributing to the transpeptidase activity of pepsin and the specificity of the renins. Comparison with the high-resolution structure of pepsinogen gives a root-mean-square deviation of 0.9 A and reveals that, in addition to local rearrangement at the active site, there appears to be a rigid group movement of part of the C-terminal lobe of pepsin towards the cleft on activation. A large proportion of the absolutely conserved residues in aspartic proteinases are polar and buried. An examination of the pepsin structure reveals that these side-chains are involved in hydrogen-bond interactions with either the main chain of the protein or other conserved side-chains of the enzyme or propart.

Refinement of the solution structure of the B DNA hexamer 5'd(C-G-T-A-C-G)2 on the basis of inter-proton distance data.

A restrained least-squares refinement of the solution structure of the self-complementary B DNA hexamer 5'd(C-G-T-A-C-G)2 is presented. The structure is refined on the basis of 190 inter-proton distances determined by pre-steady-state nuclear Overhauser enhancement measurements. Two refinements were carried out starting from two initial B DNA structures differing by an overall root-mean-square (r.m.s.) difference of 0.32 A. In both cases, the final r.m.s. difference between the experimental and calculated inter-proton distances was 0.12 A compared to 0.61 A and 0.58 A for the two initial structures. The difference between the two refined structures is small, with an overall r.m.s. difference of 0.16 A, and represents the error in the refined co-ordinates. The refined structures have a B-type conformation with local structural variations in backbone and glycosidic bond torsion angles, and base-pair propellor twist, base roll, base tilt and local helical twist angles.

X-ray analyses of aspartic proteinases. The three-dimensional structure at 2.1 A resolution of endothiapepsin.

The molecular structure of endothiapepsin (EC 3.4.23.6), the aspartic proteinase from Endothia parasitica, has been refined to a crystallographic R-factor of 0.178 at 2.1 A resolution. The positions of 2389 protein non-hydrogen atoms have been determined and the present model contains 333 solvent molecules. The structure is bilobal, consisting of two predominantly beta-sheet domains that are related by an approximate 2-fold axis. Of approximately 170 residues, 65 are topologically equivalent when one lobe is superimposed on the other. Twenty beta-strands are arranged as five beta-sheets and are connected by regions involving 29 turns and four helices. A central sheet involves three antiparallel strands from each lobe organized around the dyad axis. Each lobe contains a further local dyad that passes through two sheets arranged as a sandwich and relates two equivalent motifs of four antiparallel strands (a, b, c, d) followed by a helix or an irregular helical region. Sheets 1N and 1C, each contain two interpenetrating psi structures contributed by strands c,d,d' and c',d',d, which are related by the intralobe dyad. A further sheet, 2N or 2C, is formed from two extended beta-hairpins from strands b,c and b',c' that fold above the sheets 1N and 1C, respectively, and are hydrogen-bonded around the local intralobe dyad. Asp32 and Asp215 are related by the interlobe dyad and form an intricate hydrogen-bonded network with the neighbouring residues and comprise the most symmetrical part of the structure. The side-chains of the active site aspartate residues are held coplanar and the nearby main chain makes a "fireman's grip" hydrogen-bonding network. Residues 74 to 83 from strands a'N and b'N in the N-terminal lobe form a beta-hairpin loop with high thermal parameters. This "flap" projects over the active site cleft and shields the active site from the solvent region. Shells of water molecules are found on the surface of the protein molecule and large solvent channels are observed within the crystal. There are only three regions of intermolecular contacts and the crystal packing is stabilized by many solvent molecules forming a network of hydrogen bonds. The three-dimensional structure of endothiapepsin is found to be similar to two other fungal aspartic proteinases, penicillopepsin and rhizopuspepsin. Even though sequence identities of endothiapepsin with rhizopuspepsin and penicillopepsin are only 41% and 51%, respectively, a superposition of the three-dimensional structures of these three enzymes shows that 237 residues (72%) are within a root-mean-square distance of 1.0 A.

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.

X-ray analyses of aspartic proteinases. IV. Structure and refinement at 2.2 A resolution of bovine chymosin.

The structure of calf chymosin (EC 3.4.23.3), the aspartic proteinase from the gastric mucosa, was solved using the technique of molecular replacement. We describe the use of different search models based on distantly related fungal aspartic proteinases and investigate the effect of using only structurally conserved regions. The structure has been refined to a crystallographic R-factor of 17% at 2.2 A resolution with an estimated co-ordinate error of 0.21 A. In all, 136 water molecules have been located of which eight are internal. The structure of chymosin resembles that of pepsin and other aspartic proteinases. However, there is a considerable rearrangement of the active-site "flap" and, in particular, Tyr75 (pepsin numbering), which forms part of the specificity pockets S1 and S1'. This is probably a consequence of crystal packing. Electrostatic interactions on the edge of the substrate binding cleft appear to account for the restricted proteolysis of the natural substrate kappa-casein by chymosin. The local environment of invariant residues is examined, showing that structural constraints and side-chain hydrogen bonding can play an important role in the conservation of particular amino acids.

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.

Crystallization of 5-aminolaevulinic acid dehydratase from Escherichia coli and Saccharomyces cerevisiae and preliminary X-ray characterization of the crystals.

5-Aminolaevulinic acid dehydratase (ALAD) catalyzes the formation of porphobilinogen from two molecules of 5-aminolaevulinic acid. Both Escherichia coli and Saccharomyces cerevisiae ALADs are homo-octameric enzymes which depend on Zn2+ for catalytic activity and are potently inhibited by lead ions. The E. coli enzyme crystallized in space group I422 (unit cell dimensions a = b = 130.7 A, c = 142.4 A). The best crystals were obtained in the presence of the covalently bound inhibitor laevulinic acid. The yeast enzyme (expressed in E. coli) crystallized in the same space group (I422) but with a smaller unit cell volume (a = b = 103.7 A, c = 167.7 A). High resolution synchrotron data sets were obtained from both E. coli and yeast ALAD crystals by cryocooling to 100 K.

Crystal structure of momordin, a type I ribosome inactivating protein from the seeds of Momordica charantia.

A type I ribosome-inactivating protein, extracted and purified from M. charantia seeds, was crystallised by vapour diffusion with polyethylene glycol at pH 7.2. X-ray data were collected to 2.1 A resolution and the structure solved by molecular replacement using the A-chain coordinates of ricin. The overall fold of the protein is similar to ricin but there are differences in secondary structure, on the surface and in the active site cleft. These differences are probably due in part to the evolution of the protein without a B-chain partner. The most extensive reorganisation occurs at the C-terminus whereas Tyr70 shows the greatest change in the active site cleft.

Adaptation of plasminogen activator sequences to known protease structures.

The sequences of urokinase (UK) and tissue-type plasminogen activator (TPA) were aligned with those of chymotrypsin, trypsin, and elastase according to their 'structurally conserved regions'. In spite of its trypsin-like specificity UK was model-built on the basis of the chymotrypsin structure because of a corresponding disulfide pattern. The extra disulfide bond falls to cysteines 50 and 111d. Insertions can easily be accommodated at the surface. As they occur similarly in both, UK and TPA, a role in plasminogen recognition may be possible. Of the functional positions known to be involved in substrate or inhibitor binding, Asp 97, Lys 143 and Arg 217 (Leu in TPA) may contribute to plasminogen activating specificity. PTI binding may in part be impaired by structural differences at the edge of the binding pocket.

The use of protein homologues in the rotation function.

The success of molecular replacement depends, in part, on the degree of similarity of the target and search molecules. We have systematically investigated this effect in cross-rotation functions for members of the aspartic proteinase family of enzymes. The influence of various parameters on peak heights was investigated for six search models using magnitude of F(obs) data for two target enzymes. The beneficial effects of high-resolution data and a large radius of integration are most pronounced when target and search molecules have high-percentage identities. Correction for small differences in domain-domain orientation (typically 4-8 degrees) between search and target structures leads to only a marginal improvement in the rotation-function peak height. There is an almost linear relationship between the structural distance, D, a parameter used in cluster analysis to define differences between three-dimensional protein structures, and the height of the cross-rotation-function peaks.

MOLPACK: molecular graphics for studying the packing of protein molecules in the crystallographic unit cell.

A graphics program, MOLPACK, has been developed on the Silicon Graphics IRIS-4D computer system for displaying the packing of proteins in the crystallographic unit cell. In addition to the normal viewing operations of rotation, translation and scaling, the program has the ability to translate molecules along the cell axes while maintaining their crystallographic equivalent positions within the unit cell. This allows the user to observe the packing of protein molecules generated by molecular replacement, to create a new packing model or to locate an unknown molecule. A special feature of the program is that up to four independent molecules can be manipulated in the asymmetric unit.

Rfree and the rfree ratio. II. Calculation Of the expected values and variances of cross-validation statistics in macromolecular least-squares refinement.

The free R factor is used routinely as a cross-validation tool in macromolecular crystallography. However, without any means of deriving quantitative estimates of its expected value and variance, its application has been rather subjective and its usefulness therefore somewhat limited. In the first part of this series, estimates of the expected value of the ratio of the free R factor to the standard R factor at the convergence of the structure refinement were given. Here, estimates of the variance of this ratio are given and are compared with the observed deviations from the expected values for a selection of refined structures. It is discussed how errors in the functional form of the structure-factor model as well as other types of errors might influence this ratio.

Rfree and the rfree ratio. I. Derivation of expected values of cross-validation residuals used in macromolecular least-squares refinement.

The last five years have seen a large increase in the use of cross validation in the refinement of macromolecular structures using X-ray data. In this technique a test set of reflections is set aside from the working set and the progress of the refinement is monitored by the calculation of a free R factor which is based only on the excluded reflections. This paper gives estimates for the ratio of the free R factor to the R factor calculated from the working set for both unrestrained and restrained refinement. It is assumed that both the X-ray and restraint observations have been weighted correctly and that there is no correlation of errors between the test and working sets. It is also shown that the least-squares weights that minimize the variances of the refined parameters, also approximately minimize the free R factor. The estimated free R-factor ratios are compared with those reported for structures in the Protein Data Bank.