Crystal structure of the OPG2 Fab. An antireceptor antibody that mimics an RGD cell adhesion site.

Cell surface receptors called integrins mediate diverse cell adhesion phenomena through recognition of the sequence arginine-glycine-aspartic acid (RGD) present in proteins such as fibronectin and fibrinogen. Platelet aggregation in hemostasis is mediated by the binding of fibrinogen to the gpIIb/IIIa integrin. The OPG2 antibody binds the gpIIb/IIIa receptor and acts as a ligand mimic due to the presence of an arginine-tyrosine-aspartic acid (RYD) sequence in the CDR3 loop of the heavy chain. The RYD loop and side chains are ordered in the 2.0-A resolution crystal structure of the Fab fragment from this antireceptor antibody. Moreover, the RYD loop assumes two clearly defined conformations that may correspond to the orientations of the loop in the free state or bound to integrin. This molecule will serve as a tool for understanding protein-integrin recognition in platelet aggregation and other RGD-mediated cell adhesion interactions.

Crystal structure of the tenth type III cell adhesion module of human fibronectin.

The crystal structure of the cell adhesion module of fibronectin (FNIII10) has been determined at 1.8 A resolution. A recombinant fragment corresponding to the tenth type III module of human fibronectin was crystallized in space group P2(1) with a = 30.7, b = 35.1 and c = 37.7 A and beta = 107 degrees. The structure was determined by molecular replacement and refined by least squares methods. The crystallographic R-factor for the final model of the 91 amino acid module plus 56 solvent atoms is 0.18 for 10 to 1.8 A data. The module consists of two layers of beta-sheet, one with three antiparallel strands and the other with four antiparallel strands. The beta-sheets enclose a hydrophobic core of 24 amino acid side-chains. The module contains the RGD cell recognition sequence in a flexible loop connecting two beta-strands. The tertiary structure of the FNIII10 module has been used to develop a structure-based sequence alignment of 17 type III modules in fibronectin based on the striking conservation of homologous hydrophobic residues. A similar pattern of homologous alternating hydrophobic residues is also evident in a comparison of type III modules in proteins unrelated to fibronectin such as cytokine receptors and muscle proteins.

X-ray-crystallographic studies of complexes of pepstatin A and a statine-containing human renin inhibitor with endothiapepsin.

H-189, a synthetic human renin inhibitor, and pepstatin A, a naturally occurring inhibitor of aspartic proteinases, have been co-crystallized with the fungal aspartic proteinase endothiapepsin (EC 3.4.23.6). H-189 [Pro-His-Pro-Phe-His-Sta-(statyl)-Val-Ile-His-Lys] is an analogue of human angiotensinogen. Pepstatin A [Iva(isovaleryl)-Val-Val-Sta-Ala-Sta] is a blocked pentapeptide which inhibits many aspartic proteinases. The structures of the complexes have been determined by X-ray diffraction and refined to crystallographic R-factors of 0.15 and 0.16 at resolutions of 0.18 nm (1.8 A) and 0.2 nm (2.0 A) respectively. H-189 is in an extended conformation, in which the statine residue is a dipeptide analogue of P1 and P'1 as indicated by the conformation and network of contacts and hydrogen bonds. Pepstatin A has an extended conformation to the P'2 alanine residue, but the leucyl side chain of the terminal statine residue binds back into the S'1 subsite, and an inverse gamma-turn occurs between P'1 and P'3. The hydroxy moiety of the statine at P1 in both complexes displaces the solvent molecule that hydrogen-bonds with the catalytic aspartate residues (32 and 215) in the native enzyme. Solvent molecules originally present in the native structure at the active site are displaced on inhibitor binding (12 when pepstatin A binds; 16 when H-189 binds).

Combining experimental information from crystal and solution studies: joint X-ray and NMR refinement.

Joint refinement of macromolecules against crystallographic and nuclear magnetic resonance (NMR) observations is presented as a way of combining experimental information from the two methods. The model of interleukin-1 beta derived by the joint x-ray and NMR refinement is shown to be consistent with the experimental observations of both methods and to have crystallographic R value and geometrical parameters that are of the same quality as or better than those of models obtained by conventional crystallographic studies. The few NMR observations that are violated by the model serve as an indicator for genuine differences between the crystal and solution structures. The joint x-ray-NMR refinement can resolve structural ambiguities encountered in studies of multidomain proteins, in which low- to medium-resolution diffraction data can be complemented by higher resolution NMR data obtained for the individual domains.

Direct observation by X-ray analysis of the tetrahedral "intermediate" of aspartic proteinases.

We report the X-ray analysis at 2.0 A resolution for crystals of the aspartic proteinase endothiapepsin (EC 3.4.23.6) complexed with a potent difluorostatone-containing tripeptide renin inhibitor (CP-81,282). The scissile bond surrogate, an electrophilic ketone, is hydrated in the complex. The pro-(R) (statine-like) hydroxyl of the tetrahedral carbonyl hydrate is hydrogen-bonded to both active-site aspartates 32 and 215 in the position occupied by a water in the native enzyme. The second hydroxyl oxygen of the hydrate is hydrogen-bonded only to the outer oxygen of Asp 32. These experimental data provide a basis for a model of the tetrahedral intermediate in aspartic proteinase-mediated cleavage of the amide bond. This indicates a mechanism in which Asp 32 is the proton donor and Asp 215 carboxylate polarizes a bound water for nucleophilic attack. The mechanism involves a carboxylate (Asp 32) that is stabilized by extensive hydrogen bonding, rather than an oxyanion derivative of the peptide as in serine proteinase catalysis.

Domain flexibility in aspartic proteinases.

Comparison of the three-dimensional structures of native endothiapepsin (EC 3.4.23.6) and 15 endothiapepsin oligopeptide inhibitor complexes defined at high resolution by X-ray crystallography shows that endothiapepsin exists in two forms differing in the relative orientation of a domain comprising residues 190-302. There are relatively few interactions between the two parts of the enzyme; consequently, they can move as separate rigid bodies. A translational, librational, and screw analysis of the thermal parameters of endothiapepsin also supports a model in which the two parts can move relative to each other. In the comparison of different aspartic proteinases, the rms values are reduced by up to 47% when the two parts of the structure are superposed independently. This justifies description of the differences, including those between pepsinogen and pepsin (EC 3.4.34.1), as a rigid movement of one part relative to another although considerable distortions within the domains also occur. The consequence of the rigid body movement is a change in the shape of the active site cleft that is largest around the S3 pocket. This is associated with a different position and conformation of the inhibitors that are bound to the two endothiapepsin forms. The relevance of these observations to a model of the hydrolysis by aspartic proteinases is briefly discussed.

Functional implications of interleukin-1 beta based on the three-dimensional structure.

The molecular structure of interleukin-1 beta, a hormone-like cytokine with roles in several disease processes, has been determined at 2.0 A resolution and refined to a crystallographic R-factor of 0.19. The framework of this molecule consists of 12 antiparallel beta-strands exhibiting pseudo-3-fold symmetry. Six of the strands make up a beta-barrel with polar residues concentrated at either end. Analysis of the three-dimensional structure, together with results from site-directed mutagenesis and biochemical and immunological studies, suggest that the core of the beta-barrel plays an important functional role. A large patch of charged residues on one end of the barrel is proposed as the binding surface with which IL-1 interacts with its receptor.

X-ray analyses of aspartic proteinases. III Three-dimensional structure of endothiapepsin complexed with a transition-state isostere inhibitor of renin at 1.6 A resolution.

The aspartic proteinase, endothiapepsin (EC 3.4.23.6), was complexed with a highly potent renin inhibitor, H-261 (t-Boc-His-Pro-Phe-His-LeuOHVal-Ile-His), where OH denotes a hydroxyethylene (-(S) CHOH-CH2-) transition-state isostere in the scissile bond surrogate. Crystals were grown in a form that has the same space group P2(1) as the uncomplexed enzyme, but with a 10 A decrease in the length of the alpha-axis and a 13 degrees decrease in the beta-angle. X-ray data have been collected to a resolution of 1.6 A. The rotation and translation parameters defining the position of the enzyme in the unit cell were determined previously using another enzyme-inhibitor complex that crystallized isomorphously with that of H-261. The molecule was refined using restrained least-squares refinement and the positions of non-hydrogen atoms of the inhibitor and water molecules were defined by difference Fourier techniques. The enzyme-inhibitor complex and 322 water molecules were further refined to a crystallographic R-factor of 0.14. Apart from a small rigid group rotation of a domain comprising residues 190 to 302 and small movements in the flap, there is little difference in conformation between the complexed and uncomplexed forms of the enzyme. The inhibitor is bound in an extended conformation along the active site cleft, and the hydroxyl group of the hydroxyethylene moiety is hydrogen-bonded to 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 a series of specificity pockets along the active site cleft. The study provides useful clues as to how this potent renin inhibitor (IC50 value of 0.7 x 10(-9) M) may bind renin. In particular it defines the interactions of the hydroxyethylene transition-state isostere with the enzyme more precisely than has been previously possible and therefore provides a useful insight into interactions in the transition state complex.

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.

High-resolution X-ray diffraction study of the complex between endothiapepsin and an oligopeptide inhibitor: the analysis of the inhibitor binding and description of the rigid body shift in the enzyme.

The conformation of the synthetic renin inhibitor CP-69,799, bound to the active site of the fungal aspartic proteinase endothiapepsin (EC 3.4.23.6), has been determined by X-ray diffraction at 1.8 A resolution and refined to the crystallographic R factor of 16%. CP-69,799 is an oligopeptide transition--state analogue inhibitor that contains a new dipeptide isostere at the P1-P1' position. This dipeptide isostere is a nitrogen analogue of the well-explored hydroxyethylene dipeptide isostere, wherein the tetrahedral P1' C alpha atom has been replaced by trigonal nitrogen. The inhibitor binds in the extended conformation, filling S4 to S3' pockets, with hydroxyl group of the P1 residue positioned symmetrically between the two catalytic aspartates of the enzyme. Interactions between the inhibitor and the enzyme include 12 hydrogen bonds and extensive van der Waals contacts in all the pockets, except for S3'. The crystal structure reveals a bifurcated orientation of the P2 histidine side chain and an interesting relative rotation of the P3 phenyl ring to accommodate the cyclohexyl side chain at P1. The binding of the inhibitor to the enzyme, while producing no large distortions in the enzyme active site cleft, results in small but significant change in the relative orientation of the two endothiapepsin domains. This structural change may represent the action effected by the proteinase as it distorts its substrate towards the transition state for proteolytic cleavage.

X-ray characterisation of an additional binding site in lysozyme.

Bromophenol red (BPR) binds to lysozyme and inhibits its activity against bacterial cell walls, but not against the polysaccharide component of peptidoglycan. The binding site of BPR in the enzyme has been characterised by X-ray analysis of the complex at 5.5A resolution. The new binding site, which is outside the cleft close to subsite F, is presumably involved in interactions with the peptide component of peptidoglycan, in the action of lysozyme against bacterial cell walls.