PUBLICATIONS

30. Stura E.A., Matsumura M, Fremont D.H., Saito Y., Peterson P.A., Wilson I.A. (1992) Crystallization of multiple murine major histocompatibility complex class-I H-2K^b with single peptides. Journal of Molecular Biology., 228, 975-982.

Abstract: X-ray quality crystals of a soluble murine class I H-2Kb molecule complexed with three different peptide antigens were grown in several forms by streak seeding and macroseeding methods. Co-crystals with VSV-8 (RGYVYGQL), OVA-8 (SIINFEKL) and SEV-9 (FAPGNYPAL) peptides were grown either from NaH₂PO₄/HPO₄ or from polyethylene glycol 4000 within the pH range 5.0 to 7.5, with the use of 4-methyl-2-pentane diol (MPD) as an additive. The VSV-8 crystals grew in space groups P1, with cell dimensions a = 63.1 Å, b = 69.1 Å, c = 72.0 Å, alpha = 89.9°, beta = 77.1°, gamma = 123.3° and P2₁2₁2, with a = 138.1 Å, b = 88.6Å, c = 45.7 Å, and diffract to 2.9 and 2.3 Å, respectively. Crystals of the SEV-9 complex grew from similar crystallization conditions to those of the orthorhombic VSV-8 complex with similar cell parameters and diffract to at least 2.5 Å resolution. Crystals of the OVA-8 complex were obtained from either phosphate (space group C2, a = 118.7 Å, b = 61.6 Å, c = 85.3 Å, beta = 108.4°) or polyethylene glycol (space group P1, a = 64.5 Å, b = 71.0 Å, c = 66.3 Å, alpha = 89.7°, beta = 95.7°, gamma = 123.3°) and diffract to 3 Å resolution. The crystallization procedures used here significantly increased the rate and production of X-ray quality crystals.

31. Fremont D.H, Matsumura M., Stura E.A., Peterson P.A. and Wilson I.A. (1992) Crystal structures of two viral peptides in complex with murine MHC class I H-2K^b. Science, 257, 919-927. Comment in: Science 1992 Aug 14;257(5072):880-881

Abstract: The x-ray structures of a murine MHC class I molecule (H-2K^b) were determined in complex with two different viral peptides, derived from the vesicular stomatitis virus nucleoprotein (52-59), VSV-8, and the Sendai virus nucleoprotein (324-332), SEV-9. The H-2K^b complexes were refined at 2.3 Å for VSV-8 and 2.5Å or SEV-9. The structure of H-2K^b exhibits a high degree of similarity with human HLA class I, although the individual domains can have slightly altered dispositions. Both peptides bind in extended conformations with most of their surfaces buried in the H-2K^b binding groove. The nonamer peptide maintains the same amino- and carboxy-terminal interactions as the octamer primarily by the insertion of a bulge in the center of an otherwise beta conformation. Most of the specific interactions are between side-chain atoms of H-2K^b and main-chain atoms of peptide. This binding scheme accounts in large part for the enormous diversity of peptide sequences that bind with high affinity to class I molecules. Small but significant conformational changes in H-2K^b are associated with peptide binding, and these synergistic movements may be an integral part of the T cell receptor recognition process.

1993

32. Arevalo J.H., Stura E.A., Taussig M.J., Wilson I.A. (1993) Three-dimensional structure of an anti-steroid Fab' and progesterone Fab' complex. Journal of Molecular Biology. 321, 103-108.

Abstract: The monoclonal anti-progesterone antibody DB3 binds progesterone with nanomolar affinity (K_a approximately 10⁹ M^-1) suggesting high specificity. However, DB3 also cross-reacts with similar affinity with a subgroup of structurally distinct, progesterone-like steroids. Crystals of the unliganded Fab' and various steroid-Fab' complexes are isomorphous and belong to the hexagonal space group, P6₄22, with unit cell dimensions of a = b = 135 Å, c = 124 Å. Structures of free and progesterone-bound Fab' have been determined by X-ray crystallography at 2.7 Å resolution using molecular replacement techniques. Progesterone is bound in a hydrophobic pocket formed mainly by the interaction of three complementarity determining regions L1, H2 and H3. The orientation of the ligand in the binding site was aided by both crystallographic and biochemical analyses of substituted steroids. The indole side-chain of TrpH100 of the DB3 has two different conformations, inter-converting ``open'' and ``closed'' forms of the antibody combining site. The TrpH100 indole thus appears to be acting as an antibody-derived surrogate ligand for its own hydrophobic binding site was aided by both crystallographic and biochemical analyses of substituted steroids. The indole side-chain of TrpH100 of the DB3 has two different conformations, inter-converting ``open'' and ``closed'' forms of the antibody combining site. The TrpH100 indole thus appears to be acting as an antibody-derived surrogate ligand for its own hydrophobic binding pocket. These structures provide the first atomic view of how a steroid interacts with a protein and offer a structural explanation for the restriction of the anti-progesterone response to the VGAM3.8 family of VH genes.

33. Stura E.A., Fieser G.G., Wilson I.A. (1993) Crystallization of Antibodies and Antibody-Antigen Complexes. Immunomethods 3, 164-179.

Abstract: Although many antibodies have been crystallized, the number of structures determined in both their complexed and unliganded forms remains relatively small. With the recent improvements in the use of molecular replacement (MR), the structure determination of Fabs and Fab complex structures can proceed more rapidly, but crystallization often remains a major obstacle. Substantial improvements in methodologies have helped with the success rate in the crystallizations of Fabs and Fab-antigen complexes that are beyond previous expectations. Crystallization and structure determination has been directed mainly towards Fab fragments. The reason for this choice remains linked both to the ease with which the structure of Fabs can be determined and to the difficulties that have been presented by the crystallization of whole immunoglobulins. Such difficulty is currently believed to be due to flexibility or conformational heterogeneity of the IgG as well as the added heterogeneity from the glycosylation of the Fc fragment. Fabs share some of the same problems mainly because of the degree of heterogeneity that is the result of the proteolytic cleavage used to fragment the immunoglobulins, the flexibility in elbow regions and in some cases from glycosylation. A systematic approach to the cleavage, purification, and analysis of the resultant product can yield immunoglobulin fragments amenable to crystallization. A rational screening of crystallization conditions with extensive use of seeding can in most cases enable progress from small microcrystalline aggregates to large X-ray quality crystals. Such methodologies have become so effective that Fabs are now being used as tools to aid in the crystallization of other molecules which have been found difficult to crystallize by themselves.

34. Stura E.A., Kang, A.S, Stefanko, R.S., Calvo, J, Gaarder, K.L, Kaslow D.C., and Satterthwait, A.C. (1993) Crystallographic studies of transmission blocking antimalaria Fab 4B7 with cyclic and linear peptides from the Pfs25 protein of Plasmodium falciparum. American Journal of Tropical Medicine and Hygiene 49:Suppl.(137)-177.

Abstract: X-ray quality crystals of a transmission-blocking monoclonal antibody 4B7 (MAb 4B7) against a sexual stage protein Pfs25 of Plasmodium falciparum have been obtained. Both the intact immunoglobulin and an elastase produced Fab fragment have been crystallized both free and complexed with cyclic and linear peptides. While MAb 4B7 binds a linear peptide, cyclic peptides modeled on a predicted b-hairpin loop of the third EGF-like domain of Pfs25 bind better and are readily co-crystallized with the Fab. Several cyclic peptides and their linear counterparts have been synthesized. The affinity of MAb 4B7 for the different peptides varies widely. X-ray data have been collected from various peptide-complexed and free Fab crystals. The packing arrangement of the Fab in three independent crystal forms has been determined by molecular replacement and refinement of the structures is in progress. The genes for the variable domain of the Fab have been cloned, sequenced and the primary amino acid sequence deduced. The three-dimensional structure will aid in an understanding of the mode by which this antibody recognizes and prevents transmission of the parasite. The system presents unique opportunities to understand neutralization from the comparison between the bound forms of differently constrained cyclic peptides and the linear counterparts and from the comparison of the free and antigen-bound MAb. The study is being extended to include the structure determination of Pfs25 and its complex with MAb 4B7.

1994

35. Haynes M.R., Stura E.A., Hilvert, D., Wilson I.A. (1994) Crystallization and Preliminary Structural studies of a chorismate mutase catalytic antibody complexed with a transition state analogue. Proteins, 18,198-200.

Abstract: The Fab' fragment of a catalytic antibody with chorismate mutase activity has been crystallized as a complex with the transition-state analog hapten. The complex was crystallized by the vapor diffusion method using ammonium sulfate as the precipitant. The crystals belong to the orthorhombic space group P2₁2₁2₁ with unit cell dimensions a = 37.1 Å, b = 63.3 Å, c = 178.5 Å, and there is one Fab' molecule per asymmetric unit. The crystals diffract X-rays to at least 3.0 Å and are suitable for X-ray crystallographic studies.

36. Haynes M.R., Stura E.A., Hilvert, D., Wilson I.A. (1994) Routes to catalysis: Structure of a catalytic antibody and comparison with its natural counterpart. Science, 263:646-652.

Abstract: The three-dimensional structure of a catalytic antibody (1F7) with chorismate mutase activity has been determined to 3.0 Å resolution as a complex with a transition state analog. The structural data suggest that the antibody stabilizes the same conformationally restricted pericyclic transition state as occurs in the uncatalyzed reaction. Overall shape and charge complementarity between the combining site and the transition state analog dictate preferential binding of the correct substrate enantiomer in a conformation appropriate for reaction. Comparison with the structure of a chorismate mutase enzyme indicates an overall similarity between the catalytic mechanism employed by the two proteins. Differences in the number of specific interactions available for restricting the rotational degrees of freedom in the transition state, and the lack of multiple electrostatic interactions that might stabilize charge separation in this highly polarized metastable species, are likely to account for the observed 10⁴ times lower activity of the antibody relative to that of the natural enzymes that catalyze this reaction. The structure of the 1F7 Fab'-hapten complex provides confirmation that the properties of an antibody catalyst faithfully reflect the design of the transition state analog.

37. Arevalo J.H., Hassig C.A., Stura E.A., Sims M.J., Taussig M.J., Wilson I.A. (1994) Structural analysis of antibody specificity: detailed comparison of five Fab'-steroid complexes. Journal of Molecular Biology, 241:663-690

Abstract: Structures of the Fab' fragment of the anti-progesterone antibody DB3 in complex with five cross-reactive steroids (aetiocholanolone, 5 beta-androstane-3,17-dione, 5 alpha-pregnane-20-one-3b-ol-hemisuccinate, progesterone-11a-ol-hemisuccinate and progesterone) have been determined by X-ray crystallography to a maximum resolution of 2.7 Å. These different steroids compete with progesterone binding with affinities in the nanomolar range despite substantial differences in their three-dimensional structures. Comparison of the steroid ligands bind to an ``open'' conformation of the Fab' as defined by the orientation of the indole side-chain of TrpH100, whereas in the unliganded or ``closed'' form the binding site is occluded by TrpH100. Small but significant conformational changes take place in the antibody to maximize the physical and chemical complementarity with each ligand. The various cross-reactive ligands are accommodated in the binding site in two distinct orientations. We term these binding modes syn and anti, as they are defined by the orientation of the steroid b face relative to TrpH50. In all cases, the steroid D ring is inserted into a hydrophobic cavity formed mainly by TrpH50, TyrH97, TrpH100 and PheH100b; a hydrogen bond interaction with AsnH35 to the keto group at position C17 or C20 orients the steroid in the pocket. The AsnH35 hydrogen bond and the interaction with TrpH50 account for the restricted heavy chain response to immunization with progesterone-like steroids derivatized at the 11 a position. Cross-reactivity of the antibody with different steroids is explained by alternative binding pockets for the A ring, which generates different ligand orientations in the binding site. This study suggests orients the steroid in the pocket. The AsnH35 hydrogen bond and the interaction with TrpH50 account for the restricted heavy chain response to immunization with progesterone-like steroids derivatized at the 11 a position. Cross-reactivity of the antibody with different steroids is explained by alternative binding pockets for the A ring, which generates different ligand orientations in the binding site. This study suggests which factors are most likely to contribute to the observed antibody specificity, such as linker position and the paucity of functional groups on the immunogenic hapten.

38. Stura E.A, Satterthwait, A.C, Calvo, J.C, Kaslow, D.C, Wilson, I.A. (1994) Reverse Screening. Acta Cryst. D50 : 448-455.

Abstract: A major emphasis has been placed in recent years on ``kits'' for screening crystallization conditions of macromolecules. Such approaches have undoubtedly speeded up the initial screening and to a certain extent helped in reducing the protein required for the initial survey. Factorial screening techniques, either full factorial or sparse matrix approaches, have proved successful in the crystallization of many proteins. In cases where the amount of protein is limited a systematic approach based on a priori choice of precipitant may be preferable to an extensive search. The approach described here targets such situations.The approach consists of an initial search to determine the solubility characteristics of the macromolecule under study as a function of precipitant and macromolecule concentrations. This information is used to define a working range for the precipitant and protein concentrations. Changes in solubility as a function of pH and as a result of the introduction of additives are evaluated at a later stage. This analysis is important for establishing conditions under which the protein is highly supersaturated and hence more conducive to nucleation, one of the dominant problems that hinders successful crystallization of proteins. When ligands are available for the formation of macromolecular complexes, the screening of the different complexes of a particular macromolecule is advocated as a means to increasing the probability of nucleation. Solubility information derived from one complex that crystallizes may be used to aid in the crystallization of other complexes. Cross-seeding between complexes is an intrinsic part of the method and provides an efficient way of obtaining crystals where spontaneous nucleation is hard to achieve.

39. Stura E.A., Kang, A.S., Stefanko, R.S., Calvo, J.C., Kaslow, D.C. and Satterthwait, A.C. (1994) Crystallization, sequence and preliminary crystallographic data for transmission blocking anti-malaria Fab 4B7 with cyclic peptides from the Pfs25 protein of P. falciparum. Acta Cryst. D50 , 535-542.

Abstract: X-ray quality crystals of a Fab fragment from a transmission-blocking monoclonal antibody 4B7 (MAb 4B7) that was formed against a sexual stage protein Pfs25 of Plasmodium falciparum were grown as uncomplexed and peptide complexed forms. Because cleavage with pepsin or papain produced a non-homogeneous product, the whole immunoglobulin was crystallized (Stura et al. companion article1). Subsequently, a Fab fragment was obtained using the protease, elastase. The best complex crystals were obtained using cyclic peptide from the third EGF-like domain of Pfs25, where the N and C termini are joined with a covalent hydrogen-bond mimic. MAb 4B7 binds a linear peptide, however, the cyclic peptide shows a significant increase in binding affinity. An initial data set, complete to 3.0Å, has been collected from these complex crystals. The packing arrangement of the Fab in the crystals has been determined by molecular replacement and refinement of the structure is in progress. The genes for the variable domain of the Fab were cloned, sequenced and the primary amino acid sequence deduced. Knowledge of the three-dimensional structure of this Fab-peptide complex will be important in the understanding the mode by which this antibody recognizes and prevents transmission of the parasite.

40. Stura E.A, Satterthwait, A.C, Calvo, J.C, Stefanko, R.S, Langeveld, J.P, Kaslow, D.C (1994) Crystallization of an intact antibody(4B7) against P. falciparum with peptides from the Pfs25 protein antigen. Acta Cryst. D50 , 556-562.

Abstract: Monoclonal antibody 4B7 is a neutralizing antibody against the protein Pfs25 from the sexual stage of the malaria parasite Plasmodium falciparum. Here we report the determination of the fine specificity of MAb 4B7 within the linear epitope of Pfs25 and the crystallization of the intact murine monoclonal antibody with peptides from the Pfs25 antigen. This study highlights the importance of ligands in the crystallization of proteins. In this case peptides have been used to modulate the solubility of the peptide-IgG complex and may have provided different or additional crystal contacts to create or enhance a crystalline reticulum not favoured by the uncomplexed IgG. Multiple crystal forms characterize this crystallization and the various peptides differing both in length and sequence have been used to investigate how small changes can have a strong effect on nucleation and crystal growth.

41. Diller T.C., Shaw A, Stura E.A., Vacquier V.D., Stout C.D. (1994) Acid pH crystallization of the basic protein lysin from the spermatozoa of red abalone (Haliotis rufescens). Acta Cryst. D50 : 620-626.

Abstract: A new crystal form of dimeric red lysin, a distinctly basic protein (Mr = 16070) from the red abalone (Haliotis rufescens), has been obtained using ammonium sulfate as a precipitant with sodium citrate-boric acid-citric acid buffered pH 4.5. The acid pH crystal form resulted from a study aimed at developing conditions favorable to sitting drop vapour diffusion crystallization of other abalone lysins which do not crystallize at neutral or basic pH conditions. The space group is P222₁ with cell parameters a = 51.2, b = 47.0, c = 128.8 Å with two molecules per asymmetric unit.

42. Satterthwait A.C., Cabezas E., Calvo J.C., Chen, S.Q., Wu J.X., Wang P.L., Xie Y.L., Stura E.A., Kaslow, D.C. (1995) A peptide mimic as antigen and Immunogen. In Peptides: Biology and Chemistry, Proceedings of the Chinese 1994 Peptide Symposium. Lu, G.S. (ed.) ESCOM, Leiden, the Netherlands, pp. 229.

43. Stura E.A., Kaslow, D.C. and Satterthwait A.C. (1994) Crystallization of a neutralizing malaria immunoglobulin with linear and cyclic peptides In Peptides: Biology and Chemistry, Proceedings of the 13th American Peptide Symposium. Hodges, R., Smith, J.A. (eds.) ESCOM, Leiden, the Netherlands, pp 817.

44. Ghiara J.B., Stura E.A., Stanfield R.L. Profy A.T., Wilson I.A. (1994) Crystal Structure of the Principle Neutralizing Site of HIV-1. Science 264: 82-85.

Abstract: The crystal structure of a complex between a 24-amino acid peptide from the third variable (V3) loop of human immunodeficiency virus-type 1 (HIV-1) gp 120 and the Fab fragment of a broadly neutralizing antibody (59.1) was determined to 3 angstrom resolution. The tip of the V3 loop containing the Gly-Pro-Gly-Arg-Ala-Phe sequence adopts a double-turn conformation, which may be the basis of its conservation in many HIV-1 isolates. A complete map of the HIV-1 principal neutralizing determinant was constructed by stitching together structures of V3 loop peptides bound to 59.1 and to an isolate-specific (MN) neutralizing antibody (50.1). Structural conservation of the overlapping epitopes suggests that this biologically relevant conformation could be of use in the design of isolates. A complete map of the HIV-1 principal neutralizing determinant was constructed by stitching together structures of V3 loop peptides bound to 59.1 and to an isolate-specific (MN) neutralizing antibody (50.1). Structural conservation of the overlapping epitopes suggests that this biologically relevant conformation could be of use in the design of synthetic vaccines and drugs to inhibit HIV-1 entry and virus-related cellular fusion.

45. Churchill M.E. Stura E.A. Pinilla C. Appel J.R. Houghten R.A. Kono D.H. Balderas R.S. Fieser G.G. Schulze-Gahmen U. Wilson I.A. (1994) Crystal structure of a peptide complex of anti-influenza peptide antibody Fab 26/9. Comparison of two different antibodies bound to the same peptide antigen. Journal of Molecular Biology. 241, 534-556.

Abstract: The three-dimensional structure of the complex of a second anti-peptide antibody (Fab 26/9) that recognizes the same six-residue epitope of an immunogenic peptide from influenza virus hemagglutinin (HA1; 75-110) as Fab 17/9 with the peptide has been determined at 2.8 Å resolution. The amino acid sequence of the variable region of the 26/9 antibody differs in 24 positions from that of 17/9, the first antibody in this series for which several ligand-bound and free structures have been determined and refined. Comparison of the 26/9-peptide with the 17/9-peptide complex structures shows that the two Fabs are very similar (r.m.s.d. 0.5 to 0.8 Å) and that the peptide antigen (101-107) has virtually the same conformation (r.m.s.d. 0.3 to 0.8 Å) when bound to both antibodies. A sequence difference in the 26/9 binding pocket (L94; His in 26/9, Asn in 17/9) results in an interaction with a bound water molecule that is not seen in the 17/9 structures. Epitope mapping shows that the relative specificity of 26/9 and 17/9 antibodies for individual positions of the peptide antigen are slightly different. Amino acid substitutions in the peptide, particularly at position SerP107, are tolerated to different extents by 17/9 and 26/9. Structural and sequence analysis suggests that amino acid differences near the peptide-binding site are responsible for altering slightly the specificity of 26/9 for three peptide residues and illustrates how amino acid substitutions can modify antibody-antigen interactions and thereby modulate antibody specificity.

46. Wilson I.A., Stanfield R.L., Jewell D.A., Ghiara J.B., Fremont D.H, Stura E.A. (1994) Immune recognition of viral antigens. Infectious Agents and Disease 3,155-162.

Abstract: The response exhibited by the immune system to viral and other foreign antigens consists of antibody-mediated and T cell-mediated immunity. Structural and molecular biological studies have shown that the antibody response is tailored to provide exquisite specificity by generating binding pockets that are complementary in shape as well as in charge to the antigen. On the other hand, the cellular response uses T-cell receptors (TCRs) and the major histocompatibility complex (MHC) antigens. Structural information on the TCRs is not yet available, but the crystal structures of several MHC class I molecules have shown how one MHC molecule can bind many different peptide sequences that share only the common anchor residue positions that determine allele specificity. MHC class I interactions with the peptide backbone at the N and C termini explain the high specificity of the binding groove for peptide ligands and suggest a universal mode of recognition for peptides to MHC class I molecules. Peptide-MHC class II interactions are less well understood, although recent structural work has shown important differences in the binding clefts of MHC class I and II that lead to longer peptides being bound to class II molecules. Detailed analysis at the molecular level has indicated that conformational changes in both antibodies and MHC molecules occur upon antigen binding.

1995

47. Shalk I.J., Stura E.A., Matteson J., Wilson, I.A., Balch E.E. (1994) Crystallization and preliminary crystallographic data for Rab guanine nucleotide dissociation inhibitor (RabGDI) from Bovine Brain. Journal of Molecular Biology, 244, 469-473.

Abstract: X-ray quality crystals of Rab guanine nucleotide dissociation inhibitor (RabGDI) from bovine brain expressed in Escherichia coli have been obtained from 1.73 M ammonium sulfate. The crystals are prismatic long rods and belong to the monoclinic space group P21 with approximate cell dimensions a = 91.9 Å, b = 43.5 Å, c = 63.2 Å, beta = 104.5° and one molecule per asymmetric unit. The crystals are stable in the X-ray beam and diffract to at least 2.3 Å. Reverse screening, streak seeding and macroseeding methods were used to obtain and improve the crystals.

48. Fremont D.H, Stura E.A., Matsumura M., Peterson P.A. and Wilson I.A. (1995) Crystal structure of an H-2K^b-ovalbumin peptide complex reveals interplay of primary and secondary anchor positions in the major histocompatibility complex binding groove. Proceedings of the National Academy of Sciences of the United States of America. 92:2479-2483.

Abstract: Sequence analysis of peptides naturally presented by major histocompatibility complex (MHC) class I molecules has revealed allele-specific motifs in which the peptide length and the residues observed at certain positions are restricted. Nevertheless, peptides containing the standard motif often fail to bind with high affinity or form physiologically stable complexes. Here we present the crystal structure of a well-characterized antigenic peptide from ovalbumin [OVA-8, ovalbumin-(257-264), SIINFEKL] in complex with the murine MHC class I H-2K^b molecule at 2.5 Å resolution. Hydrophobic peptide residues Ile-P2 and Phe-P5 are packed closely together into binding pockets B and C, suggesting that the interplay of peptide anchor (P5) and secondary anchor (P2) residues can couple the preferred sequences at these positions. Comparison with the crystal structures of H-2K^b in complex with peptides VSV-8 (RGYVYQGL) and SEV-9 (FAPGNYPAL), where a Tyr residue is used as the C pocket anchor, reveals that the conserved water molecule that binds into the B pocket and mediates hydrogen bonding from the buried anchor hydroxyl group could not be likewise positioned if the P2 side chain were of significant size. Based on this structural evidence, H-2K^b has at least two submotifs: one with Tyr at P5 (or P6 for nonamer peptides) and a small residue at P2 (i.e., Ala or Gly) and another with Phe at P5 and a medium-sized hydrophobic residue at P2 (i.e., Ile). Deciphering of these secondary submotifs from both crystallographic and immunological studies of MHC peptide binding should increase the accuracy of T-cell epitope prediction.

49.Wien M.W, Filman, D.J, Stura, E.A, Guillot, S, Delpeyroux, Crainic, R, Hogle, M. (1995) Structure of the complex between the Fab fragment of a neutralizing antibody for type 1 poliovirus and its viral epitope. Nature, Structural Biology 2, 232-243.

Abstract: The crystal structure of the complex between the Fab fragment of C3, a neutralizing antibody for poliovirus, and a peptide corresponding to the antibody was originally raised to heat inactivated (noninfectious) virus particles, it strongly neutralizes the Mahoney strain of type 1 poliovirus. Eleven peptide residues are well-defined in the electron-density map and form two type I b-turns in series. At the carboxyl end, the peptide is bound snugly in the antibody-combining site and adopts a conformation that differs significantly from the structure of the corresponding residues in the virus. Structural comparisons between he peptide in the complex and the viral epitope suggests that on binding to infectious virions, this antibody may induce structural changes important for neutralization.

50. Stura E.A, Ghosh S, Garcia-Junceda E, Chen L, Wong C-H, Wilson I.A. (1995) Crystallization and preliminary crystallographic data for class I Deoxy-5-Phosphate aldolase from Escherichia coli: An Application of Reverse Screening. Proteins, 22, 67-72.

Abstract: X-ray quality crystals of class I deoxyribose-5-phosphate aldolase from Escherichia coli have been obtained for the unliganded enzyme and in complex with its substrate, 2-deoxyribose-5-phosphate. The enzyme catalyzes the reversible cleavage of 2-deoxyribose-5-phosphate to acetaldehyde and D-glyceraldehyde-3-phosphate.The unliganded and complex crystals are prismatic long rods and belong to the orthorhombic space group P2₁2₁2₁ with cell dimensions a = 183.1 Å, b = 61.4 Å, c = 49.3 Å and a = 179.2 Å, b = 60.5, Å, c = 49.1 Å, respectively. Two molecules in the asymmetric unit are related by a non-crystallographic 2-fold axis. The crystals are stable in the X-ray beam and diffract to at least 2.6 Å. A new method, reverse screening, designed to minimize protein utilization during the screening process was used to determine supersaturation and crystallization conditions.

51. Klein C. Chen P. Arevalo J.H. Stura E.A. Marolewski A. Warren M.S. Benkovic S.J. Wilson I.A. (1995) Towards structure-based drug design: crystal structure of a multisubstrate adduct complex of glycinamide ribonucleotide transformylase at 1.96 Å resolution. Journal of Molecular Biology. 249. 53-75.

Abstract: An inhibitor complex structure of glycinamide ribonucleotide transformylase (GAR-Tfase; EC 2.1.2.2) from Escherichia coli has been determined with a multisubstrate adduct BW1476U89 to an R-value of 19.1% at 1.96 Å resolution. The structure was determined by a combination of molecular and single isomorphous replacement using data from two different monoclinic crystal lattices and collecting data from crystals soaked in 20% (w/v) methyl-pentanediol as cryoprotectant for shock-freezing at -150o C. The multisubstrate adduct is bound in an extended crevice at the interface between the two functional domains of the enzyme. This inhibitor is positioned in the binding site by three sets of tight interactions with its phosphate, glutamate and pyrimidone ring moieties, while its intervening linker atoms are more flexible and adopt two distinct sets of conformations. The highly conserved Arg103, His108 and Gln170 residues that are key in ligand binding and catalysis (His108), have compensatory conformational variation that gives some clues as to their role in substrate specificity and in the formyl transfer. The molecular design of 1476U89 as a multisubstrate adduct inhibitor (K_i approximately 100 pM at pH 8.5), is confirmed as it closely mimics the shape, molecular interaction and combined binding constants of the natural 10-formyltetrahydrofolate (10-CHO-H4F; Km approximately 77.4 mM at pH 8.5) and glycinamide-ribonucleotide (GAR; K_m approximately 8.1 mM at pH 8.5) substrates. The stereochemistry of this ligand complex suggests that His108 may act as an electrophile stabilizing the oxyanion of the tetrahedral intermediate that is formed as a result of the direct attack on the 10-CHO-H4F by the amino group of GAR. Structural comparison of the folate binding modes among GAR-Tfase, dihydrofolate reductase and thymidylate synthase reveals that folate derivatives bound to GAR-Tfase differentially adopt the trans conformation for the dihedral angle between atoms C-6 and C-9 providing a handle for targeting specific folate-dependent enzymes. The structural information derived from two different discrete conformations of the ligand in the binding site also suggests several leads for the de novo design of inhibitors of GAR-Tfase that may develop into useful chemotherapeutic agents.

52. Livnah, O., Stura, E. A., Johnson, D.L., Mulcahy, L.S., Wrighton, N.C., Dower, W.J., Jolliffe, L.K. and Wilson I. A. (1996) Functional mimicry of a protein hormoneby a peptide agonist: The EPO receptor at 2.8 Å. Science 273:464-471.

Abstract: The 2.8Å structure of the extracellular domain of the EPO receptor with the RWJ-61233 peptide reveals the first active complex between a class-I cytokine receptor and a synthetic naturally-unrelated ligand. The complex consists of two receptors and two peptide molecules that interact forming an almost perfect two-fold dimer. Unlike the hGH-hGHR complex, which is the other active system of the cytokine receptor family, the two EPO-receptor molecules hardly interact with each other, and the EPOR-peptide assembly is mainly mediated by the two peptides. Although different dimer configurations and ligand size and shape, the two receptor systems share a similar construct of binding determinants. The analysis of the crystal structure provides valuable data towards a design of a non peptidic anemia drug, and supports the importance of receptor dimerization as a mechanism for signal induction.

53. Shalk, I., Zeng, K., Wu, S.-K., Stura, E.A., Matteson J., Huang M., Tandon, A., Wilson, I.A. and Balch, W.E. (1996) Structure and mutational analysis of Rab GDP-dissociation inhibitor. Nature 381:42-48.

Abstract: The crystal structure of the bovine alpha-isoform of guanine nucleotide dissociation inhibitor (GDI), which functions in vesicle membrane transport to recycle and regulate Rab GTPases, has been determined to a resolution of 1.81 Å. GDI is constructed of two main structural units, a large complex multi-sheet domain I and a smaller a-helical domain II. The fold organization of domain I is surprisingly closely related to FAD-containing mono-oxygenases and oxidases. Sequqnce-conserved regions comon to GDI and the choroideremia gene product, which delivers Rab to catalytic subunits of Rab geranyltransferase II, are clustered on one face of the molecule.The two most sequqnce-conserved regions, which form a compact structure at the apex of GDI, are shown by site directed mutagenesis to play a critical role in the binding of Rab proteins.

54. Stura, E. A., Ruf, W., Wilson I. A. (1996) Crystallization and preliminary crystallographic data for a ternary complex between tissue factor, factor VIIa and a BPTI-derived inhibitor. J. Cryst. Growth 168:260-269.

Abstract: The binding of tissue factor (TF) with the serine protease coagulation factor VIIa (VIIa) is the initial trigger for activation of the coagulation cascades. In complex with TF, VIIa has greatly enhanced proteolytic function in the activation of the natural substrate factors X and IX. A structural understanding of the interaction of VIIa with TF and how VIIa can be inhibited may help in the development of an anticoagulant strategy. Here we report the screening and identification of crystallization conditions to produce diffraction quality crystals of the complex between TF-VIIa and a potent inhibitor (5L15) derived from bovine pancreatic trypsin inhibitor (BPTI) by random mutagenesis and selection by phage display. The crystals were obtained from the soluble extracellular domain of tissue factor, expressed in Escherichia coli as a fusion protein, VIIa expressed in mammalian cells and recombinant 5L15. Because only 1.5 mg of complex were available for this work, a reverse screening based strategy was used in the search and optimization of the crystallization conditions. Two different crystal forms were obtained from polyethylene glycol 4,000 and monomethyl polyethylene glycol 2,000 with cacodylate buffer at pH 6.5 in the presence of sodium and calcium ions. The addition of magnesium, zinc and other divalent metals has profound effects on the crystallization. Both crystal forms are trigonal with cell parameters a = b = 129.3 Å, c=100.8 Å and a = b = 67.4 Å, c=317.1 Å diffracting to 6 Å and 3 Å resolution with one molecule in the asymmetric unit. Complete data sets have been collected from each of these to the resolution to which the crystals diffract.

55. Prasad, G.S., Stura, E.A., McRee D.E., Stout, C. D. and Elder, J. H. (1996) Crystal Structure of FIV dUTP pyrophosphatase. Protein Science 5:2429-2437.

Abstract: The crystal structure of the dUTP pyrophosphatase (dUTPase) from the feline immunodeficiency virus (FIV) has a similar fold to the E. coli enzyme in which the C-terminal strand of an anti-parallel beta-sandwich participates in the beta-sheet of an adjacent subunit to form an interdigitated, biologically functional trimer. dUTPase hydrolyzes dUTP to dUMP and pyrophosphate and by maintaining a low cellular concentration of dUTP the enzyme prevents the incorporation of uracil into DNA, which if unchecked leads to numerous strand breaks due to excessive excision repair by uracil-DNA-glycosylase. As an essential enzyme of nucleotide metabolism dUTPase is a potential target for drug design. The structure provides a basis for understanding the substrate specificity and the Mg²⁺ dependence and the enzymatic mechanism of dUTPase. Three crystal forms of FIV dUTPase have been obtained using recombinant protein expression in both E. coli and baculovirus (monomer 15 kDa, 134 residues). The structure has been solved by SIRAS using the P6₃ crystal form containing two monomers per asymmetric unit along the 6₃ screw axis. Data for this form have been collected to 1.8Å at 90oK. The magnesium position at a site along the 6₃ axis is deduced from the position of the samarium, gadolinium and ytterbium derivatives. A second crystal form, P212121 with one trimer in the asymmetric unit was grown in the presence of dUDP, an inhibitor of the enzyme. Another organization of the dUTPase trimers is found in a third crystal form, P6₁, with 12 monomers arranged as a dimer of dimers of trimers.

56. Prasad, G.S., McRee D.E., Stura, E.A., Levitt, D.G. Lee, H.C. and Stout, C. D. (1996) Crystal structure of Aplysia ADP ribosyl cyclase, a homologue of the bifunctional ectozyme CD38. Nature Strutural Biology 3:957-964

Abstract: ADP ribosyl cyclase synthesizes the novel secondary messenger cyclic ADP ribose (cADPR) from NAD via the intermediate ADP ribose (ADPR). cADPR is an endogenous regulator of calcium induced calcium release. ADP ribosyl cyclase is highly homologous with two extra cellular antigens and enzymes associated with lymphocyte differentiation, CD38 and BST-1. The crystal structure reveals that the enzyme has two domains separated by a large cleft and connected by a hinge region. The N-terminal domain is comprised of a five helical bundle, contains three disulfides and represents a unique fold; the C-terminal domain contains two disulfides and a five stranded, parallel b-sheet flanked by helices. In two crystal forms ADP ribosyl cyclase exists as a dimer; the cleft of each monomer is oriented towards the other, creating a cavity at the dimer interface. The structure of the dimer provides a model for CD38 and an explanation for the ability of CD38 to hydrolyze as well as synthesize cADPR. Residual electron density in two distinct binding pockets, one associated with each domain, suggests that the enzyme catalyzes the formation of cADPR by displacement of nicotinamide in one pocket and cyclization of ADPR in the other, requiring diffusion of the intermediate between sites within the monomer or dimer.

57. He. M., Gani, M., Livnah, O., Stura, E.A., Beale, D., Coley. J., Wilson. I.A., Taussig, M.J. (1997) Sequence, specificity and crystallization of an oestrone-3-glucuronide antibody (3910) Immunology 90: 632-639. We describe the specificity profile and V region sequences of a high-affinity monoclonal antibody (mAb), 3910, directed against oestrone-3-glucuronide (E3G). Inhibition studies show that the D-ring is critical for steroid specificity, while the glucuronic acid attached to the A ring is required for high binding affinity, suggesting that both "ends' of the E3G ligand are recognized. The VH domain is encoded by a gene from the VH7183 family, while VL appears to be encoded by the Vk5.1 gene (kappa II subgroup) with a deletion of six residues from complementarity-determining region-1 (CDR1). The VH CDR3 is 10 amino acid residues in length, of which D/N contributes five residues. Comparison of VH CDR of 3910 with those of mAb against progesterone (DB3) and digoxin (26-10, 40-50), for which crystal structures have been determined, suggests that aromatic side chains are important for E3G binding and that tyrosine residues H50, H97 and H100 may interact with the ligand. The Fab fragment of 3910 has been crystallized in its native and steroid (E3G and oestriol-3-glucuronide) complexed forms. An X-ray diffraction data set to 3 A resolution has been collected for the native Fab. 58. Satterthwait, A.C., Cabezas, E., Calvo, J.C., Wu, J.X., Chen, S.Q., Kaslow, D.C., Livnah, O., and Stura, E.A., (1996) Constrained Synthetic Peptide Vaccines in "Peptides: Chemistry, Structure & Biology" Proceedings of the 14th American Peptide Symposium. Kaumaya P.T.P., Hodges, R.S. eds. pp. 772-773.

Abstract: A critical issue involves the development of a strategy for identifying constrained peptide vaccines. We systematically vary the size and cadence of natural peptide sequences constrained with hydrogen mimics while using neutralizing monoclonal antibodies to identify the best mimetic. Since every other amino acid in protein engages in a hydrogen bond and most hydrogen bonds in proteins are localized, many potential conformations can be screened. Here we report on the use of this strategy for the identification of a constrained peptide corresponding to an epitope on Pfs25, a protein found on malaria sexual stages and recognized by a neutralizing monoclonal antibody, Mab 4B7.

1997 and in preparation

59. DiScipio R.G. and Stura, E.A. (1997) Crystallization of human complement component C5. Acta Cryst. D (in preparation).

Abstract: Human complement C5 has been crystallized using a low salt batch technique. Hexagonal bipyramidal crystals of over 1.5 mm in each dimension have been obtained. The crystals are stable to dissolution in aqueous buffers and under up to 2M NaCl. The space group has been tentatively identified as P622 (screw axis not determined) with cell parameters a = b = 158.9 Å, c = 260.5 Å. The resolution to which the crystals diffract is limited to 6Å at best.

60. Stura, E.A., Livnah, O., Johnson, D., Jolliffe, L. K., Mulcahy, L.S., Wilson, I.A., and Satterthwait, A.C. (1997) Design of a peptide for the crystallization of the erythropoietin receptor: Preliminary crystallographic report. Acta Cryst D. (Prepared)

Abstract: Erythropoietin (EPO) plays a critical role in stimulating the proliferation and differentiation of erythroid precursor cells. EPO is heavily glycosylated with three asparagine N-linked tetraantennary oligosaccharides and a single serine O-linked oligosaccharide. Removal of the N-linked sugars results in low solubility and a strong tendency to aggregate. Although we were able to obtained complex crystals of fully glycosylated Epo with the extra-cellular portion of its receptor expressed in Escherichia coli (EPO-binding protein EBP), these crystals which are tetrahedral pyramids up to 0.5 mm in size do not diffract. We report here the approach taken to design a specific peptide for the crystallization of EBP and its successful completion. Crystals that diffract to up to 3.0Å, belonging to the space group C2, with cell parameters a = 168.6 Å, b = 61.0 Å, c = 110.5 Å, beta = 109.4° are grown from ammonium sulfate. The structure is being determined by molecular replacement.

61. Stura, E. A., Livnah, O., Johnson, D.L., Mulcahy, L.S., Wrighton, N.C., Dower, W.J., Jolliffe, L.K. and Wilson I. A. (1997) Crystallization of an active complex between the extracellular domain of the human erythropoietin receptor and an agonist peptide. Acta Cryst. D. (in preparation).

Abstract. The external domain of the erythropoietin receptor expressed in Escherichia coli has been crystallized in complex with synthetic cyclic agonist peptides. The crystals have been obtained in three different orthorhombic crystal forms either from ammonium sulfate or using a combination of monomethyl ether polyethylene glycol 5,000, polyethylene glycol 600 and sodium chloride as co-precipitants. The three crystal forms display virtually identical cell dimensions but are morphologically and crystallographically distinct. The use of reverse screening and streak seeding have been instrumental in the analysis of the crystallization properties of the many peptide complexes. The three crystals forms diffract to 3.3 Å, 2.4 Å and 2.2 Å resolution with cell parameters P22₁2₁, a = 59.4 Å, b = 74.9 Å, c = 137.6 Å; P212121, a = 59.2 Å, b = 75.5 Å, c = 132.2 Å; and P2₁2₁2₁, a = 58.8 Å, b = 75.0 Å, c = 130.5 respectively. In each case the asymmetric unit contains 2 molecules with the two-fold axis relating them closely aligned with the c-axis. The crystallization, data collection and heavy atom search have been carried out simultaneously with synergism, utilizing information which had been acquired previously in the crystallization of the receptor with linear peptides.

62. Stura, E.A., Diller, T.C., Prasad, G.S., Hasselkus-Light, C., Laco, G.S., Stout, C.D. and Elder, J.H. (1997) Crystallization and preliminary crystallographic studies of Feline Immunodeficiency Virus dUTPase in the absence and in complex with dUMP. FEBS Lett. (In Preparation)

Abstract: Crystals of the feline immunodeficiency virus that diffract to 1.8Å resolution, and crystals in the presence of the inhibitor, dUDP that diffract to 2.3Å have been obtained. A second crystal form, which is orthorhombic containing one trimer per asymmetric unit have been grown in the presence of the natural inhibitor dUDP. The results show that the enzyme is an interdigitated trimer, with marked structural similarity to E. Coli dUTPase. With respect to viral protein expression, it is particularly interesting that DU is encoded in the middle of the Pol gene, the enzyme cassette of the retroviral genome. Other enzymes encoded in the Pol polyprotein, including protease (PR), reverse transcriptase (RT), and most likely integrase (IN) are dimeric enzymes, which implies that the stochiometry of expression of active trimeric DU is distinct from the other Pol-encoded enzymes. The P63 crystals have enabled the structure to be solved by the SIRAS method. The molecules pack in the crystal form as a dimer of trimers, one trimer on the 63 screw axis and one trimer on the 3-fold axis). For two HG derivatives, the figure of merit was 0.67 and 2.7 Å resolution (phasing power 2.90 and 2.97).

63. Zeng, Z., Castaño, A.R., Segelke, B.W., Stura E.A., Peterson P.A. and Wilson I.A. (1997) Crystal structure of murine CD1 reveals a hydrophobic closed binding groove. Science. (submitted)

Abstract: The three-dimensional structure of murine CD1 has been determined in its empty form. Despite its low level of homology and the prediction from secondary structural studies, the overall structure of murine CD1 (mCD1) is similar to that of MHC class I molecules. It folds into the classical three domains that associate with b2m. The a1 and a2 fold ina an analogous manner to MHC class I forming a platform of eight anti parallel b-sheets and two lines of a-helices. All secondary structure elements of MHC class I are conserved in CD1 except the H1 helix of a1 which deforms into a coil. As a result a little different structure is generated with a quite different binding groove. The overall structure and in particular the a2 domain is most similar to the neonatal Fc receptor (FcRn) while other domains considered individually are better related to either MHC class I and class II. The structure maintains a ligand binding groove, or elongated pocket, lined by hydrophobic residues, 18Å at the entrance, 6-7Å wide and a maximal longitudinal distance of 28Å In mCD1 the binding pocket delineates a surface area of 966Å2 the most extensive area of any antigen presenting molecule. The topology of the groove does not appear to be the most appropriate for peptide binding, however the peptide binding motif defined for mCD1 matches the biochemical characteristics of the pocket with the anchor residues at positions P1, P7 and a long aliphatic residue at position P4. Although there appears to be a discrepancy between the length of the peptide and the size of the groove, an empty MHC molecule is presumably very different from a peptide filled complex, and it may be possible that conformational changes upon interaction with appropriate peptides would open the binding groove in such a way that long peptides are bound. Overall mCD1 is most related to class I MHC than class II or FcRn.

64. Livnah, O., Wang, P.L., Wilson I.A., Kaslow, D.C., Satterthwait, A.C. and Stura, E.A., (1996) Anti-malaria transmission blocking: Crystal structures of Fab 4B7 unbound and complexed with cyclic peptides. Science. (In preparation)

65. Satterthwait, A.C., Cabezas, E., Calvo, J.C., Wu, J.X., Chen, S.Q., Kaslow, D.C., Livnah, O., and Stura, E.A., (1996) Constrained Synthetic Peptide Vaccines in ``Peptides: Chemistry, Structure & Biology'' Proceedings of the 14th American Peptide Symposium. Kaumaya P.T.P., Hodges, R.S. eds. pp. 772-773.

66. Stanfield, R.L., Cabezas, E., Satterthwait, A.C., Stura, E.A., Profy, A.,Wilson I.A. (1997) Structures of linear and cyclic peptides in complex with an HIV-1 neutralizing Fab. (In preparation)

Abstract: Antibodies raised against peptides corresponding in sequence to the principal neutralizing determinant of HIV-1 gp120, neutralize the virus and inhibit syncytia formation. Thus, these peptides may be of use as vaccines or anti-HIV-1 drugs. In an effort to develop a useful peptide-based vaccine, we have tried to design constrained peptides that mimic the conformation of the loop in solution. Crystal structures have been determined for Fab 58.2, a broadly neutralizing antibody, in complex with a linear and cyclic peptide containing the sequence of the MN isolate of the principal neutralizing determinant from gp120. The peptide conformation is very similar in the linear and cyclic forms. However, this conformation differs from that seen for the same linear peptide bound to a different neutralizing antibody, Fab 59.1.

67. Heine, A., Stura, E.A., Fieser, G.G., Rosenblum, J., Cravatt, B., Lerner R.A., Wilson I.A. (1997) Crystallization, sequence and preliminary structural studies structural studies of a syn elimination catalytic antibody obtained by a new approach in Fab purification (In preparation)

Abstract: A Fab fragment of a monoclonal antibody (1D4), which catalyzes a disfavored syn elimination reaction has been crystallized with and without its transition state analogue (TSA). The Fab was purified with protein A followed by protein G resulting in a fraction of high purity, which can be crystallized readily under various PEG conditions. The two crystal forms are monoclinic P21 with cell parameters a = 71.22, b = 67.90, c = 93.35, b = 100.19o and a = 70.50, b = 48.76, c = 67.55, b = 103.06 respectively. Complete data sets to 3Å have been collected for both crystal forms in its free and hapten bound states. The genes encoding the 1D4 light and heavy chains of the variable domains have been cloned and sequenced

68. Stura E.A. (1997) Seeding Techniques. In "Crystallization of Nucleic Acids and Proteins: a practical approach" Second Edition (A. Ducruix and G.Gieg� eds.) IRL Press, Oxford, in revision.

69. Stura E.A. (1997) Soaking of Crystals. In "Crystallization of Nucleic Acids and Pro teins: a practical approach" Second Edition (A. Ducruix and G.Gieg� eds.) IRL Press, Oxford, in revision.

Abstract : Once crystals of a macromolecule are obtained there are many circumstances were it is necessary to change the environment in which the macromolecule is bathed. Such changes include the addition of inhibitors, activators, substrates, products and heavy atoms to the bathing solution to achieve their binding to the macromolecule, which may have sufficient freedom to undergo some conformational changes in response to these effectors. In fact, macromolecular crystals have typical ly a high solvent content which ranges from 27% to 95% (1,2), and although, part of this solvent, "bound solvent", (typically 10%) is tightly associated with the protein matrix consisting of both water molecules and other ions that occupy well defined positions in refined crystal structure it can be be replaced in soaking experiments, at a slower rate compared to the "free solvent". In this chapter we will consider the relative merits of various methods for modifying crystals, the restaints that the lattice may impose on the macromolecule and the relative merits of soaking com pared to cocrystallization.

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D. Phil Thesis: Glycogen Phosphorylase b, Oxford University

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1997 and in preparation

Last Modified: June 12, 1997