Crystallization of Fab from Immunoglobulins

Screening for Crystals:
Footprint Screen number 1 was designed mainly for antibodies:

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 he 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 conformat tional 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 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 app roach 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.

Review on crystallization and Structure determination:

22. Wilson I.A., Rini J.M., Fremont D.H. Fieser G.G., Stura E.A.(1991) Use of X-ray Diffraction analysis of free and antigen complexed Fab fragments in studying the structural basis of antigen-antibody interaction. [Review] Meth. Enz. 203: 270.

Use of PEG and salt combinations:

27. Stura E.A., Stanfield R.L., Rini J.M., Fieser G.G., Silver S., Roguska M., Hincapie L.M., Simmerman H.K.B., Profy A.T., Wilson I.A. (1992) Crystallization, Sequence and Preliminary Crystallographic data for an anti-peptide Fab 50.1 and peptide complexes with the principal neutralizing determinant of HIV-1. Proteins, 14, 499-508.

Abstract: X-ray quality crystals of an Fab fragment from an antipeptide monoclonal antibody (R/V3-50.1) that recognizes the principal neutralizing determinant (PND) of the gp120 glycoprotein of human immunodeficiency virus type 1 (HIV-1) (MN isolate) were grown as uncomplexed and peptide complexed forms. Crystals of the free Fab grew from high salt in orthorhombic space groups P2₁2₁2₁ and I222 and from polyethylene glycol in space groups P1 and P22₁. Seeds from either the P1 and P22₁ native (uncomplexed) Fab crystals induced nucleation of crystals of the Fab complexed to a 16-residue synthetic peptide corresponding to the PND when streak seeded into pre-equilibrated solutions of this complex. Data were collected from these complex crystals and from each of the four native Fab forms to at least 2.8 Å resolution. The genes for the variable domain of the Fab were cloned and sequenced and the primary amino acid sequence was deduced from this information. Knowledge of the were collected from these complex crystals and from each of the four native Fab forms to at least 2.8 Å resolution. The genes for the variable domain of the Fab were cloned and sequenced and the primary amino acid sequence was deduced from this information. Knowledge of the three-dimensional structure of this Fab-peptide complex will be important in the understanding of the PND of HIV-1 and its recognition by neutralizing monoclonal antibodies.

Crystallization of different cyclic peptides:

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.

Crystallization of Complexes with Steroids: Solubilization of low solubility compounds.

11. Stura E.A., Feinstein A., Wilson I.A. (1986) Crystallization and Preliminarycrystallographic data for an anti-progesterone monoclonal antibody Fab' and steroid-Fab' complexes. Journal of Molecular Biology. 193:299-231.

Abstract: Crystals of an Fab' from an antiprogesterone monoclonal antibody (IgG1) have been grown from 1.5 M-ammonium sulfate, pH 5.5 to 8.5. The single crystals are hexagonal rods, space group P6₂22 (or P6₄22), with cell dimensions a = b = 135.2 Å, and c = 124.2 Å. Cocrystals of the Fab' with progesterone, pregnanedione and other related steroids have been grown. The complex crystals have different morphology but are isomorphous with the native crystals. A hydroxy-progesterone derivative obtained by substituting an iodo-benzoyl group at the 11 a-hydroxyl position looks promising as a suitable heavy-atom candidate in addition to other potential conventional heavy-atom derivatives. All crystals diffract to at least 2.8 Å resolution and are suitable for high-resolution X-ray diffraction studies.

12. Stura E.A., Arevalo J.H., Feinstein A., Heap R.B., Taussig M.J., Wilson I.A. (1987) Analysis of an anti-progesterone antibody: variable crystal morphology of the Fab' and steroid-Fab' complexes. Immunology 62:511-521.

Abstract: Anti-progesterone monoclonal antibodies are being used for structural studies of antibody-antigen interaction, for their ability to block pregnancy shortly after fertilization, and for hormone immunoassay. A mouse anti-progesterone monoclonal Fab' fragment has been crystallized in its native form and co-crystallized with seven different, but structurally its native form and co-crystallized with seven different, but structurally related, steroids. The crystals show interesting preferences in their crystal morphology, depending on the bound steroid ligand. The X-ray crystallographic analysis of this Fab', complexed with a series of related steroid ligands, should reveal details of the chemistry of antibody-antigen union and provide insights into how steroids interact with proteins.

Fab peptide complexes:

16. Stura E.A., Stanfield R.L., Fieser T.M., Balderas, R.S., Smith, L.R., Lerner R.A., Wilson I.A. (1989) Preliminary Crystallographic Data and Primary Sequence for an Anti-peptide Fab' B13I2 and Its Complex with the C-helix Peptide from Myohemerythrin. Journal of Biological Chemistry 264:15721-15725.

Abstract: Crystals of the Fab' fragment from the monoclonal anti-peptide antibody B1312 and of the Fab'-peptide antigen complex have been characterized. The monoclonal antibodies were raised against a synthetic homologue of the C-helix of myohemerythrin (residues 69-87 in myohemerythrin). The Fab'-peptide complex crystallizes in space group P6₃22 with unit cell dimensions a = b = 142.5 Å, c = 101.5 Å, alpha = beta = 90°, gamma = 120°, and Z = 1. The native Fab' crystallizes in space group P2₁2₁2₁ with unit cell dimensions a = 98.0 Å, b = 151.7 Å, c = 80.8 Å, alpha = beta = gamma = 90°, and Z = 2. Both crystal forms diffract to beyond 2.6 Å resolution. We also report the cDNA and predicted amino acid sequences for the variable regions of both the light and heavy chains dimensions a = b = 142.5 Å, c = 101.5 Å, alpha = beta = 90°, gamma = 120°, and Z = 1. The native Fab' crystallizes in space group P2₁2₁2₁ with unit cell dimensions a = 98.0 Å, b = 151.7 Å, c = 80.8 Å, alpha = beta = gamma = 90°, and Z = 2. Both crystal forms diffract to beyond 2.6 Å resolution. We also report the cDNA and predicted amino acid sequences for the variable regions of both the light and heavy chains of this anti-peptide antibody.

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.

Catalytic Antibody crystallization:

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.

Andreas Heine has recently solved the structure of an Aldolase Abzyme