Anti-Haemaggluttinin antibodies:

Fab 17/9:

Fab 26/9:

Fab 17/9:


Crystallization:

13. Schulze-Gahmen U., Rini J.M., Arevalo J.H., Stura E.A., Kenten J.H. and Wilson I.A. (1988)

Preliminary crystallographic data, primary sequence, and binding data for an anti-peptide Fab and its complex with a synthetic peptide from influenza virus hemagglutinin.

Journal of Biological Chemistry 263:17100-17105.

Abstract: X-ray quality crystals which diffract to high resolution (less than or equal to 1.9-2.1 Å) have been grown of an anti-peptide Fab and its complex with a 9-residue peptide antigen. Both crystals are monoclinic P21, with unit cell dimensions a = 90.3 Å, b = 82.9 Å, c = 73.4 Å, beta = 122.5° for the native Fab and a = 63.9 Å, b = 73.0 Å, c = 49.1 Å, beta = 120.6° for the complex. The peptide sequence corresponds to residues 100-108 of all influenza virus hemagglutinins (HA1) of the H3 subtype (1968-1987). The peptide antigen has been well characterized immunologically (Wilson, I.A., Niman, H.L., Houghton, R.A., Cherenson, A.R., Connolly, M.L., and Lerner, R.A. (1984) Cell 37, 767-778; Wilson, I.A., Bergmann, K.F., and Stura, E.A. (1986) in Vaccines '86 (Channock, R.M., Lerner, R.A., and Brown, F., eds) pp. 33-37, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), structurally, as a free peptide by NMR (Dyson, J.H., Cross, K.J., Houghton, R.A., Wilson, I.A., Wright, P.E., and Lerner, R.A. (1985) Nature 318, 480-483; Dyson, J.H., Lerner, R.A., and Wright, P.E., (1988) Annu. Rev. Biophys. Chem. 17, 305-324), as part of the intact antigen by x-ray crystallography (Wilson, I.A., Skehel, J.J., and Wiley, D. C. (1981) Nature 289, 366-373) and by binding studies to the HA molecule (White, J.M., and Wilson, I.A. (1987) J. Cell Biol. 105, 2887-2896). Knowledge of the three-dimensional structure of the complex will elucidate the details of how anti-peptide antibodies complex will elucidate the details of how anti-peptide antibodies recognize a small peptide antigen and provide insights into the recognition of the same sequence in the intact protein antigen. As both native Fab and the peptide-Fab complex have been crystallized, we can also determine in addition whether changes in the structure of the antibody accompany antigen binding. The nucleotide sequence of the mRNA coding region of the anti-peptide Fab has been determined to provide the amino acid sequence ultimately required for the high resolution three-dimensional structure determination.

Crystallization conditions Peptide sequence Cell parameters Resolution (Å)
30-39% PEG 600, 200 mM imidazole malate, pH 5.6-6.5 None P21 a = 90.3 Å b = 82.9 Å c = 116.0 Å, beta = 122.5° 1.9
7-12% PEG 600, 200 mM imidazole malate, 100mM NaCl, pH 5.6-6.5 YDVPDYASL P21 a = 63.9 Å b = 73.0 Å c = 49.1 Å, beta = 120.6° 2.1


Structure:

The Structure of the Native Fab was determined by Jim Rini:

23. Wilson I.A. Stanfield R.L. Rini J.M. Arevalo J.H. Schulze-Gahmen U. Fremont D.H. Stura E.A. (1991)

Structural aspects of antibodies and antibody-antigen complexes. [Review]

Ciba Foundation Symposium. 159:13-28; discussion 28-39.


Structural Evidence for Induced Fit as a Mechanism for Antibody-Antigen Recognition. Rini, J.M., Schulze-Gahmen, U. and Wilson, I.A. (1992) Science, 255, 959-965. [Abstract].

Fab 26/9:

Crystallization & Structure:

Crystallization conditions Peptide sequence Cell parameters Resolution (Å)
17% PEG 10,00, 200 mM imidazole malate, pH 7.0-9.0 YDVPDYASL P212121 a = 72.0 Å b = 103.5 Å c = 66.0 Å 2.6
17% PEG 10,000, 200 mM imidazole malate, pH 7.0-9.0 DVPDYASL P21212121 a = 66.6 Å b = 115.1 Å c = 73.3 Å 2.8
17% PEG 10,000, 100 mM imidazole malate, pH 7.0-8.3 DVPDYASL Form 2: Orthorhombic a = 66.4 Å b = 103.2 Å c = 73.1 Å 2.8
14-17% PEG 10,000, 100 mM imidazole malate, pH 6.5-8.0 YPYDVPDYASLRS Orthorhombic a = 73.6 Å b = 104.4 Å c = 67.0 Å 2.8


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.