1.85 A structure of anti-fluorescein 4-4-20 Fab

Area of Science:

• Structural Biology
• Immunology
• Crystallography

Background:

• High-affinity antibody-antigen complexes are crucial for immune response and therapeutic development.
• The 4-4-20 Fab fragment recognizes fluorescein with high affinity (Ka = 10^10 M-1).
• Previous structural data at lower resolution existed for this complex.

Purpose of the Study:

• To determine the high-resolution crystal structure of fluorescein bound to the 4-4-20 Fab fragment.
• To analyze the molecular interactions between fluorescein and the antibody's complementarity-determining regions (CDRs).
• To compare the new structure with previously reported lower-resolution data and identify structural differences.

Main Methods:

• X-ray crystallography was used to determine the structure of the fluorescein-4-4-20 Fab complex.
• Isomorphous crystals of two isoelectric forms of the antibody were grown.
• The crystal structure was refined to an R value of 0.188 at 1.85 A resolution using 26,328 unique reflections.

Main Results:

• The crystal complex of fluorescein bound to the 4-4-20 Fab was determined at 1.85 A resolution.
• Significant differences in backbone conformation were observed compared to a previously reported 2.7 A structure, particularly in the light chain CDR1.
• Root-mean-square deviations between variable and constant domains were 0.77 A and 1.54 A, respectively.

Conclusions:

• The high-resolution structure provides a detailed atomic model of fluorescein binding to the 4-4-20 antibody.
• Observed structural differences highlight the importance of resolution and data quality in interpreting antibody-antigen interactions.
• Further analysis of interactions between bound fluorescein, CDRs, and active-site mutants will be discussed.