Linda J. Harris

Conformational Similarity and Systematic Displacement of Complementarity Determining Region Loops in High Resolution Antibody X-ray Structures

Comparison of seven high resolution x-ray structures shows that the conformations of canonical complementarity determining region (CDR) loops, which are shared by these antibodies, are very similar. However, large spatial displacements (up to 2.7 Å) of the essentially identical CDR loops become evident when the antibody β-sheet frameworks, to which the loops are attached, are least-squares superposed. The loop displacements follow, and amplify, small positional differences in framework/loop splice points. Intradomain structural variability and, to a lesser extent, domain-domain orientation appear to cause the observed loop divergences. The results suggest that the selection of framework regions for loop grafting procedures is more critical than previously thought. Immunoglobulin variable domains, VL 1 and VH, associate noncovalently to form the Fv, a dimer of antiparallel, eight-stranded β-sandwiches(1 2). The VL and VH β-sheets from different antibodies are nearly identical in three dimensions. However, the six complementarity determining region (CDR) loops (L1-L3, H1-H3), which connect the β-strands of the conserved framework and encode antigenic specificities, are much more variable in both sequence and conformation (2 3). Chothia, Lesk, and colleagues (4 5) identified sets of similar “canonical” conformations for all CDR loops except H3. Between 50 and 95% of antibody sequences are consistent with the classified canonical conformations(4), which are determined by conserved interactions of only a few key residues (“structural determinants”) within the loop and/or the framework regions. Some differences in the position of canonical CDR loops relative to superposed framework regions by comparing x-ray structures (4) or x-ray and modeled structures (5) were previously observed. However, these effects were observed in structures determined at medium resolution, generally considered minor(4 5), and not systematically explored. We have compared, via least-squares superpositions, the Fvs of seven x-ray structures refined to high resolution (better than 2 Å) available in the Brookhaven Protein Data Bank(6). It was anticipated that a systematic comparison of structures determined to such high precision would (i) shed light on the general relation between CDR loop conformation and position and (ii) help to assess the limitations of a procedure widely used in comparative model building, i.e. splicing of loops from a known x-ray structure onto the conserved structural scaffold of an antibody model(7 8). Fig. 1 shows a comparison (9) of the amino acid sequences of the seven antibodies, which include various heavy and light chains (both κ and ) from free as well as antigen-complexed antibodies. The 4-4-20 Fv, the highest resolution structure with κ light chain, was used as the template on which backbone segments of the other Fvs were superposed. Cumulative backbone root mean square (rms) deviations of the β-strands were determined after superposing each of these antibodies on the 4-4-20 Fv. Two alternative least-squares superpositions were used, employing different pairs of equivalence residues. The S1 set of residues consisted of only the most conserved regions of the Fvs, i.e. the four short 4-residue segments (10) of the central β-sheets. The S2 set consisted of a more extended set of residues and included the majority of the β-sheet framework (Fig. 1) akin to Stanfield et al.(11). As can be seen in Table 1 the cumulative backbone rms deviations were small and the results obtained with the two superposition sets were similar.