Destabilizing loop swaps in the CDRs of an immunoglobulin V_L domain

Abstract

It is generally believed that loop regions in globular proteins, and particularly hypervariable loops in immunoglobulins, can accommodate a wide variety of sequence changes without jeopardizing protein structure or stability. We show here, however, that novel sequences introduced within complementarity determining regions (CDRs) 1 and 3 of the immunoglobulin variable domain REI VL can significantly diminish the stability of the native state of this protein. Besides their implications for the general role of loops in the stability of globular proteins, these results suggest previously unrecognized stability constraints on the variability of CDRs that may impact efforts to engineer new and improved activities into antibodies.Keywords: complementarity determining regions; hypervariable loops; immunoglobulin stability; protein stability; Stern-Volmer plots; unfolding In the structures of antibody heavy and light chain variable domains, complementarity determining region (CDR) loops of diverse sequence and conformation project from P-sandwich frameworks to determine the affinity and specificity of antigen binding. These diverse sequences arise in the development of an antibody response via a complex combination of gene rearrangements and somatic mutations (Branden & Tooze, 1991). Protein engineers have transferred antigen binding activity from one framework to another by swapping hypervariable CDR loops between frameworks (Jones et al., 1986;Riechmann et al., 1988). In addition, totally artificial loop sequences have been introduced into variable domain frameworks to generate novel binding functions (Barbas et al., 1993;Fisch et al., 1994). This apparent tolerance of the immunoglobulin fold for both sequence diversity and loop swaps is consistent with the view that the major determinants of structural stability of globular proteins lie in the sequence-specific formation and packing of regular secondary structure elements (Shortle, 1992;Matthews, 1993), whereas surface loops are more forgiving of sequence changes (EL Hawrani et al., 1994).Although the CDR loops differ greatly in sequence, they are not infinitely variable in either sequence or conformation. Positions with highly conserved amino acids are found within many CDR sequences (Kabat et al., 1991 of "canonical structures" appear to be available for each CDR. For example, in an examination of the structural database available in 1989, only four different loop conformations were identified in the light chain crystallographic database for CDRl, and only three for CDR3 . If these patterns d o suggest constraints on the sequence variability of CDRs, however, it remains difficult from examination of evolved antibodies to either gauge the severity of these constraints or to understand their structural or functional basis.In this report we describe the preparation of a number of mutants of the immunoglobulin light chain variable domain REI, in which wild-type CDR sequences were replaced by loops of differing sequence and length. These replacements cause dramat...

Sequence replacements in the central β‐turn of plastocyanin

“…Most substitutions are not tolerated, suggesting that sequence information in this turn plays a significant role in dictating the overall structure of the 0-barrel. These results are consistent with recent work by Helms and Wetzel(1995), who reported that substitutions in the hypervariable loops of an immunoglobulin V, domain destabilize the structure of that P-barrel as well. Mutagenesis studies of a &turn in staphylococcal nuclease have led to similar conclusions (R.O.…”

Functional Consequences of Insertions and Deletions in the Complementarity-determining Regions of Human Antibodies

“…4). Destabilizing effects of loop grafting into an antibody framework have been reported previously (40), but in that particular case, the grafted sequences were totally unrelated to antibody hypervariable loops. The use of naturally occurring CDR sequences for grafting into immunoglobulin frameworks often ensures that the inserted loops are optimally functional as they have been proofread and selected for functionality during the formation of the B cell receptors.…”

The Folding Pathway of the Antibody VL Domain

“…Apparent differences may be due to the nature of CDR loops that are, by necessity, different in all V L domains. A systematic study of the contribution of different CDR loops has not been carried out; however, significant differences in the stability of isolated V L domains have been observed with different length loops, 76 and replacement of the loops in one V L domain has significantly increased stability. 77 Loop regions can be important in the initial stages of protein folding, bringing different regions of the protein into close proximity.…”