The nature of antibody heavy chain residue H6 strongly influences the stability of a VH domain lacking the disulfide bridge.
Monoclonal antibody mAb 03/01/01, directed against the musk odorant traseolide, carries a serine residue instead of the conserved Cys H92 in the heavy chain variable domain, and is thus lacking the highly conserved disulfide bridge. We investigated the energetic consequence of restoring the disulfide bond and the nature of residue H6 (Glu or Gln), which is poised to interact with Ser H92 in the recombinant scFv fragment obtained from this antibody. In the scFv fragment derived from this antibody, the stabilizing effect of Gln H6 over Glu was found to be as large as the effect of reintroducing the disulfide bond. We have analyzed the conformation and hydrogen bond pattern of Gln H6 and Glu H6 in antibodies carrying these residues and suggest mechanisms by which this residue could contribute to VH domain stability. We also show that the unpaired cysteine H22 is buried, and conforms to the expected VH structure. The antibody appears to have acquired two somatic mutations (Ser H52 and Arg H66), which had been previously characterized as having a positive effect on VH stability. The overall domain stability is the decisive factor for generating functional, disulfide-free antibody domains, and several key residues play dominant roles.
Biochemisches Institut Universität Zürich, Winterthurerstrasse 190, Zürich, CH-8057, Switzerland.
A C
AC
Langedijk
A
A
Honegger
J
J
Maat
R J
RJ
Planta
R C
RC
van Schaik
A
A
Plückthun
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry; D006860:Hydrogen Bonding; D007128:Immunoglobulin Fragments / Q000737:chemistry; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry*; D007135:Immunoglobulin Variable Region / Q000737:chemistry*; D007189:Indans / Q000276:immunology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D012694:Serine / Q000737:chemistry; D014508:Urea
C110695:5-acetyl-1,1,2,6-tetramethyl-3-isopropylindane; D000911:Antibodies, Monoclonal; D004220:Disulfides; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D007189:Indans; D012694:Serine; D014508:Urea