A single-domain antibody fragment in complex with RNase A: non-canonical loop structures and nanomolar affinity using two CDR loops.
BACKGROUND
Camelid serum contains a large fraction of functional heavy-chain antibodies - homodimers of heavy chains without light chains. The variable domains of these heavy-chain antibodies (VHH) have a long complementarity determining region 3 (CDR3) loop that compensates for the absence of the antigen-binding loops of the variable light chains (VL). In the case of the VHH fragment cAb-Lys3, part of the 24 amino acid long CDR3 loop protrudes from the antigen-binding surface and inserts into the active-site cleft of its antigen, rendering cAb-Lys3 a competitive enzyme inhibitor.


RESULTS
A dromedary VHH with specificity for bovine RNase A, cAb-RN05, has a short CDR3 loop of 12 amino acids and is not a competitive enzyme inhibitor. The structure of the cAb-RN05-RNase A complex has been solved at 2.8 A. The VHH scaffold architecture is close to that of a human VH (variable heavy chain). The structure of the antigen-binding hypervariable 1 loop (H1) of both cAb-RN05 and cAb-Lys3 differ from the known canonical structures; in addition these H1 loops resemble each other. The CDR3 provides an antigen-binding surface and shields the face of the domain that interacts with VL in conventional antibodies.


CONCLUSIONS
VHHs adopt the common immunoglobulin fold of variable domains, but the antigen-binding loops deviate from the predicted canonical structure. We define a new canonical structure for the H1 loop of immunoglobulins, with cAb-RN05 and cAb-Lys3 as reference structures. This new loop structure might also occur in human or mouse VH domains. Surprisingly, only two loops are involved in antigen recognition; the CDR2 does not participate. Nevertheless, the antigen binding occurs with nanomolar affinities because of a preferential usage of mainchain atoms for antigen interaction.
Decanniere
K
K
Laboratorium voor Ultrastructuur, Vlaams Interuniversitair Instituut voor Biotechnologie, Vrije Universiteit Brussel, Paardenstraat 65, B-1640 St. Genesius-Rode, Belgium. klaas@ultr.vub.ac.be
Desmyter
A
A
Lauwereys
M
M
Ghahroudi
M A
MA
Muyldermans
S
S
Wyns
L
L
D000595:Amino Acid Sequence; D000818:Animals; D000915:Antibody Affinity; D000918:Antibody Specificity; D000936:Antigen-Antibody Complex / Q000737:chemistry*; D000937:Antigen-Antibody Reactions; D001666:Binding Sites, Antibody; D002162:Camelus / Q000276:immunology*; D002417:Cattle; D018360:Crystallography, X-Ray; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry* / Q000276:immunology; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010179:Pancreas / Q000201:enzymology; D011487:Protein Conformation; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000276:immunology; D012984:Software; D013045:Species Specificity
D000936:Antigen-Antibody Complex; D007143:Immunoglobulin Heavy Chains; D012259:Ribonuclease, Pancreatic