The X-ray structure of a ternary complex between human chorionic gonadotropin hormone (hCG) and two Fvs recognizing its α and β subunits has been recently determined. The Fvs recognize the elongated hCG molecule by its two ends, one being the Leu-12–Cys-29 loop of the α subunit. We have designed and synthesized a 17-amino-acid peptide (named PepH14) derived from the sequence of this antigenic loop with the purpose of mimicking its three-dimensional structure and its affinity for antibodies. We have determined the solution structure of PepH14 by homonuclear NMR spectroscopy and derived distance restraints. Comparison of this structure with that of the corresponding antigenic loop of α-hCG reveals strong conformational similarities. In particular, the two pairs of residues that establish crucial contacts with the Fv fragment share the same conformation in PepH14 and in the authentic hormone loop. We propose a three-dimensional model of interaction of PepH14 with a llama VHH (VHH-H14) fragment cloned from a single-chain llama immunoglobulin raised against α-hCG. This model has been constrained by the chemical shift variations of the H14 1HN and 15N resonances monitored upon binding with PepH14. Mapping of the backbone chemical shift variations on the VHH structure determined by NMR indicates that PepH14 binds to VHH-H14 and forms a complex using the three complementary determining regions (CDRs). They define a shallow groove encompassing residues Thr-31, Ala-56, Tyr-59 and Trp-104 which have been shown to be in conformational exchange [Renisio, Pérez, Czisch, Guenneugues, Bornet, Frenken, Cambillau and Darbon (2002) Proteins 47, 546–555] and also Phe-37 and Ala-50. This groove is close to the hydrophobic interface area observed between VH and VL domains in Fvs from classical antibodies, which explains the rather lateral binding of PepH14 on the VHH.
Abbreviations used: α-hCG, α subunit of human chorionic gonadotropin hormone; CDR, complementary determining region; DQF-COSY, double-quantum-filtered correlation spectroscopy; HSQC, heteronuclear single-quantum correlation; nOe, nuclear Overhauser effect; RMSD, root-mean-square distance.
These authors contributed equally to this work.
Present address: Laboratoire de Bioénergétique et Ingénierie des Protéines, UPR9036, CNRS-IBSM. 31 chemin Joseph Aiguier, 13402 Marseille CEDEX 20, France.