Structural basis for chemically-induced homodimerization of a single domain antibody

Chemically-induced dimerization (CID) systems are essential tools to interrogate and control biological systems. AcVHH is a single domain antibody homo-dimerizing upon caffeine binding. AcVHH has a strong potential for clinical applications through caffeine-mediated in vivo control of therapeutic gene networks. Here we provide the structural basis for caffeine-induced homo-dimerization of acVHH.

Tyr104 (N7-methyl) and the methyl group of threonines Thr101 and Thr101′. These interactions features explain the observed specificity against closely related biomolecules such as theophylline or guanine. The structure was refined to resolution limit of 2.25 Å, and many water molecules were identified. Among them, 12 were present in all the four independent dimers interface of the asymmetric crystallographic unit (Fig. 2  -area B). Caffeine-induced dimerization engulfs complex network of water molecules that are shielded from the bulk solvent. The ligand is in direct contact with 2 water molecules (see above) and these water molecules are themselves connected with 2 water molecules and the lateral chain of the both Ser35 and Ser35′ (Fig. 2 -area B).  The VHH/VHH interaction surface comprises a hydrophobic surface of 850 A 2 that comprise the residues Phe39, Phe49, Met63, Tyr61, Tyr108 and Trp111 from each monomer. The hydrophobic packing (composed of Phe37, Tyr100B, and Trp103) around the boundaries of the CDR3 is conserved among VHH antibodies and has been described as important for VHH domain stability 10 . In addition, the VHH/VHH interaction surface also comprises residue forming H-bounds and 2 salt-bridges (Arg40, Glu46, Glu48 and Ser65) (Fig. 2 -area C).
One remarkable difference between acVHH and conventional VHHs is its short CDR3 loop region. It is composed of only 10 amino acids, which is far shorter than the average 16 amino acid camelid CDR3 11 (Fig. 3). Therefore, the short CDR3 loop of AcVHH do not to shields this hydrophobic surface like in conventional VHHs 12 . As a consequence, the acVHH residues Phe39, Phe49, Tyr108 and Trp111 become solvent accessible and are able to interact within a dimer (Fig. 2 -area C).
We further investigated the conformational flexibility of the system by performing atomistic, explicit solvent Molecular Dynamics (MD) simulations of the acVHH monomer and of the apo and caffeine-bound homo-dimers. Surprisingly, the caffeine-binding CDR loop is rather rigid in VHH monomer in this timescale and inter-chain interactions and/or the presence of the caffeine do not further hamper the limited conformational dynamics of VHH, with the exception of few tyrosine (Tyr34, Tyr61, Tyr104). For the caffeine-binding homodimer simulations, the most relevant structural fluctuations correspond to a transient yet sizable opening of the VHH/VHH interaction area, which suggest that other regions of the interface, such as caffeine/VHH interaction surface and water molecule cavity, may ultimately be more relevant for stabilizing the overall dimeric architecture.
This study provides a deeper understanding of the structural determinant for caffeine-induced dimerization of acVHH. AcVHH binding mode is different from previously known anti-hapten-VHH structures (i.e. PDB1I3U, PDB1QD0, PDB3QXV), in which the hapten molecule binds into a pockets formed by multiple CDR loops belonging to a single VHH [13][14][15] . For instance, Rr1 dye accommodates in a groove formed by CDR2 and CDR3 of the VHH. In acVHH, caffeine binds into a cavity formed by highly rigid CDR1 and CDR3 from two different VHH molecules, and triggers their dimerization. The binding mode between acVHH and caffeine shares similarities with conventional hapten-antibodies interactions in which haptens bind into a pocket formed by CDRs from both variable heavy and light chain 16 .
A key feature promoting acVHH dimerization is the shorter CDR3 loop. Consequently, hydrophobic residues are exposed and compose most of the VHH/VHH interaction surface. The hydrophobic surface of the acVHH dimer interface is composed by conserved residues involved in the hydrophobic interface between the VH domain and the VL domain in scFV. These residues are also conserved between acVHH and human consensus VHH. On the other hand, the conserved interaction between Pro-L44 and Leu-H45 in antibodies variable domain is replaced by residues Arg40, Glu46, Glu48, and Ser65 which form H-bonds and 2 salt-bridges in acVHH dimer (Fig. 2 -area C). However, this hydrophobic surface neither promotes self-dimerization of the VHH nor changes its stability 7 . The homodimerisation is a direct consequence of the caffeine binding and the set-up of an important and unusual network of interaction between water molecules and the two VHH molecules.
One limitation of current CID systems is their lack of scalability. AcVHH, as a single domain antibody amenable to mutagenesis and selection approaches, could serve as a suitable platform to generate CID systems responding to new ligands. The structure presented here suggests that the large water cavity created by acVHH dimerization and stabilized by caffeine could accommodate other molecules with different size and geometries. The short CDR3 loop of acVHH allows a patch of conserved hydrophobic residues to be exposed and dimerization to happen. (C) In "classic" nanobodies, the longer CDR3 loop masks those hydrophobic residues, for the llama nanobody PorM_01 the Leu100 shield the hydrophobic residues.