Three Camelid VHH Domains in Complex with Porcine Pancreatic α-Amylase: Inhibition and Versatility of Binding Topology

Camelids produce functional antibodies devoid of light chains and CH1 domains .The antigen-binding fragment of such heavy-chain antibodies is therefore comprised in one single domain, the VHH. We report here on the structures of three dromadery VHH domains in complex with porcine pancreatic αamylase. Two VHHs bind outside the catalytic site and do not inhibit or inhibit only partially the amylase activity. The third one, AMD9, interacts with the active site crevice and is a strong amylase inhibitor (Ki=10 nM). In contrast with complexes of other proteinaceous amylase inhibitors, amylase keeps its native structure. The VHHs water accessible surface areas covered by amylase range between 850 and 1150 Å2, values similar or even larger to those observed in the complexes between proteins and classical antibodies. These values could certainly be reached because a surprisingly high extent of framework residues are involved in the interactions of VHHs with amylase. The framework residues that participate in the antigen recognition represents 25-40% of the buried surface. The inhibitory interaction of AMD9 involves mainly its CDR2 loop whereas the CDR3 loop is small and certainly not protruding as in the cAb-Lys3, a VHH inhibiting lysozyme. AMD9 inhibits amylase although it is outside direct reach of the catalytic residues, therefore it is to be expected that inhibiting VHH’s might also be elicited against proteases. These results illustrate the versatility and efficiency of VHH domains as protein binders and enzyme inhibitors and are arguments in favor of their use as drugs against diabetes. Abbreviations: Ig, immunoglobulin; VHH: camelid heavy-chain antibody VH; PPA: porcine pancreatic α-amylase; CDR: complementarity determining region; NCS: Non crystallographic symmetry; CNS: Crystallography and NMR System; RMSD: Root mean square deviation; vdW: van der Waals. 2 by gest on Sptem er 1, 2017 hp://w w w .jb.org/ D ow nladed from