Three camelid VHH domains in complex with porcine pancreatic alpha-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 camelid heavy chain antibody VH (VHH). Here we report on the structures of three dromedary VHH domains in complex with porcine pancreatic alpha-amylase. Two VHHs bound outside the catalytic site and did not inhibit or inhibited only partially the amylase activity. The third one, AMD9, interacted with the active site crevice and was a strong amylase inhibitor (K(i) = 10 nm). In contrast with complexes of other proteinaceous amylase inhibitors, amylase kept its native structure. The water-accessible surface areas of VHHs covered by amylase ranged between 850 and 1150 A(2), values similar to or even larger than 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 represented 25-40% of the buried surface. The inhibitory interaction of AMD9 involved mainly its complementarity-determining region (CDR) 2 loop, whereas the CDR3 loop was small and certainly did not protrude as it does in cAb-Lys3, a VHH-inhibiting lysozyme. AMD9 inhibited amylase, although it was outside the direct reach of the catalytic residues; therefore it is to be expected that inhibiting VHHs 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.
Desmyter
Aline
A
Vlaams Interuniversitair Instituut Biotechnologie, Vrije Universiteit Brussel, Paardenstraat 65, B-1640 Sint Genesius Rode, Belgium.
Spinelli
Silvia
S
Payan
Francoise
F
Lauwereys
Marc
M
Wyns
Lode
L
Muyldermans
Serge
S
Cambillau
Christian
C
D000595:Amino Acid Sequence; D000818:Animals; D002161:Camelids, New World / Q000276:immunology*; D020134:Catalytic Domain; D022801:Complementarity Determining Regions; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry*; D007135:Immunoglobulin Variable Region / Q000737:chemistry*; D008969:Molecular Sequence Data; D013552:Swine; D000516:alpha-Amylases / Q000037:antagonists & inhibitors / Q000737:chemistry*
D022801:Complementarity Determining Regions; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D000516:alpha-Amylases