Crystal structure of snake venom acetylcholinesterase in complex with inhibitory antibody fragment Fab410 bound at the peripheral site - Evidence for open and closed states of a back door channel
Résumé
The acetylcholinesterase found in the venom of Bungarus fas-
ciatus (BfAChE) is produced as a soluble, non-amphiphilic
monomer with a canonical catalytic domain but a distinct C
terminus compared with the other vertebrate enzymes. More-
over, the peripheral anionic site of BfAChE, a surface site
located at the active site gorge entrance, bears two substitutions
altering sensitivity to cationic inhibitors. Antibody Elec410,
generated against Electrophorus electricus acetylcholinesterase
(EeAChE), inhibits EeAChE and BfAChE by binding to their
peripheral sites. However, both complexes retain significant
residual catalytic activity, suggesting incomplete gorge occlu-
sion by bound antibody and/or high frequency back door open-
ing. To explore a novel acetylcholinesterase species, ascertain
the molecular bases of inhibition by Elec410, and document the
determinants and mechanisms for back door opening, we solved
a 2.7-Å resolution crystal structure of natural BfAChE in com-
plex with antibody fragment Fab410. Crystalline BfAChE forms
the canonical dimer found in all acetylcholinesterase structures.
Equally represented open and closed states of a back door chan-
nel, associated with alternate positions of a tyrosine phenol ring
at the active site base, coexist in each subunit. At the BfAChE
molecular surface, Fab410 is seated on the long -loop between
two N-glycan chains and partially occludes the gorge entrance, a
position that fully reflects the available mutagenesis and bio-
chemical data. Experimentally based flexible molecular docking
supports a similar Fab410 binding mode onto the EeAChE anti-
gen. These data document the molecular and dynamic peculiar-
ities of BfAChE with high frequency back door opening, and the
mode of action of Elec410 as one of the largest peptidic inhibi-
tors targeting the acetylcholinesterase peripheral site.
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