Structural basis of inhibition of lipid-linked oligosaccharide flippase PglK by a conformational nanobody

• DOI: 10.1038/srep46641
• Publication Date: 2017-04-19T09:36:44Z
• Authors: Camilo Perez; Martin Köhler; Daniel Janser; Els Pardon; Jan Steyaert; Renato Zenobi; Kaspar P. Locher
• Keywords: 0303 health sciences; X-Ray Crystallography; Single-Domain Antibodies; Antibodies, Bacterial; Biochemistry; Article; X-Ray Crystallography; Biochemistry; Campylobacter jejuni; 03 medical and health sciences; Bacterial Proteins; ATP-Binding Cassette Transporters; Protein Multimerization; Protein Structure, Quaternary

AbstractPglK is an ABC transporter that flips a lipid-linked oligosaccharide (LLO) that serves as a donor in protein N-glycosylation. Previous structures revealed two inward-facing conformations, both with very large separations of the nucleotide binding domains (NBDs), and a closed, ADP-bound state that featured an occluded cavity. To investigate additional states, we developed conformation-sensitive, single-domain camelid nanobodies (Nb) and studied their effect on PglK activity. Biochemical, structural, and mass spectrometric analyses revealed that one inhibitory Nb binds as a single copy to homodimeric PglK. The co-crystal structure of this Nb and ADP-bound PglK revealed a new, narrowly inward-open conformation. Rather than inducing asymmetry in the PglK homodimer, the binding of one Nb results in steric constraints that prevent a second Nb to access the symmetry-related site in PglK. The Nb performed its inhibitory role by a “sticky-doorstop” mechanism, where inhibition of ATP hydrolysis and LLO flipping activity occurs due to impaired closing of the NBD interface, which prevents PglK from converting to an outward-open conformation. This inhibitory mode suggests tight conformational coupling between the ATPase sites, which may apply to other ABC transporters.