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

TitleStructural basis of inhibition of lipid-linked oligosaccharide flippase PglK by a conformational nanobody.
Publication TypeJournal Article
Year of Publication2017
AuthorsPerez, C., M. Köhler, D. Janser, E. Pardon, J. Steyaert, R. Zenobi, and K. P. Locher
JournalSci Rep
Volume7
Pagination46641
Date Published2017 Apr 19
ISSN2045-2322
Abstract

PglK 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.

DOI10.1038/srep46641
Alternate JournalSci Rep
PubMed ID28422165
PubMed Central IDPMC5395944
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