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

Perez, Camilo and Köhler, Martin and Janser, Daniel and Pardon, Els and Steyaert, Jan and Zenobi, Renato and Locher, Kaspar P.. (2017) Structural basis of inhibition of lipid-linked oligosaccharide flippase PglK by a conformational nanobody. Scientific Reports, 7. p. 46641.

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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.
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Structural Biology & Biophysics
UniBasel Contributors:Perez, Camilo
Item Type:Article, refereed
Article Subtype:Research Article
Publisher:Nature Publishing Group
Note:Publication type according to Uni Basel Research Database: Journal article
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Last Modified:13 Oct 2017 09:38
Deposited On:13 Oct 2017 09:38

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