|Title||Structural basis of inhibition of lipid-linked oligosaccharide flippase PglK by a conformational nanobody.|
|Publication Type||Journal Article|
|Year of Publication||2017|
|Authors||Perez, C., M. Köhler, D. Janser, E. Pardon, J. Steyaert, R. Zenobi, and K. P. Locher|
|Date Published||2017 Apr 19|
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.
|Alternate Journal||Sci Rep|
|PubMed Central ID||PMC5395944|
Structural basis of inhibition of lipid-linked oligosaccharide flippase PglK by a conformational nanobody.