5NBD

PglK flippase in complex with inhibitory nanobody

Summary for 5NBD

• Entry DOI: 10.2210/pdb5nbd/pdb
• Descriptor: WlaB protein, Nanobody, ADENOSINE-5'-DIPHOSPHATE (3 entities in total)
• Functional Keywords: abc transporter flippase, nanobody, transport protein
• Biological source: Campylobacter jejuni; More
• Total number of polymer chains: 3
• Total formula weight: 143151.23

Authors

Perez, C.,Pardon, E.,Steyaert, J.,Locher, K.P. (deposition date: 2017-03-01, release date: 2017-05-03, Last modification date: 2024-11-06)

Primary citation

Perez, C.,Kohler, M.,Janser, D.,Pardon, E.,Steyaert, J.,Zenobi, R.,Locher, K.P.
Structural basis of inhibition of lipid-linked oligosaccharide flippase PglK by a conformational nanobody.
Sci Rep, 7:46641-46641, 2017

PubMed 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.

PubMed: 28422165
DOI: 10.1038/srep46641

Experimental method

X-RAY DIFFRACTION (3.9 Å)