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

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.

The extracellular gate shapes the energy profile of an ABC exporter

“…Molecular clamping of the closed extracellular gate was also achieved by a cyclic peptide raised against CmABCB1 19 . Further examples of binders that prevent the IF-OF conversion are nanobodies raised against ABCB1 and PglK, which both sterically clash with NBD closure 20,21 . In contrast, our binders are specific for the OF state and consequently impede the OF-IF conversion.…”

Structure of Outward-Facing PglK and Molecular Dynamics of Lipid-Linked Oligosaccharide Recognition and Translocation

“…Proteins mediating this process are passive or active transporters. Well-studied examples include the calcium-activated nhTMEM16 scramblase that catalyzes the shuffling of lipids between the inner and outer leaflets of the bilayer (Bethel and Grabe, 2016;Brunner et al, 2014); the flippase MurJ (Kuk et al, 2017), which catalyzes the translocation of the peptidoglycan building block undecaprenyl-diphosphate-MurNAcpentapeptide-GlcNAc (lipid II) in bacteria; type IV P-type ATPases (Lopez-Marques et al, 2014;Vestergaard et al, 2014), which are ATP-driven transporters that catalyze the translocation of phospholipids from the exoplasmic to the cytosolic leaflet of cell membranes; the ATP binding cassette (ABC) flippase MsbA (Mi et al, 2017;Ward et al, 2007), an essential inner membrane transporter in Gram-negative bacteria that exports lipid A core (Doshi and van Veen, 2013;Eckford and Sharom, 2010); and the ABC flippase PglK (Perez et al, 2015(Perez et al, , 2017 that translocates the lipid-linked oligosaccharide (LLO) undecaprenyl-diphosphate-BacGalNAc 5 Glc, essential for bacterial protein N-glycosylation in the human pathogen Campylobacter jejuni (Alaimo et al, 2006). A similar LLO translocation process occurs in the endoplasmic reticulum membrane, where the LLO Man 5 GlcNAc 2 -PP-dolichol is flipped from the cytosolic side to the luminal side; however, the nature of the responsible flippase remains elusive (Burda and Aebi, 1999;Helenius et al, 2002).…”

Intramembranal disulfide cross-linking elucidates the super-quaternary structure of mammalian CatSpers

“…Although the mechanism of translocation is still unknown, the crystal structure of PglK flippase contains a salt bridge cluster that suggests a hypothetical translocation intermediate containing a charge-stabilized buried lipid head group. A large conformational change is required for flippase activity, as demonstrated by the inhibition of its function by a cross-binding nanobody [39] In other studies, it was experimentally demonstrated that same side positioning of multiple Arg or His on a transmembrane α-helix was sufficient to generate flippase activity, using only short peptides [40]. The crystal structure of MurJ flippase (Supplementary Fig.…”