Camilo Perez1, Martin Köhler2, Daniel Janser1, Els Pardon3,4, Jan Steyaert3,4, Renato Zenobi2 & Kaspar P. Locher1
- Department of Biology, Institute of Molecular Biology and Biophysics, ETH Zürich, CH-8093 Zürich, Switzerland.
- Department of Chemistry and Applied Biosciences, ETH Zürich, CH-8093 Zürich, Switzerland.
- VIB Center for Structural Biology, VIB, 1050 Brussels, Belgium.
- Structural Biology Brussels, Vrije Universiteit Brussel, 1050 Brussels, Belgium.
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.
CovalX Technology Used (Click each option to learn more)
Samples of PglK (18 μM) and Nbs (18 μM) in buffer (10 mM HEPES, 100 mM NaCl, 0.016% LMNG, 5 nM MgCl2 and 5 mM ADP or 5 mMATP) were incubated for 10 minutes on ice. Following incubation, the crosslinking reaction was performed for 30 minutes using 0.1% glutaraldehyde. Ultra-centrifugal filters were used to remove unbound nanobodies and unreacted glutaraldehyde in the samples before they were washed 5 times with 100 μL of buffer and then centrifuged at 8000 x g for 15 minutes at 4 °C. Using the sandwich method, samples were spotted on a stainless steel MALDI target with sinapic acid used as the matrix. Analysis of the samples was performed in a MALDI-TOF/TOF mass spectrometer modified with a CovalX HM2 detection system. Using this technique, the researchers were able to determine that PglK-Nb87 had reduced crosslinking efficiency when the PglK was incubated with ATP. It was determined that one inhibitory Nb binds to a homodimeric PglK as a single copy. Thus, it can be suggested that when PglK is in the native membrane, it will mostly adopt conformations that are closed NBD dimers.
H2O2 sample preparation: tissues were covered in a 3% solution of H2O2 before being incubated in a saturated vapor pressure chamber for 30 minutes and then dried in a vacuum desiccator. Finally, the samples were prepared using a matrix solution (20 mg/ml sinapic acid in acetonitrile:0.1% TFA (7:3, v/v)).