posted on 2024-02-09, 12:36authored byMaryam Yekefallah, Evan J. van Aalst, Roy A. M. van Beekveld, Isaac R. Eason, Eefjan Breukink, Markus Weingarth, Benjamin J. Wylie
Lipids adhere to
membrane proteins to stimulate or suppress molecular
and ionic transport and signal transduction. Yet, the molecular details
of lipid–protein interaction and their functional impact are
poorly characterized. Here we combine NMR, coarse-grained molecular
dynamics (CGMD), and functional assays to reveal classic cooperativity
in the binding and subsequent activation of a bacterial inward rectifier
potassium (Kir) channel by phosphatidylglycerol (PG), a common component
of many membranes. Past studies of lipid activation of Kir channels
focused primarily on phosphatidylinositol bisphosphate, a relatively
rare signaling lipid that is tightly regulated in space and time.
We use solid-state NMR to quantify the binding of unmodified 13C-PG to the K+ channel KirBac1.1 in liposomes.
This specific lipid–protein interaction has a dissociation
constant (Kd) of ∼7 mol percentage
PG (ΧPG) with positive cooperativity (n = 3.8) and approaches saturation near 20% ΧPG.
Liposomal flux assays show that K+ flux also increases
with PG in a cooperative manner with an EC50 of ∼20%
ΧPG, within the physiological range. Further quantitative
fitting of these data reveals that PG acts as a partial (80%) agonist
with fivefold K+ flux amplification. Comparisons of NMR
chemical shift perturbation and CGMD simulations at different ΧPG confirm the direct interaction of PG with key residues,
several of which would not be accessible to lipid headgroups in the
closed state of the channel. Allosteric regulation by a common lipid
is directly relevant to the activation mechanisms of several human
ion channels. This study highlights the role of concentration-dependent
lipid–protein interactions and tightly controlled protein allostery
in the activation and regulation of ion channels.