Alanine Scanning to Define Membrane Protein–Lipid Interaction Sites Using Native Mass Spectrometry

Lipids surrounding membrane proteins interact with different
sites
on the protein with varying specificities, ranging from highly specific
to weak interactions. These interactions can modulate the structure,
function, and stability of membrane proteins. Thus, to better understand
membrane protein structure and function, it is important to identify
the locations of lipid binding and the relative specificities of lipid
binding at these sites. In our previous native mass spectrometry (MS)
study, we developed a single and double mutant analysis approach to
profile the contribution of specific residues toward lipid binding.
Here, we extend this method by screening a broad range of mutants
of AqpZ to identify specific lipid binding sites and by measuring
binding of different lipid types to measure the selectivity of different
lipids at selected binding sites. We complemented these native MS
studies with molecular dynamics (MD) simulations to visualize lipid
interactions at selected sites. We discovered that AqpZ is selective
toward cardiolipins (CL) but only at specific sites. Specifically,
CL orients with its headgroup facing the cytoplasmic side, and its
acyl chains interact with a hydrophobic pocket located at the monomeric
interface within the lipid bilayer. Overall, this integrative approach
provides unique insights into lipid binding sites and the selectivity
of various lipids toward AqpZ, enabling us to map the AqpZ protein
structure based on the lipid affinity.