Abramsson, M. L.; Corey, R. A.; Skerle, J. L.; Persson, L. J.; Anden, O.; Oluwole, A. O.; Howard, R. J.; Lindahl, E.; Robinson, C. V.; Strisovsky, K.; Marklund, E. G.; Drew, D.; Stansfeld, P. J.; Landreh, M.

Integral membrane proteins carry out essential functions in the cell, and their activities are often modulated by specific protein-lipid interactions in the membrane. Here, we elucidate the intricate role of cardiolipin (CDL), a regulatory lipid, as a stabilizer of membrane proteins and their complexes. Using the in silico-designed model protein TMHC4_R (ROCKET) as a scaffold, we employ a combination of molecular dynamics simulations and native mass spectrometry to explore the protein features that facilitate preferential lipid interactions and mediate stabilization. We reveal that the spatial arrangement of positively charged residues as well as local conformational flexibility are pivotal in distinguishing stabilizing from non-stabilizing CDL interactions. Extending our insights to naturally occurring proteins, we identify a stabilizing CDL site within the E. coli rhomboid intramembrane protease GlpG and uncover its regulatory influence on enzyme substrate preference. This work establishes a framework for engineering functional lipid interactions, paving the way for the design of proteins with membrane-specific properties or functions.