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 find that the spatial arrangement of positively charged residues as well as local conformational flexibility are factors that distinguish stabilizing from non-stabilizing CDL interactions. However, we also find that even in this controlled, artificial system, a clear-cut distinction between binding and stabilization is difficult to attain, revealing that overlapping lipid contacts can partially compensate for the effects of binding site mutations. 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. In this work, we establish a framework for engineering functional lipid interactions, paving the way for the design of proteins with membrane-specific properties or functions.

53BP1 is a key player in DNA repair and together with BRCA1 regulate selection of DNA double strand break repair mechanisms. Localization of DNA repair factors to sites of DNA damage by 53BP1 is controlled by its oligomerization domain (OD) and binding to LC8, a hub protein that functions to dimerize >100 clients. Here we show that 53BP1 OD is a trimer, an unusual finding for LC8 clients which are all dimers or tetramers. As a trimer, 53BP1 forms a heterogeneous mixture of complexes when bound to dimeric LC8 with the largest mass corresponding to a dimer-of-trimers bridged by 3 LC8 dimers. Analytical ultracentrifugation and isothermal titration calorimetry demonstrate that only the second of the three LC8 recognition motifs is necessary for a stable bridged complex. The stability of the bridged complex is tuned by multivalency, binding specificity of the second LC8 site, and the length of the linker separating the LC8 binding domain and OD. 53BP1 mutants deficient in bridged species fail to impact 53BP1 focus formation in human cell culture studies, suggesting that the primary role of LC8 is to bridge 53BP1 trimers which in turn promotes recruitment of 53BP1 at sites of DNA damage. We propose that the formation of higher-order oligomers of 53BP1 explains how LC8 elicits an improvement in 53BP1 foci and affects the structure and functions of 53BP1.