CHARACTERIZATION OF THE INTERACTIONS BETWEEN BAR DOMAIN PROTEINS AND MODEL LIPID MEMBRANES

Many cellular activities such as, cell division, cell migration, cell trafficking, exocytosis, and endocytosis, crucially depend on dynamic membrane remodeling that is achieved by the interplay between lipids and proteins [1]. Members of the BAR domain family proteins are key players in these processes. They can sense, stabilize and induce membrane curvature. BAR domains are self-dimerized to form homo-dimers via a helical coiled-coil motif and create elongated crescent or banana shape structures.BAR domain proteins can be categorized into 3 subgroups: N-BAR, F-BAR, and I-BAR according to their dimer curvature and direction [2–4].

Here we focus on the relations between the type of BAR domain protein and the composition of model membrane systems (liposomes). We found by TEM and cryo-TEM that all the BAR proteins were able to create tubes, however, the structural details vary significantly. Extended investigations were done with the F-BAR protein Nervous wreck (Nwk) and the I-BAR protein missing in metastasis (MIM).

Nwk was autoregulated by a C-terminal SH3 domain module that interacts directly with its F-BAR domain. Surprisingly, this autoregulation does not mediate a simple “on-off” switch for membrane remodeling. Instead, the isolated Nwk F-BAR domain efficiently assembles into higher-order structures and deforms membranes only within a limited range of negative membrane charge, and autoregulation elevates this range. Thus, autoregulation could either reduce membrane binding or promote higher order assembly, depending on local cellular membrane composition [5].

With MIM we focused on the high-order assembly. High-resolution cryo-TEM imaging of MIM-decorated lipid tubes using a sensitive direct detector (K2, Gatan) and high accelerating voltage microscope (300kV) showed that the protein is located inside the tubes and it creates a uniform pattern upon the tube. Conformational changes in the secondary structures of the proteins upon binding were identified by circular dichroism (CD). The affinity of the protein to certain lipids was further analyzed by calorimetry (ITC).

References:

¹ McMahon HT, Gallop JL. Membrane curvature and mechanisms of dynamic cell membrane remodelling. Nature 2005; 438: 590–596

² Mim C, Unger VM. Membrane curvature and its generation by BAR proteins. TRENDS BIOCHEMSCI 2012; 37: 526–533

³ Peter BJ, Kent HM, Mills IG, Vallis Y, Butler PJG, Evans PR, McMahon HT. BAR domains as sensors of membrane curvature: the amphiphysin BAR structure. Science 2004; 303: 495–499

⁴ Suetsugu S, Toyooka K, Senju Y. Subcellular membrane curvature mediated by the BAR domain superfamily proteins. Seminars in Cell & Developmental Biology 2010; 21: 340–349

⁵ Kelley CF, Messelaar EM, Eskin TL, Wang S, Song K, Vishnia K, Becalska AN, Shupliakov O, Hagan MF, Danino D, Sokolova OS, Nicastro D, Rodal AA. Membrane charge directs the outcome of F-BAR domain lipid binding and autoregulation. Cell reports 2015; 13: 2597–2609