Understanding Periperal Membrane Protein Interactions | BPS Thematic Meeting

Understanding Peripheral Membrane Protein Interactions: Structure, Dynamics, Function and Therapy

Monday Speaker Abstracts

NOVEL TWO-STEP PERIPHERAL MEMBRANE RECRUITMENT MECHANISM OF BRUTON'S TYROSINE KINASE THROUGH NATIVE MASS SPECTROMETRY

Rachel McAllister 1,2,3 ; Kallol Gupta 2 ; Moitrayee Bhattacharyya 1 ; 1 Yale University, Department of Pharmacology, New Haven, CT, USA 2 Yale University, Department of Cell Biology, New Haven, CT, USA 3 Yale University, Nanobiology Institute, West Haven, CT, USA

Peripheral membrane proteins are critical in propagating signaling cascades from organellar membranes throughout the cell. The ability to interact with membranes dynamically in response to rapidly changing cellular conditions is absolutely crucial for their function. For many peripheral membrane proteins, membrane interactions are mediated via high-affinity specificity towards certain lipids, yet countless others form low-affinity transient interactions with other membrane lipids. These are often weak and momentary, remaining challenging to capture despite their crucial role in regulating the protein. Taking Bruton's Tyrosine Kinase (BTK), a non receptor tyrosine kinase essential for B cell maturation and activation, we demonstrate a native mass spectrometry (nMS) methodology to understand the recruitment mechanism of peripheral membrane proteins by directly studying it from lipid bilayers customized to a target organellar membrane. We demonstrate that BTK directly binds phosphatidylserine (PS) through sites distinct from those used for its activating lipid, phosphatidylinositol (3,4,5) phosphate (PIP 3 ) and that PS-bound BTK is capable of binding simultaneously to PIP 3 . Contrary to the current understanding of BTK, this suggests a PIP 3 -independent basal recruitment of BTK to the plasma membrane inner leaflet. Using auto-phosphorylation assays, we demonstrate that at physiological concentrations of PIP 3 , PS on the membrane surface enhances the amplitude of BTK activation. Our nMS and biochemical data render a model of membrane recruitment where a low-affinity interaction with abundant PS enables recruitment of BTK to the plasma membrane independent of PIP 3 . This increases the local concentration of BTK on the membrane, which, upon genesis of PIP 3 , leads to greater kinase amplification. This weak lipid-binding mediated regulation likely extends to a broader set of peripheral membrane proteins. This nMS platform can be broadly extended to further peripheral membrane proteins and diverse lipid targets – simultaneously determining tight and weak lipid binding interactions as well as their specificity and stoichiometry.

13