Biology subjects

McAllister, R.

Publications and source records attributed to McAllister, R..

3 recordsLinked to original sources

bioRxiv2025

Two-Step Mechanism of Bruton's Tyrosine Kinase MembraneRecruitment and Activation

Peripheral membrane proteins (PMPs) are critical mediators of signaling cascades initiated via activation of cell-surface receptors. Their functions rely on their innate ability to interact with membranes dynamically in response to rapidly changing cellular conditions. This membrane recruitment may occur via high-affinity interactions with specific lipids or transient, low-affinity membrane interactions. These weak and dynamic interactions that are critical regulators of protein function are challenging to capture. Taking Brutons Tyrosine Kinase (BTK), a non-receptor tyrosine kinase essential for B cell activation, as an example, we demonstrate a native mass spectrometry (nMS) platform to understand the recruitment of PMPs by directly studying it from lipid bilayers customized to target membranes. Our data demonstrates that BTK recognizes phosphatidylserine (PS) through sites distinct from phosphatidylinositol (3,4,5) phosphate (PIP3) binding. We show PS-bound BTK retains PIP3 binding simultaneously via high-affinity sites, exhibiting PIP3-independent basal membrane recruitment of BTK. Biochemical assays show that this PS-mediated recruitment sensitizes BTK to PIP3-mediated activation under near-physiological concentrations of PIP3. Thus, we propose a two-step model for BTK membrane recruitment and activation. A low-affinity interaction with high-copy number PS enables plasma membrane recruitment of BTK and increases its membrane-bound concentration. Upon B-cell activation, this pre-recruited, membrane-bound BTK population localizes to PIP3-rich domains through electrostatic gliding along the membrane driven by low-affinity PS and high-affinity PIP3 binding. This indicates a cooperative mechanism where PS can amplify B-cell signaling through increased membrane-bound BTK concentration. Our work demonstrates a general model of PH-domain-containing protein regulation by weak protein-lipid interactions, which can be extended to many other PMPs.

biophysics↗

bioRxiv2024

A proteome-wide quantitative guide for nanoscale spatially resolved extraction of membrane proteins into native nanodiscs

The intricate molecular environment of the native membrane profoundly influences every aspect of membrane protein (MP) biology. Despite this, the most prevalent method of studying MPs uses detergent-like molecules that disrupt and remove this vital local membrane context. This severely impedes our ability to quantitatively decipher the local molecular context and comprehend its regulatory role in the structure, function, and biogenesis of MPs. Using a library of membrane-active polymers we have developed a platform for the high-throughput analysis of the membrane proteome. The platform enables near-complete spatially resolved extraction of target MPs directly from their endogenous membranes into native nanodiscs that maintain the local membrane context. We accompany this advancement with an open-access database that quantifies the polymer-specific extraction variability for 2065 unique mammalian MPs and provides the most optimized condition for each of them. Our method enables rapid and near-complete extraction and purification of target MPs directly from their endogenous organellar membranes at physiological expression levels while maintaining the nanoscale local membrane environment. Going beyond the plasma membrane proteome, our platform enables extraction from any target organellar membrane including the endoplasmic reticulum, mitochondria, lysosome, Golgi, and even transient organelles such as the autophagosome. To further validate this platform, we took several independent MPs and demonstrated how our resource can enable rapid extraction and purification of target MPs from different organellar membranes with high efficiency and purity. Further, taking two synaptic vesicle MPs, we show how the database can be extended to capture multiprotein complexes between overexpressed MPs. We expect these publicly available resources to empower researchers across disciplines to efficiently capture membrane nano-scoops containing a target MP and interface with structural, functional, and other bioanalytical approaches. We demonstrate an example of this by combining our extraction platform with single-molecule TIRF imaging to demonstrate how it can enable rapid determination of homo-oligomeric states of target MPs in native cell membranes.

biophysics↗

bioRxiv2023

Direct determination of oligomeric organization of integral membrane proteins and lipids from intact customizable bilayer

Hierarchical organization of integral membrane proteins (IMP) and lipids at the membrane is essential for regulating myriad downstream signaling. A quantitative understanding of these processes requires both detections of oligomeric organization of IMPs and lipids directly from intact membranes and determination of key membrane components/properties that regulate them. Addressing this, we have developed a platform that enables native mass spectrometry (nMS) analysis of IMP-lipid complexes directly from intact and customizable lipid membranes. Both the lipid composition and membrane properties (such as curvature, tension, fluidity) of these bilayers can be precisely customized to a target membrane. Subsequent direct nMS analysis of these intact proteo-lipid vesicles can yield the oligomeric states of the embedded IMPs, identify bound lipids, and determine the membrane properties that can regulate the observed IMP-lipid organization. Applying this, we show how lipid binding regulates neurotransmitter release and how membrane composition regulates the functional oligomeric state of a transporter.

biophysics↗