Structure-Function Studies of Apolipoprotein B100

About This Grant

Project Summary/Abstract Lipoproteins (LPs) are heterogeneous macromolecular nanoparticles that play a central role in transporting lipids and cholesterol between the gut, liver, and other tissues. Apolipoprotein B (apoB), one of the largest proteins known, serves three main functions: (1) coordinating the synthesis of LP particles; (2) acting as the primary structural component of all non-high-density LPs to maintain particle integrity; and (3) providing the binding domain for receptors, enabling cellular uptake. Dysregulation of apoB-containing LP metabolism and mutations in apoB contribute to atherosclerosis, metabolic diseases, and a range of inherited lipid disorders. Despite its pivotal role in fundamental lipid biochemistry and physiology, significant gaps remain in our understanding of apoB structure and function, hindering progress toward a comprehensive understanding of lipid and cholesterol metabolism and associated disease mechanisms. Progress toward understanding apoB's structure and function has been slow due to its large size, complex membrane associations, and the inherent heterogeneity of LPs. The Berndsen group recently made a seminal contribution by solving the structure of apoB, revealing an unexpected multi-domain architecture and complex arrangement on the LP surface. This breakthrough uniquely positions us to address some of the most pressing unanswered questions about apoB, including: How does apoB change conformation to accommodate LPs of varying size and composition, and how do these changes influence its interactions with receptors? What roles do the individual apoB domains play in its three primary functions? How do naturally occurring genetic variants impact apoB’s structure and function? Our approach will be primarily biophysical, with a focus on state-of-the-art electron microscopy, including both single-particle analysis and tomographic techniques, which were instrumental in resolving the apoB structure. Secondary objectives include the continued development and dissemination of these experimental methods, as well as the application of advanced computational modeling techniques. To probe the structure- function relationship of apoB, we will build on insights gained from our recently solved structure and leverage extensive resources cataloging the phenotypes of naturally occurring mutations. We will determine the structure of apoB from heterogeneous LPs isolated from human serum and mutant apoB-containing LPs generated through recombinant expression, both alone and in complex with their cellular receptor. To complement these structural studies, we will measure LP size, mass, lipid composition, receptor-binding thermodynamics, and the efficiency of cellular assembly and secretion to construct a comprehensive understanding apoB function. The outcomes of these experiments and the technologies we develop will advance our fundamental understanding of apoB structure and LP metabolism, provide valuable tools and knowledge to the broader research community, and yield critical insights into the molecular mechanisms underlying various diseases.