# Project 1 - Structural basis of HDL assembly

> **NIH NIH P01** · VANDERBILT UNIVERSITY MEDICAL CENTER · 2020 · $221,075

## Abstract

We hypothesize that apoA-I is the major HDL platform and functions as a conformationally dynamic scaffold
that facilitates the interaction of a host of other apolipoproteins and lipid remodeling factors. It follows from this
hypothesis that a detailed understanding of HDL assembly is not possible through traditional unidisciplinary
approaches but requires an integrated multidisciplinary team approach. Our objective in this project is to use
that approach to understand, in unprecedented detail, the structural basis for HDL assembly. We will combine in
silico approaches with more traditional in-solution experimental approaches: i) high resolution mass
spectrometry combined with thiol-cleavable cross-linking chemistry (Core D), ii) site-directed mutagenesis (Core
D), and iii) functional studies (Core C). To achieve this objective, we propose three specific aims: Aim 1: Three
early steps in HDL assembly: i) ApoA-I monomer structure and dynamics. We will experimentally test our
models using cross-linking and site-directed mutagenesis (Core D) and assays of function (Core C). ii) ABCA1
structure and dynamics. We will use computational methods to develop the extracellular- and intracellular-facing
conformations and the molecular basis for this conformational cycle of ABCA1 and how this cycle produces a
phospholipid pump (Core B). Using purified and mammalian cell-derived ABCA1, we will experimentally test the
models by, respectively, mass spectroscopy-chemical cross-linking (Cores C and D) and site-directed
mutagenesis (Core D) using cell-based assays of HDL function and quantification of HDL particles (Core C). iii)
Physical basis for assembly of nascent HDL. We will test the monolayer pleating hypothesis by MD simulations,
examining, for example, the effects of lysoPC and FFA on disc-membrane fission and mechanisms of lipidation
of dimeric apoA-I (Core B). Mutagenesis will target key Lys residues in the extracellular domain of ABCA1
where pleating and apoA-I association is propose to occur. High resolution EM tomography will be used to
examine 3D images of budding discs under various conditions of lipid composition. Aim 2: Two middle steps in
HDL assembly: i) Simulations of the interactions of LCAT with apoA-I and dHDL (Core B). The models will be
experimentally tested using cross-linking and mutagenesis (Heinecke). ii) Simulations of the interactions of
apoA-II with apoA-I and HDL. The models will be experimentally tested using cross-linking (Davidson). Aim 3:
Three later steps in HDL assembly. i) ABCG1 structure and dynamics. The models will be developed using
homology modeling with MODELLER, double state normal mode analysis and MD simulations (Core B). ii)
Detailed molecular models of PLTP and its interactions with HDL (Davidson). The models will be
experimentally tested using homology modeling with MODELLER (Core B), assays of function (Core C), cross-
linking (Cores C and D) and high resolution EM tomography. iii) Detailed molecular models of th...

## Key facts

- **NIH application ID:** 9951098
- **Project number:** 5P01HL128203-05
- **Recipient organization:** VANDERBILT UNIVERSITY MEDICAL CENTER
- **Principal Investigator:** Jere P Segrest
- **Activity code:** P01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $221,075
- **Award type:** 5
- **Project period:** — → 2022-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9951098

## Citation

> US National Institutes of Health, RePORTER application 9951098, Project 1 - Structural basis of HDL assembly (5P01HL128203-05). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9951098. Licensed CC0.

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