Understanding the function of high-density lipoprotein (HDL) requires detailed knowledge of the structure of its primary protein, apolipoprotein A-I (APOA1). However, APOA1 flexibility and HDL heterogeneity have confounded decades of efforts to determine high-resolution structures and consistent models. Here, molecular dynamics simulations totaling 30 μs on two nascent HDLs, each with 2 APOA1 and either 160 phospholipids and 24 cholesterols or 200 phospholipids and 20 cholesterols, show that residues 1-21 of the N-terminal domains of APOA1 interact via strong salt bridges. Residues 26-43 of one APOA1 in the smaller particle form a hinge on the disc edge, which displaces the C-terminal domain of the other APOA1 to the phospholipid surface. The proposed structures are supported by chemical cross-linking, Rosetta modeling of the N-terminal domain, and analysis of the lipid-free ∆185APOA1 crystal structure. These structures provide a framework for understanding HDL maturation and revise all previous models of nascent HDL.

Apolipoprotein (apo)A-I is an organizing scaffold protein that is critical to high density lipoprotein (HDL) structure and metabolism, likely mediating many of its cardioprotective properties. However, HDL biogenesis is poorly understood as lipid-free apoA-I has been notoriously resistant to high resolution structural study. Published models from low resolution techniques share certain features but vary considerably in shape and secondary structure. To tackle this central issue in lipoprotein biology, we assembled an unprecedented team of lipoprotein structural biologists and set out to build a consensus model of monomeric lipid-free human apoA-I. Combining novel and published cross-link constraints, small angle X-ray scattering (SAXS), hydrogen-deuterium exchange (H-DX) and crystallography data, we propose a time averaged model consistent with much of the experimental data published over the last 40 years. The model provides a long sought platform for understanding and testing details of HDL biogenesis, structure and function.

Adenylate kinase (AdK) is a phosphoryl-transfer enzyme with important physiological functions. Based on a ligand-free open structure and a ligand-bound closed structure solved by crystallography, here we use molecular dynamics simulations to examine the stability and dynamics of AdK conformations in the absence of ligands. We first perform multiple simulations starting from the open or the closed structure, and observe their free evolutions during a simulation time of 100 or 200 nanoseconds. In all seven simulations starting from the open structure, AdK remained stable near the initial conformation. The eight simulations initiated from the closed structure, in contrast, exhibited large variation in the subsequent evolutions, with most (seven) undergoing large-scale spontaneous conformational changes and approaching or reaching the open state. To characterize the thermodynamics of the transition, we propose and apply a new sampling method that employs a series of restrained simulations to calculate a one-dimensional free energy along a curved pathway in the high-dimensional conformational space. Our calculated free energy profile features a single minimum at the open conformation, and indicates that the closed state, with a high (∼13 kcal/mol) free energy, is not metastable, consistent with the observed behaviors of the unrestrained simulations. Collectively, our simulations suggest that it is energetically unfavorable for the ligand-free AdK to access the closed conformation, and imply that ligand binding may precede the closure of the enzyme.

Rationale: High-density lipoprotein (HDL) may be cardioprotective because it accepts cholesterol from macrophages via the cholesterol transport proteins ABCA1 and ABCG1. The ABCA1-specific cellular cholesterol efflux capacity (ABCA1 CEC) of HDL strongly and negatively associates with cardiovascular disease (CVD) risk, but how diabetes impacts that step is unclear. Objective: To test the hypothesis that HDL's cholesterol efflux capacity is impaired in subjects with type 2 diabetes. Methods and Results: We performed a case-control study with 19 subjects with type 2 diabetes and 20 control subjects. Three sizes of HDL particles, small-HDL, medium-HDL and large-HDL, were isolated by high-resolution size exclusion chromatography from study subjects. Then we assessed the ABCA1 CEC of equimolar concentrations of particles. Small-HDL accounted for almost all of ABCA1 CEC activity of HDL. ABCA1 CECbut not ABCG1 CECof small-HDL was lower in the subjects with type 2 diabetes than the control subjects. Isotope dilution tandem mass spectrometry demonstrated that the concentration of serpin family A member 1 (SERPINA1) in small-HDL was also lower in subjects with diabetes. Enriching small-HDL with SERPINA1 enhanced ABCA1 CEC. Structural analysis of SERPINA1 identified 3 amphipathic alpha-helices clustered in the N-terminal domain of the protein; biochemical analyses demonstrated that SERPINA1 binds phospholipid vesicles. Conclusions: The ABCA1 CEC of small-HDL is selectively impaired in type 2 diabetes, likely because of lower levels of SERPINA1. SERPINA1 contains a cluster of amphipathic α-helices that enable apolipoproteins to bind phospholipid and promote ABCA1 activity. Thus, impaired ABCA1 activity of small HDL particles deficient in SERPINA1 could increase CVD risk in subjects with diabetes.