**2. Structure and function of HDL**

HDL, a plasma lipoprotein, plays an important role in cholesterol metabolism [21–23], with several potentially anti-atherogenic properties (remove cholesterol from macrophages) [24–26]. Knowing the assembly mechanism and spatial information is of great importance to mediate cholesterol transport. HDLs exit three main steadier state during the cholesterol transport process: lipid-free apoA-I (apoA-I, the major protein component of HDL particles), discoidal and spherical HDL, with highly heterogeneous and differences of density, size, shape, as well as composition of lipid and protein.

#### **2.1. Lipid-free apoA-I**

important role in the transport of cholesterol [3]. Based on density and size, lipoproteins can be classified as ultra-low- (chylomicrons), very low- (VLDL), intermediate- (IDL), low- (LDL), and high- density lipoproteins (HDL) [4]. The last two might be the significant sections of cholesterol transport and metabolism: (1) LDL could transfer lipids into the blood vessel walls, and contribute to the atherosclerosis, which causally be associated with CVD and all-cause mortality; (2) HDL could remove the lipids and carry them back to the liver, being regarded as "good" one [5, 6]. Hence, the lipoprotein-mediated cholesterol metabolism (cholesterol transport) has aroused great attention and showed the benefit for the in-depth understanding

As shown in **Figure 1**, the lipoprotein-mediated cholesterol metabolism can be divided into exogenous and endogenous pathways [7]. Exogenous pathway is one of crucial ways to transport cholesterol to the body tissues (chylomicrons → VLDL → IDL → LDL) [8, 9], under the co-action of lipoprotein lipase (LPL) and hepatic lipase (HL) [10, 11]. While the higher plasma LDL level might drive the process of atherosclerosis [12]. Endogenous pathway delivers cholesteryl esters back to the liver, working cooperatively in a concurrent manner with ATPbinding cassette transporter A1 (ABCA1) [13], enzyme lecithin-cholesteryl acyltransferase (LCAT) [14], as well as HDL receptors scavenger receptor B1 (SR-BI) [15] or other unidentified HDL receptor (HDLR) [16]. It is widely accepted that HDL protein particles alleviate atherosclerosis with better cardiovascular health (reverse cholesterol transport, RCT) [6, 17, 18]. Besides, cholesteryl ester transfer protein (CETP) does a heteroexchange of triglycerides and cholesteryl esters between VLDL/ LDL and HDL, with the lessen of cholesterol eliminations [19, 20]. Therefore, the functions of HDL, LDL and CETP play the important roles during the cholesterol transport (lipoprotein particle metabolism), and pharmacological inhibition of

of CVDs, as well as the prevention and treatment of CVDs.

4 Cholesterol - Good, Bad and the Heart

CETP is being regarded as a way to prevent CVDs [19, 20].

**Figure 1.** Lipoprotein-mediated cholesterol metabolism in human body.

Structure of full-length lipid-free apoA-I (28-kD, 243 residues) at native states still remains unclear due to its high flexibility. The initial X-ray crystal structure revealed that N-terminal truncated (Δ(1–43)) lipid-free apoA-I features "horseshoe-shape" antiparallel helical dimers [27], being regarded as a vital initial model ("double-belt" model) for comprehending the structure of apoA-I on HDL subclasses (**Figure 2b**) [28]. Subsequent crystal organization of lipid-free Δ(1–43)apoA-I accommodated a four-helix bundle [29–31]. However, the structural information is out of step with some physical biochemical measurements, hinting the conformation dynamics of lipid-free apoA-I. The crystal structures of the N- and C-terminally truncated

**Figure 2.** Three structures of lipid-free apoA-I: (a) full-length lipid-free apoA-I, [36] (b) N-terminal truncated Δ(1–43) apoA-I dimer, [27] and (c) C-terminal truncated Δ[185–243] apoA-I dimer [32].

proteins presented antiparallel helical dimers, with inherent properties (e.g., 5/5 repeat register, **Figure 2b** and **c**) in the lipid-bound and intermediate states [27, 32]. Amphipathic α-helix enables apoA-I to stabilize all HDL subclasses via the conformation change, and N-terminal two thirds constitute a dynamic, four-helix bundle, and the helical segments unfold and refold in seconds. While the C-terminal third, an intrinsically disordered domain, mediates initial binding to phospholipid surfaces. These structural motifs are important for the remodeling of apoA-I during the formation of various HDL particles. Nowadays, there remains some confusions for the structure of full length free apoA-I, especially the dynamic conformations in solutions. The dynamic helical structure is unfolding and refolding in seconds, and the helices bundle at the N-terminal of apoA-I is far more stable than could be achieved in isolation, with mutually stabilizing interactions [33, 34]. The highly dynamic apoA-I molecules are capable of adopting an array of conformations through remodeling HDL that is crucial to lipid transport during the RCT process. Further studies show that mutations in apoA-I induce varied types of dyslipidemias [35].

[34, 46]. Above descriptions were further confirmed by the structures of reconstituted discoidal HDL particles via nuclear magnetic resonance (NMR), electron paramagnetic resonance (EPR) and transmission electron microscopy (TEM) methods [51]. Based on the structures of lipid-free and lipid-bound apoA-I, we can speculate that the monomeric apoA-I forms a helix bundle in which the C-terminal domain binds the lipid to form a helical structure (**Figure 3**). Discoidal HDL are stabilized by two apoA-I molecules wrapped around the edge of the disc in an antiparallel, double-belt arrangement so that the hydrophobic PL acyl chains are protected from exposure to water [52]. These apoA-I molecules are in a highly dynamic state and adapt to discs of different sizes by certain segments forming loops that detach reversibly from

Structural Basis and Functional Mechanism of Lipoprotein in Cholesterol Transport

http://dx.doi.org/10.5772/intechopen.76015

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Due to the complexity of spherical HDL particles in human plasma, the spherical HDL structures are rarely known compared with lipid-free apoA-I and discoidal HDL. Recent developments in native and reconstituted spherical HDL supported a trefoil model, using by the elegant chemical cross-linking and mass spectrometry [53]. In this model, half of each apoA-I molecule in the double-belt arrangement is bent 60° out of the plane of the particle, suggesting the hinging of the Δ(1–43)apoA-I molecule is occur near residues 133 and 233 [53] which is different from the hinging of the full-length protein conformation, meanwhile, trefoil model is assumed to occur near residues 65 and 185 [54]. Determined by small angle neutron scattering method, the helical dimer with a hairpin (HdHp) model was proposed, associated with the

The first LpA-I HDL model at molecular level was proposed, with only apoA-I fractions isolated from human plasma [56]. These isolated human plasma HDL particles range in diameter from 8.8 to 11.2 nm and contain 3–5 apoA-I molecules. It was found that apoA-I adopts intermolecular interactions in plasma HDL which is very similar to those of the double-belt and trefoil models derived from reconstituted systems. Thus, apoA-I might adopt a common structural organization, characterized by distinct intermolecular contacts, regardless of size and shape or natural versus synthetic method of production [57]. Furthermore, circulating

**Figure 3.** The monomer open conformation transfer to dimer conformation of apoA-I (intermediate state) and final HDL

intramolecular interactions within the hairpined apoA-I [55].

state in solution regulated by the H5 region.

the particle surface.

**2.3. Spherical HDL**

#### **2.2. Discoidal HDL**

Human plasma HDL is high heterogeneous, and exists as a short-lived heterogeneous substrate for LCAT in human plasma. Hence, reconstituted HDL particle (rHDL) is a powerful in vivo model system to study its structure and function, with most of the properties of native lipoprotein complexes (e.g., LCAT activation, lipid transfer, and receptor binding) [37–39]. Based on the crystal structure of Δ(1–43)apoA-I, [27] the original double-belt model features two antiparallel monomers, where each helix 5 segments directly oppose each other [40, 41], and the closely contact involved hydrophobic face of amphipathic α-helix with the fatty acid acyl chains [42]. In refined "looped belt" model, N- and C-terminal 40–50 residues doubled back as the "belt and buckle" [43], and residues 134–145 were coincide with a looping region, resulting in partial opening of the parallel belts. It is consistent with the accession between LCAT and the cholesterol and phospholipid acyl chains [44], With the aid of mass spectrometry (MS) and rHDL, lipid-free and lipid-bound apoA-I structures were solved at 104 Å resolution, and resulted in a "solar flares" model, where C-terminal of both apoA-I molecules interacted with each other, and 159–178 loop might be the LCAT binding site, with reduced deuterium exchange [45, 46], Different from normal discoidal shape, double superhelix (DSH) apoA-I model [47] has an open helical shape, with the similar interface interaction between two apoA-I molecules (5/5 double-belt). While, the DSH model is not stable, and could rapidly collapse to a disc-shaped structure during the molecular dynamics (MD) simulations [48].

In according to the rapid growth of transmission electron microscopy (EM) technique, the directly imaging particle's structure can be performed on individual particles, in order to preferably investigate lipoprotein structures. Negative stain EM combined with cryo-EM tomography have been applied to uncover the discoidal shape of apoA-I/HDL particles (both plasma HDL and 7.8, 8.4, 9.6 nm of rHDLs) [49, 50]. In these rHDL particles, the double belt was formed in an antiparallel fashion, with a gross "right-to-right" rotation of the helices after lipidation. The nonhelical regions in lipid-free apoA-I (residues 45–53, 66–69, 116–146, and 179–236) change conformation from random coil to α-helix, to adjust a hydrophobic interior [34, 46]. Above descriptions were further confirmed by the structures of reconstituted discoidal HDL particles via nuclear magnetic resonance (NMR), electron paramagnetic resonance (EPR) and transmission electron microscopy (TEM) methods [51]. Based on the structures of lipid-free and lipid-bound apoA-I, we can speculate that the monomeric apoA-I forms a helix bundle in which the C-terminal domain binds the lipid to form a helical structure (**Figure 3**). Discoidal HDL are stabilized by two apoA-I molecules wrapped around the edge of the disc in an antiparallel, double-belt arrangement so that the hydrophobic PL acyl chains are protected from exposure to water [52]. These apoA-I molecules are in a highly dynamic state and adapt to discs of different sizes by certain segments forming loops that detach reversibly from the particle surface.

#### **2.3. Spherical HDL**

proteins presented antiparallel helical dimers, with inherent properties (e.g., 5/5 repeat register, **Figure 2b** and **c**) in the lipid-bound and intermediate states [27, 32]. Amphipathic α-helix enables apoA-I to stabilize all HDL subclasses via the conformation change, and N-terminal two thirds constitute a dynamic, four-helix bundle, and the helical segments unfold and refold in seconds. While the C-terminal third, an intrinsically disordered domain, mediates initial binding to phospholipid surfaces. These structural motifs are important for the remodeling of apoA-I during the formation of various HDL particles. Nowadays, there remains some confusions for the structure of full length free apoA-I, especially the dynamic conformations in solutions. The dynamic helical structure is unfolding and refolding in seconds, and the helices bundle at the N-terminal of apoA-I is far more stable than could be achieved in isolation, with mutually stabilizing interactions [33, 34]. The highly dynamic apoA-I molecules are capable of adopting an array of conformations through remodeling HDL that is crucial to lipid transport during the RCT process. Further studies show that mutations in apoA-I induce varied types

Human plasma HDL is high heterogeneous, and exists as a short-lived heterogeneous substrate for LCAT in human plasma. Hence, reconstituted HDL particle (rHDL) is a powerful in vivo model system to study its structure and function, with most of the properties of native lipoprotein complexes (e.g., LCAT activation, lipid transfer, and receptor binding) [37–39]. Based on the crystal structure of Δ(1–43)apoA-I, [27] the original double-belt model features two antiparallel monomers, where each helix 5 segments directly oppose each other [40, 41], and the closely contact involved hydrophobic face of amphipathic α-helix with the fatty acid acyl chains [42]. In refined "looped belt" model, N- and C-terminal 40–50 residues doubled back as the "belt and buckle" [43], and residues 134–145 were coincide with a looping region, resulting in partial opening of the parallel belts. It is consistent with the accession between LCAT and the cholesterol and phospholipid acyl chains [44], With the aid of mass spectrometry (MS) and rHDL, lipid-free and lipid-bound apoA-I structures were solved at 104 Å resolution, and resulted in a "solar flares" model, where C-terminal of both apoA-I molecules interacted with each other, and 159–178 loop might be the LCAT binding site, with reduced deuterium exchange [45, 46], Different from normal discoidal shape, double superhelix (DSH) apoA-I model [47] has an open helical shape, with the similar interface interaction between two apoA-I molecules (5/5 double-belt). While, the DSH model is not stable, and could rapidly collapse to a disc-shaped structure during the molecular dynamics (MD)

In according to the rapid growth of transmission electron microscopy (EM) technique, the directly imaging particle's structure can be performed on individual particles, in order to preferably investigate lipoprotein structures. Negative stain EM combined with cryo-EM tomography have been applied to uncover the discoidal shape of apoA-I/HDL particles (both plasma HDL and 7.8, 8.4, 9.6 nm of rHDLs) [49, 50]. In these rHDL particles, the double belt was formed in an antiparallel fashion, with a gross "right-to-right" rotation of the helices after lipidation. The nonhelical regions in lipid-free apoA-I (residues 45–53, 66–69, 116–146, and 179–236) change conformation from random coil to α-helix, to adjust a hydrophobic interior

of dyslipidemias [35].

6 Cholesterol - Good, Bad and the Heart

**2.2. Discoidal HDL**

simulations [48].

Due to the complexity of spherical HDL particles in human plasma, the spherical HDL structures are rarely known compared with lipid-free apoA-I and discoidal HDL. Recent developments in native and reconstituted spherical HDL supported a trefoil model, using by the elegant chemical cross-linking and mass spectrometry [53]. In this model, half of each apoA-I molecule in the double-belt arrangement is bent 60° out of the plane of the particle, suggesting the hinging of the Δ(1–43)apoA-I molecule is occur near residues 133 and 233 [53] which is different from the hinging of the full-length protein conformation, meanwhile, trefoil model is assumed to occur near residues 65 and 185 [54]. Determined by small angle neutron scattering method, the helical dimer with a hairpin (HdHp) model was proposed, associated with the intramolecular interactions within the hairpined apoA-I [55].

The first LpA-I HDL model at molecular level was proposed, with only apoA-I fractions isolated from human plasma [56]. These isolated human plasma HDL particles range in diameter from 8.8 to 11.2 nm and contain 3–5 apoA-I molecules. It was found that apoA-I adopts intermolecular interactions in plasma HDL which is very similar to those of the double-belt and trefoil models derived from reconstituted systems. Thus, apoA-I might adopt a common structural organization, characterized by distinct intermolecular contacts, regardless of size and shape or natural versus synthetic method of production [57]. Furthermore, circulating

**Figure 3.** The monomer open conformation transfer to dimer conformation of apoA-I (intermediate state) and final HDL state in solution regulated by the H5 region.

sHDL contains similar amount of core lipid in reconstituted sHDL and has obviously less surface lipid monolayers, indicating that the apoA-I package on native spheres is much closer than the typical recombinant particles [46]. When a HDL disc alters to a sphere (LCAT converts free cholesterol to cholesteryl ester), global apoA-I conformation does not change significantly between particles of different shapes or origins, with similar protein–protein contacts.
