Structures of Angptl3 and Angptl4, modulators of triglyceride levels and coronary artery disease

Abstract

Coronary artery disease is the most common cause of death globally and is linked to a number of risk factors including serum low density lipoprotein, high density lipoprotein, triglycerides and lipoprotein(a). Recently two proteins, angiopoietin-like protein 3 and 4, have emerged from genetic studies as being factors that significantly modulate plasma triglyceride levels and coronary artery disease. The exact function and mechanism of action of both proteins remains to be elucidated, however, mutations in these proteins results in up to 34% reduction in coronary artery disease and inhibition of function results in reduced plasma triglyceride levels. Here we report the crystal structures of the fibrinogen-like domains of both proteins. These structures offer new insights into the reported loss of function mutations, the mechanisms of action of the proteins and open up the possibility for the rational design of low molecular weight inhibitors for intervention in coronary artery disease.

Figure 1

Crystal structure of the fibrinogen-like domains of Angptl3 and Angptl4. (A) Schematic of Angiopoietin-like protein domain architecture. Each angiopoietin and angiopoietin-like protein (except Angptl8) contains an N-terminal region predicted to be intrinsically disordered, a coiled-coil region and a C-terminal fibrinogen-like domain. (B) Structure of Angptl3 (left) and Angptl4 (right) fibrinogen-like domains. Each can be split into three subdomains A, B and P. Each structure is colored according to secondary structure elements with α-helices in gold (Angptl3) and violet (Angptl4), β-strands in cyan (Angptl4) and green (Angptl4) and loops in grey. Missing sections in the loops are indicated by dashed lines. Secondary structure elements are labeled and the N- and C-termini are indicated. (C) Electrostatic surface potential of Angptl3, the orientation is equivalent to that in panel B and rotated 90° around horizontal axis to see the underside of the P subdomain. Negative charge is shown in red and positive in blue. (D) As panel C but Angptl4.

Figure 2

Comparison of the Angptl3 and Angptl4 fibrinogen-like domains with angiopoietins. (A) Overlay of the Cα-trace of Angptl3 (blue, left) and Angptl4 (green, right) with those of Ang1 (yellow) and Ang2 (violet). (B) Comparison with Ca²⁺ binding site of Ang1 (light yellow) and Ang2 (light violet) of the equivalent region in Angptl3 (blue, top) and Angptl4 (green, bottom). Residues involved in Ca²⁺ binding are shown in stick representation and colored similarly to the protein they belong. Ca²⁺ ions are shown in ball representation. (C) Alignment of the proteins shown in panel A. Cysteines are highlighted in yellow and conserved amino acids in blue. The P subdomain is boxed and the Asp involved in calcium binding in Ang1 and Ang2 are indicated with arrowheads.

Figure 3

Structural analysis of mutations in Angptl3 which cause loss of function. (A) Angptl3 fibrinogen-like domain crystal structure with the sites of loss of function mutations depicted in ball and stick representation and highlighted in red. (B) Close up views of the environment of the mutated amino acids in panel A. Mutated residues are shown in ball and stick representation and neighboring or interacting residues are in stick representation. Residues are colored according to their location in the secondary structure elements with nitrogen atoms in blue, oxygen in red and sulfur in yellow. Potential hydrogen bonds are depicted as solid black lines.

Figure 4

Structural analysis of mutations in Angptl4 which cause loss of function. (A) Angptl4 fibrinogen-like domain crystal structure with the sites of mutations resulting in lower triglyceride levels are indicated in red, mutations leading to the increase of triglyceride levels are shown in blue. (B) Close up views of the environment of the mutated amino acids in panel A. Residues are depicted and colored as in Fig. 3.

Figure 5

Analysis of loss of function mutations (A) Coomassie stained SDS-PAGE gel of IMAC purified proteins. Upper gel Angptl3, lower gel Angptl4 (Supplementary Fig. 1 shows the full gels) (B) Thermal stability of purified wild-type Angptl3 determined by thermofluor. The insert panel shows the derivative of the change in fluorescence signal. Angptl3 shows cooperative unfolding.