Crystal structure of human mitochondrial trifunctional protein, a fatty acid β-oxidation metabolon

Abstract

Membrane-bound mitochondrial trifunctional protein (TFP) catalyzes β-oxidation of long chain fatty acyl-CoAs, employing 2-enoyl-CoA hydratase (ECH), 3-hydroxyl-CoA dehydrogenase (HAD), and 3-ketothiolase (KT) activities consecutively. Inherited deficiency of TFP is a recessive genetic disease, manifesting in hypoketotic hypoglycemia, cardiomyopathy, and sudden death. We have determined the crystal structure of human TFP at 3.6-Å resolution. The biological unit of the protein is α₂β₂ The overall structure of the heterotetramer is the same as that observed by cryo-EM methods. The two β-subunits make a tightly bound homodimer at the center, and two α-subunits are bound to each side of the β₂ dimer, creating an arc, which binds on its concave side to the mitochondrial innermembrane. The catalytic residues in all three active sites are arranged similarly to those of the corresponding, soluble monofunctional enzymes. A structure-based, substrate channeling pathway from the ECH active site to the HAD and KT sites is proposed. The passage from the ECH site to the HAD site is similar to those found in the two bacterial TFPs. However, the passage from the HAD site to the KT site is unique in that the acyl-CoA intermediate can be transferred between the two sites by passing along the mitochondrial inner membrane using the hydrophobic nature of the acyl chain. The 3'-AMP-PPi moiety is guided by the positively charged residues located along the "ceiling" of the channel, suggesting that membrane integrity is an essential part of the channel and is required for the activity of the enzyme.

Keywords: fatty acid oxidation; metabolon; mitochondrial trifunctional protein; substrate channeling.

Fig. 1.

Schematic diagram of fatty acid β-oxidation in mitochondria. TFP and VLCAD are associated with the mitochondrial inner membrane. LCAD, MCAD, and SCAD represent long-, medium-, and short-chain acyl-CoA dehydrogenase, respectively.

Fig. 2.

(A) Enzymatic activity of various TFP proteins. The overall reaction activity is in milliunit/mg (purified TFP), Numbers in parentheses are the numbers of repeated measurements. (B) Chromatographic elution profile of wild type (green), ∆β(170-209) (blue), and F277K (cyan). Purified proteins were applied to a size-exclusion column (Bio-Rad Enrich SEC 650) running buffer containing 25 mM Tris·HCl, pH 7.5, 150 mM NaCl and 5% glycerol at a flow rate of 0.4 mL/min using a Shimadzu Prominence HPLC system. Wild-type TFP in the same running buffer plus 24 mM octyl-β-d-glucopyranoside (black) or plus 0.4 mM n-dodecyl-β-d-maltoside (red) is also shown. The apparent molecular mass of the major peaks 1 and 2; and the shoulder peak 3 are calculated at about 317, 554, and 813 kDa, respectively, corresponding to TFP α₂β₂ (258 kDa), α₄β₄ (516 kDa), and α₆β₆ (774 kDa) oligomeric states.

Fig. 3.

Surface representation of the structure of the α₆β₆ form. One asymmetric unit of the human TFP crystal contains three α₂β₂ units, which form a ball with a 32 symmetry (three twofold axes are perpendicular to the threefold axis as marked). All α-subunits are shown in green or orange/yellow shades, and β-subunits are shown in blue or magenta. (A) View down a twofold axis located at the middle of a α₂β₂ unit. (B) View down the threefold axis, where three α-subunits meet. (C) Enlarged view of the hydrophobic interactions among Ile275 and Phe277 of the three α-subunits at the threefold axis.

Fig. 4.

Cartoon representation of the α₂β₂ structure. The ECH domain (Thr37-Thr333) and HAD domain (Lys334-Gln763) of α1 are shown in light and dark green, respectively. The corresponding domains of α2 are in light and dark orange, respectively. β1 is in blue and β2 in magenta. The shaded area in Left represents possible hydrophobic interactions with a curved membrane, and four red dashed ellipses in Right indicate interfaces between the α- and β-subunits.

Fig. 5.

Proposed substrate channels between the three active sites. The same color scheme is used as in Fig. 4, unless stated otherwise. (A) Proposed channel from the ECH to HAD binding sites in the α-subunit (pink) and from the HAD to KT binding sites in the β-subunit (gray). The channels were created using the program HOLLOW (hollow.sourceforge.net). When creating the channel from HAD/α1 to KT/β1, a set of lipid bilayer molecules was also included to mimic the bound membrane. The dashed line represents the other possible substrate pathway from HAD/α1 to KT/β2, that has a large portion open to the solvent (see text for details). Substrates/intermediates are shown with cyan sticks. For clarity, the substrate acyl binding cavities are not included. (B) A different view from A with a 90° rotation along the x axis. For clarity, the α2-subunit is not shown. The alternative channel from HAD/α1 to KT/β2 (dashed line) clearly shows the solvent-exposed passage. (C) Schematic drawing of the substrate channeling between the three active sites. The negatively charged 3′- AMP-PPi moiety (shown as red octagons labeled as AMP-PPi) and the fattyacyl group (black zigzags) are linked by a pantetheine group. The channel between the ECH and HAD sites (pink) has a length of about 27 Å, a width of ∼12 Å at the widest section, and a depth of ∼20 Å (receding into the plane of the diagram). Its volume is ∼2,900 ų. The channel between the HAD and KT sites (gray) is ∼49 Å long with a cross-section of ∼10 × 10 Å, excluding the hydrophobic membrane bilayer (beneath the plane of the diagram). Its volume is ∼6,000 ų. The overall substrate/product channeling events, after 2-enoyl-CoA binds to the ECH active site and 3-hydroxyacyl-CoA is formed, are as follows: (1) The negatively charged AMP-PPi is relocated from the ECH active site to the positively charged area situated at the interface between the ECH/HAD domains (dotted red octagon). (2) The hydrophobic acyl group is released from the ECH active site and relocated to the HAD active site. (3) The AMP-PPi then binds to the HAD active site. The above three steps complete the channeling from the ECH to HAD sites. (4) Then, the negatively charged AMP-PPi moves from the HAD binding site back to the positively charged area (dotted red octagon). (5) The hydrophobic ketoacyl group is released from the HAD active site and moves into the hydrophobic membrane bilayer. (6) The 3-ketoacyl-CoA intermediate wades through the HAD-to-KT channel and finally binds to the KT active site.