Structure and active site
Thioesterase superfamily members 4 (Them4) and 5 (Them5) are proteins found in human mitochondria.
Our text is mainly focused on the crystal structure solved by X-ray crystallography at 2.3Å resolution of the complex between the recombinant Δ39Them4 protein and the inhibitor undecan-2-one-CoA.
To start a precise analysis of Them4, it is interesting to switch the representation to the cartoon representation, which reveals the secondary structure elements that are present within this protein's folding. Furthermore, we shall start with a tertiary structure.
These proteins' name come from the single hotdog-fold thioesterase domain in their tertiary structure.
The hotdog fold is characterized by a curved antiparallel beta sheet around a long alpha helix. In the case of Them4, this domain encompasses residues 119 to 231. The core beta sheet in Them4 is six-stranded. It is possible to identify that the topological structural elements of Them4 and its paralog Them5 are arranged as α1-β1-β2-α2-β3-β4-β5-β6.
Notwithstanding that hotdog-fold thioesterases are mainly grouped by their atomic structure since there is little similarity in their primary structure, it is notable that Them4 and Them5 possess a conserved HGG…D…T motif also observed in orthologs.
In Them4, the catalytic residues are Asp161 and Thr177, which interact through a hydrogen bond between the carboxylate in aspartate and the hydroxyl in threonine. For Them5, the catalytic residues are Asp167 and Thr183.
In the proposed catalytic mechanism, the deprotonated aspartate residue abstracts a proton from a water molecule, making it very reactive and prone to a nucleophilic attack on the thioester bond.
As observed in other single hotdog-fold thioesterases, the biological assembly of Them4 and Them5 is a homodimer with a 2-fold symmetry axis. This dimer is maintained mainly by a network of hydrogen bonds between the residues from strand 6 in each monomer. Notably, this network involves the backbone in strand 6 between the beta sheets as well as the side chain of Asn179, Asn181 and Asn183 from the same strand. As a result, the homodimer has a continuous antiparallel 12-stranded beta sheet.
In this quaternary structure, for Them4 the catalytic residues from one monomer are in proximity to His152, Gly153 and Gly154 from the other monomer, which are proposed to accommodate the thioester substrate within the active site. For Them5, Asp167 and Thr183 from one monomer are close to His158, Gly159 and Gly160 from the other monomer.
As a direct consequence, in each catalytically competent Them4 and Them5 there are two active sites located in the interface between monomers of the obligatory homodimer.
Besides the core hotdog-fold, in both Them4 and Them5 there is another alpha helix in each monomer. This element of secondary structure is tightly attached to the convex side of the curved beta sheet owing to the hydrophobic effect. More specifically, it is an amphiphilic alpha helix whose apolar residues are spatially collapsed over apolar residues in strands 1 and 2 of the core beta sheet. For Them4, this alpha helix is formed by residues 55 to 68, while for Them5 the respective residues are 64 to 79.
Interestingly, at each of the flanking regions of the additional alpha helix, there is a pi-stacking interaction that also contributes to maintaining the local folding. Fort Them4, Trp53 and Phe61 make this interaction at the N-terminal side of the alpha helix while Phe64 and Trp73 are the analogues at the C-terminal side.
Them4 also has turns and coils, which is also observed in Them5.
Interaction with the substrate
In order to study the binding of acyl-CoA's to Them4, Zhao et al. (2012) obtained by X-ray crystallography the atomic structure of the human Them4 complexed with undecan-2-one-CoA, which is a structural analog of acyl-CoA's and inhibitor of this protein. Since there are two active sites per dimer, two molecules of Andean-2-one-CoA can bind to Them4. It was reported from the crystal structure that the phosphate groups in the coenzyme A moiety establish electrostatic interactions with Arg206 and Lys207. Furthermore, there is a hydrogen bond seen between the C(6)NH2 from the adenine ring in coenzyme A and Asn193. Nonetheless, Zhao et al. (2012) point out that such interactions may be minimized by the polar solvent.
Function
From enzymatic activities in vitro, it was shown that Them4 (Zhao et al., 2009) and Them5 (Zhuravleva et al., 2012) have higher kcat/KM for acyl-CoA's with medium and long hydrocarbon chain, such as myristoyl-CoA (14:0), palmitoyl-CoA (16:0), oleoyl-CoA (18:1) and linoleoyl-CoA (18:2). According to Zhuravleva et al. (2012), linoleoyl-CoA (18:2) was a preferred substrate for Them5.
From studies with Them5−/− mice, it was identified by mass spectrometry (MS) that loss of Them5 is related to an increase in the levels of monolysocardiolipin (MLCL), which is a metabolite upstream of the cardiolipin remodeling process in mitochondria.
Furthermore, the lipidomics analysis by MS for Them5−/− mice also revealed a 2-fold decrease of free fatty acids, notably linoleic (18:2) and linolenic (18:3) acids. This is consistent with the in vitro assay for the recombinant ∆34Them5 which revealed higher kcat/KM for linoleoyl-CoA (18:2).
Moreover, it is observed by two-dimensional electron microscopy (2D-EM) and subsequent 3D reconstruction that in hepatocytes from Them5−/− mice, mitochondria were more elongated and interconnected, with a 2-fold increase in volume.
With these data, Zhuravleva et al. (2012) propose that Them5 might be a regulator of cardiolipin remodeling through modulation of the unsaturated acyl-CoA pool in mitochondria. This modulation in turn seems to affect mitochondrial morphology.
Zhao et al. (2012) observed that Them4 shows very weak binding affinity (Ki > 1 mM) for carboxylic acids generated after the thioester bond hydrolysis, suggesting that this enzyme is not regulated by product inhibition.
Them4 is also called Akt C-Terminal Modulator Protein (CTMP), owing to previous data suggesting that it interacts with the serine-threonine protein kinase Akt1 in an inferred mechanism of regulating apoptosis. However, this putative activity is not well defined yet. Through pull-down assays, Zhao et al. (2012) verified that Them4 and Akt1 form a stable complex and that Them4 inhibits Akt1 activity in vitro, but Akt1 does not inhibit Them4.
