Mitochondrial hotdog-fold thioesterase

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Overview of thioesterases

Thioesterases are enzymes that catalyze the hydrolysis of thioester bonds, which are the linkage between a carbonyl and a sulfur atom.

The ATP-dependent formation of a thioester bond from a carboxylate and a thiol in biomolecules makes them more reactive and is particularly an important commitment step in lipid metabolism. Therefore, thioesterases counteract this activation by releasing upon hydrolysis a molecule with the more stable carboxylate group. For this reason, thioesterases are found at the end of some metabolic pathways but they also may act as regulators of flux. Besides lipid metabolism, thioester bonds also occur in biosynthetic pathways for polyketide and non-ribosomal peptide production, as well as in main metabolites of carbon metabolism such as acetyl-CoA and succinyl-CoA.

There are two main families of thioesterases which are distinguished by their folding, named the α/β-hydrolases and the hotdog-fold hydrolases. Notably, these two different families are evolutionarily distant, so the thioesterase activity is a shared feature owing to convergent evolution.

spinqualitylabelsall models Human Them4 (PDB entry 4gah) Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image Contents 1 Structure and active site 2 Interaction with the substrate 3 Comparison between the mammalian paralogs 4 Comparison with the bacterial PaaI ortholog Structure and active site Thioesterase superfamily members 2 (Them2) 4 (Them4) and 5 (Them5) are proteins found in mammalian mitochondria, but may also occur in other compartments such as the endoplasmic reticulum and the citosol. This section of 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 (PDB 4GAH). We shall represent structural details from 4GAH using JSmol [1] from Proteopedia. To start a precise analysis of Them4, it is interesting to switch the space filling representation to the , which reveals the secondary structure elements that are present within this protein's folding. Furthermore, we shall start with a , represented here by the subunit B. This protein's name comes from the 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 232. The in Them4 is six-stranded. There is also an packed within this secondary structure. It is possible to identify that the topological structural elements of the hotdog-fold in Them4 are arranged as . 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 possesses a conserved motif also observed in orthologs and its paralogs. In Them4, the catalytic residues are , which through a hydrogen bond between the carboxylate in aspartate and the hydroxyl in threonine. 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 Them4 is a homodimer with a 2-fold symmetry axis. This is maintained mainly by a network of hydrogen bonds between the residues from the in each monomer. Notably, this network involves the backbone in strand 6 between the beta sheets as well as the side chain of from the same strand. As a result, the homodimer has a around oriented antiparallel to one another. Additionally, there are other interactions that contribute in stabilizing the homodimer. As an example from Them4, there is a - involving Phe111, Phe115 and Phe121 from both subunits - in another interface region which is stabilized by hydrophobic effect. In this quaternary structure, for Them4 the catalytic residues from one monomer are to His152, Gly153 and Gly154 from the other monomer, which are proposed to accommodate the thioester substrate within the active site. As a direct consequence, in each catalytically competent Them4 there are located in the interface between monomers of the obligatory homodimer. Besides the core hotdog-fold, Them4 has in each monomer. In Them4 and Them5, this element of secondary structure is tightly attached to the convex side of the curved beta sheet over owing to the hydrophobic effect. More specifically, it is an whose Hydrophobic residues are spatially over Hydrophobic residues in those strands of the core beta sheet, whereas the Polar residues interact with the bulk water. 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 of the additional alpha helix, there is a pi-stacking interaction. For 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. From the PDB 4GAH crystal structure, Zhao et al. (2012) [2] noted that subunit B was better defined, as exemplified by one more alpha helix from residues , whereas this same region was structurally disordered in subunit A and could not be observed. Them4 also has . Interaction with the substrate Substrate binding site in Them4 In order to study the binding of acyl-CoA's to Them4, Zhao et al. (2012) [2] obtained by X-ray crystallography the of the , which is a structural analog of acyl-CoA's and inhibitor of this protein. The authors identified . Of those, two were bound to the active sites whereas the third was partially disordered and interacting with chain B. Here we shall focus on those . Firstly, with residues identified as Polar or Hydrophobic in the representation of Them4 complexed with the two molecules of undecan-2-one-CoA in the active sites, we can . Cavity in Them4 that allows the hydrocarbon chain from the acyl-CoA substrate (represented here by the analog undecan-2-one-CoA) to access the hydrophobic pocket and active site meanwhile the coenzyme A moiety remains on the protein's surface Now it is easier to analyze the interface region where the substrate binds. It is observed that the hydrocarbon chain of the substrate is located within a hydrophobic pocket as expected. As observed in the 2D image obtained with PyMOL, it happens that there is a cavity that fits the hydrocarbon chain as well as the thioester bond, where it is available for the catalytic carboxylate within the protein. Meanwhile, the polar coenzyme A moiety remain bound to the protein's surface. It was reported from the that the coenzyme A moiety establish salt bridges with through between the negatively charged phosphate groups in the substrate and these positively charged residues. Furthermore, there is a seen between the C(6)NH2 from the adenine ring in coenzyme A and Asn183. Nonetheless, Zhao et al. (2012) [2] point out that such interactions may be minimized by the polar solvent. Within the active site, it was observed that the -derived amide in the backbone also interacts with the substrate. Zhao et al. (2012) [2] suggest that the Gly153-derived amide acts by polarizing the carbonyl from the thioester during catalysis. At last, the from the catalytic aspartate residue promotes the thioester hydrolysis. Given the main residues interacting with the substrate, we can have a at undecan-2-one-CoA bound to Them4. Comparison between the mammalian paralogs Now that we introduced the mammalian mitochondrial hotdog-fold thioesterases through Them4 as a model, we are interested in presenting the similarities and differences between this protein and its paralogs Them5 and Them2. It is known by sequence alignment that also has the conserved , with the catalytic residues being . In the case of , the motif occurs as the variant where the catalytic residues are . It is important to mention that both threonine and serine are polar residues with a hydroxyl in the side chain, which conserves the hydrogen bond with the carboxylate from aspartate (or glutamate, as occurs in some orthologs). Interestingly, Them4 and Them5 have an N-terminal region, not present in the crystal structure, that may act as the mitochondrial targeting sequence. This portion does not occur in Them2, which explains why this protein seems smaller when aligned with its paralogs. Besides the catalytic residues, as briefly mentioned, all these paralogs have the HGG portion of the conserved motif. For Them5, Asp167 and Thr183 from one monomer are close to His158, Gly159 and Gly160 from the other monomer. Meanwhile, forThem2, Asp65 and Ser83 are close to His56, Gly57 and Gly58. Here it is the , which is quite similar to what was previously shown in Them4. Specifically for Them2, there is evidence from x-ray crystallography that this protein may also form a through back-to-back association of two homodimers. the interaction between the two dimers for tetramerization is mainly dependent on residues . The additional alpha helix is also present in Them2 and Them5. For , its spacial organization is quite similar to Them4. On the other hand, for the additional alpha helix is not located interacting with the convex side of the core beta sheet since this very region is involved with the back-to-back interaction that stabilizes the Them2 homotetramer. In this case, the additional alpha helix is located over the concave side of strand 1 from the beta sheet. Comparison with the bacterial PaaI ortholog As an example of how conserved the hotdog-fold thioesterases are, we bring the protein from the thermophilic Gram-negative bacterium Thermus thermophilus HB8. Although PaaI does not have the conserved threonine from the active side of other orthologs, it has the motif variant with the highly conserved catalytic carboxylate from aspartate. We observe that this protein is a owing to back-to-back interaction between homodimers, which is quite similar to mammalian Them2.

Variable sequences within a conserved folding

SSM superposition of Them5 (4AE7), Them2 (2H4U) and PaaI (1WM6) on Them4 (4GAH) as an example to reveal the conserved folding amongst these proteins

As mentioned previously, although the hotdog-fold thioesterases bear little similarity between their primary structure, they all share their characteristic conserved folding, which is exemplified by a comparison between the human paralogs Them4 (PDB 4GAH), Them5 (PDB 4AE7) and Them2 (PDB 2H4U) and the bacterial ortholog PaaI (PDB 1WM6) through a secondary-structure matching (SSM) superposition ^[3] using Coot ^[4] followed by spacial alignment using PyMOL. For this analysis, only the chain A from each structure is represented. It is clearly noticeable that their tertiary structures match precisely as the hotdog-fold domain.

SSM superposition of Them5 (4AE7), Them2 (2H4U) and PaaI (1WM6) on Them4 (4GAH) with the HGG...D...T motif (and its variants) highlighted

More specifically, by highlighting the residues from the HGG...D...T motif in Them4 and Them5 and its variants in Them2 and PaaI, it is observed that their orientations are also compatible, which is important for a putative binding of some acyl-CoA substrate in a similar way. Furthermore, the Them4 structure presented here was obtained in complex with the acyl-CoA structural analog undecan-2-one-CoA, whereas the other structures were obtained without a substrate nor such analog (the undecan-2-one-CoA molecules were omitted in this spacial alignment for a clearer comparison between the proteins's folding); although there is this difference, the residues from the catalytic motif in Them5, Them2 and PaaI crystallized without the ligand have a very similar orientation to the corresponding ones in Them4 with undecan-2-one-CoA. On one hand, this might suggest that the catalytic motif does not show a significant conformational change upon binding of the substrate; on the other hand, this might be a consequence of the fact that undecan-2-one-CoA is an structural analog of the acyl-CoA's substrates and not the acyl-CoA's themselves. In this latter case, in the presence of undecan-2-one-CoA, there might be a subtle difference upon binding of this molecule to the active site that does not cause a putative conformational change for catalysis.

Function

From enzymatic activities in vitro, it was shown that Them4^[5] and Them5^[6] have higher k_cat/K_M 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)^[6], 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 k_cat/K_M 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) ^[6] 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) ^[2] observed that Them4 shows very weak binding affinity (K_i > 1 mM) for carboxylic acids generated after the thioester bond hydrolysis, suggesting that this enzyme is not regulated by product inhibition.

Interestingly, it was revealed that Them2 interacts physically with the protein StarD2/PC-TP, which has a START (StAR-related lipid-transfer, with StAR standing for Steroidogenic Acute Regulatory protein) domain. More specifically, Kanno et al. (2007) ^[7] observed that StarD2 stimulates myristoyl-CoA hydrolysis catalyzed by Them2. It becomes even more curious when considering that Them1/ACOT11, another mammalian hotdog-fold thioesterase, has in its sequence an additional START domain.

Them4 is also called Akt Carboxyl-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. At the plasma membrane, CTMP inhibits Akt by preventing its phosphorylation on key residues, threonine 308 and serine 473. This inhibition by CTMP reduces Akt's activity, which is crucial in insulin signaling, cellular survival, and transformation^[8] . However, this activity is not well defined yet. Through pull-down assays, Zhao et al. (2012) ^[2] verified that Them4 and Akt1 form a stable complex and that Them4 inhibits Akt1 activity in vitro, but Akt1 does not inhibit Them4. In pathological contexts, such as skeletal muscle atrophy, CTMP has been shown to exacerbate muscle degeneration by reducing Akt signaling, thereby increasing muscle catabolism and decreasing protein synthesis^[9] . In glioblastomas, CTMP expression is often downregulated due to promoter hypermethylation, removing its inhibitory effects on Akt and contributing to tumorigenesis^[10] . Conversely, in head and neck squamous cell carcinoma (HNSCC), elevated CTMP levels enhance Akt phosphorylation, promoting tumor growth and metastasis, and correlate with poor prognosis^[11] .

References

1. ↑ Hanson, R. M., Prilusky, J., Renjian, Z., Nakane, T. and Sussman, J. L. (2013), JSmol and the Next-Generation Web-Based Representation of 3D Molecular Structure as Applied to Proteopedia. Isr. J. Chem., 53:207-216. doi:http://dx.doi.org/10.1002/ijch.201300024
2. ↑ ^{2.0 2.1 2.2 2.3 2.4 2.5} Zhao H, Lim K, Choudry A, Latham JA, Pathak MC, Dominguez D, Luo L, Herzberg O, Dunaway-Mariano D. Correlation of Structure and Function in the Human Hotdog-fold Enzyme hTHEM4. Biochemistry. 2012 Aug 21;51(33):6490-2. Epub 2012 Aug 9. PMID:22871024 doi:10.1021/bi300968n
3. ↑ Krissinel E, Henrick K. Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions. Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2256-68. PMID:15572779 doi:10.1107/S0907444904026460
4. ↑ Emsley P, Cowtan K. Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2126-32. Epub 2004, Nov 26. PMID:15572765 doi:10.1107/S0907444904019158
5. ↑ Zhao H, Martin BM, Bisoffi M, Dunaway-Mariano D. The Akt C-terminal modulator protein is an acyl-CoA thioesterase of the Hotdog-Fold family. Biochemistry. 2009 Jun 23;48(24):5507-9. doi: 10.1021/bi900710w. PMID:19453107 doi:http://dx.doi.org/10.1021/bi900710w
6. ↑ ^{6.0 6.1 6.2} Zhuravleva E, Gut H, Hynx D, Marcellin D, Bleck CK, Genoud C, Cron P, Keusch JJ, Dummler B, Esposti MD, Hemmings BA. Acyl coenzyme a thioesterase them5/acot15 is involved in cardiolipin remodeling and Fatty liver development. Mol Cell Biol. 2012 Jul;32(14):2685-97. Epub 2012 May 14. PMID:22586271 doi:10.1128/MCB.00312-12
7. ↑ Kanno K, Wu MK, Agate DS, Fanelli BJ, Wagle N, Scapa EF, Ukomadu C, Cohen DE. Interacting proteins dictate function of the minimal START domain phosphatidylcholine transfer protein/StarD2. J Biol Chem. 2007 Oct 19;282(42):30728-36. PMID:17704541 doi:10.1074/jbc.M703745200
8. ↑ Maira SM, Galetic I, Brazil DP, Kaech S, Ingley E, Thelen M, Hemmings BA. Carboxyl-terminal modulator protein (CTMP), a negative regulator of PKB/Akt and v-Akt at the plasma membrane. Science. 2001 Oct 12;294(5541):374-80. PMID:11598301 doi:http://dx.doi.org/10.1126/science.1062030
9. ↑ Wang J, Tierney L, Wilson C, Phillips V, Goldman L, Mumaw C, Muang E, Walker CL. Carboxyl-terminal modulator protein (CTMP) deficiency mitigates denervation-induced skeletal muscle atrophy. Biochem Biophys Res Commun. 2023 Feb 12;644:155-161. PMID:36652767 doi:10.1016/j.bbrc.2023.01.023
10. ↑ Knobbe CB, Reifenberger J, Blaschke B, Reifenberger G. Hypermethylation and transcriptional downregulation of the carboxyl-terminal modulator protein gene in glioblastomas. J Natl Cancer Inst. 2004 Mar 17;96(6):483-6. PMID:15026474 doi:10.1093/jnci/djh064
11. ↑ Chang JW, Jung SN, Kim JH, Shim GA, Park HS, Liu L, Kim JM, Park J, Koo BS. Carboxyl-Terminal Modulator Protein Positively Acts as an Oncogenic Driver in Head and Neck Squamous Cell Carcinoma via Regulating Akt phosphorylation. Sci Rep. 2016 Jun 22;6:28503. PMID:27328758 doi:10.1038/srep28503