Mitochondrial hotdog-fold thioesterase

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. 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 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 and Them5 possess a conserved motif also observed in orthologs.

In Them4, the catalytic residues are , which through a hydrogen bond between the carboxylate in aspartate and the hydroxyl in threonine. For Them5, the catalytic residues are Asp167 and Thr183 whereas for Them2, a smaller paralog, the catalytic residues are Asp65 and Ser83 (noticing that both threonine and serine are polar residues with a hydroxyl in the side chain). 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, Them2, Them4 and Them5 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. For Them5, Asp167 and Thr183 from one monomer are close to His158, Gly159 and Gly160 from the other monomer. Meanwhile, for Them2, Asp65 and Ser83 are close to His56, Gly57 and Gly58. Specifically for Them2, there is evidence from x-ray crystallography that this protein may also form a homotetramer through back-to-back association of two homodimers. As a direct consequence, in each catalytically competent Them4 and Them5 there are located in the interface between monomers of the obligatory homodimer, whereas for Them2 there are four active sites.

Besides the core hotdog-fold, in both Them2, Them4 and Them5 there is 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) noted that chain B was better defined, as exemplified by one more alpha helix from residues , whereas this same region was structurally disordered in chain A and could not be observed. Them4 also has , which is also observed in Them5 and Them2.

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 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 in order 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. 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) 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) 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.