Substrate-induced dimerization of engineered monomeric variants of triosephosphate isomerase from Trichomonas vaginalis

"The dimeric nature of triosephosphate isomerases (TIMs) is maintained by an extensive surface area interface of more than 1600 angstrom 2. TIMs from Trichomonas vaginalis (TvTIM) are held in their dimeric state by two mechanisms: a ball and socket interaction of residue 45 of one subunit that fits into the hydrophobic pocket of the complementary subunit and by swapping of loop 3 between subunits. TvTIMs differ from other TIMs in their unfolding energetics. In TvTIMs the energy necessary to unfold a monomer is greater than the energy necessary to dissociate the dimer. Herein we found that the character of residue I45 controls the dimer-monomer equilibrium in TvTIMs. Unfolding experiments employing monomeric and dimeric mutants led us to conclude that dimeric TvTIMs unfold following a four state model denaturation process whereas monomeric TvTIMs follow a three state model. In contrast to other monomeric TIMs, monomeric variants of TvTIM1 are stable and unexpectedly one of them (I45A) is only 29-fold less active than wild-type TvTIM1. The high enzymatic activity of monomeric TvTIMs contrast with the marginal catalytic activity of diverse monomeric TIMs variants. The stability of the monomeric variants of TvTIM1 and the use of cross-linking and analytical ultracentrifugation experiments permit us to understand the differences between the catalytic activities of TvTIMs and other marginally active monomeric TIMs. As TvTIMs do not unfold upon dimer dissociation, herein we found that the high enzymatic activity of monomeric TvTIM variants is explained by the formation of catalytic dimeric competent species assisted by substrate binding.

Virtual and In Vitro Screens Reveal a Potential Pharmacophore that Avoids the Fibrillization of Aβ1-42.

Among the multiple factors that induce Alzheimer's disease, aggregation of the amyloid β peptide (Aβ) is considered the most important due to the ability of the 42-amino acid Aβ peptides (Aβ1-42) to form oligomers and fibrils, which constitute Aβ pathological aggregates. For this reason, the development of inhibitors of Aβ1-42 pathological aggregation represents a field of research interest. Several Aβ1-42 fibrillization inhibitors possess tertiary amine and aromatic moieties. In the present study, we selected 26 compounds containing tertiary amine and aromatic moieties with or without substituents and performed theoretical studies that allowed us to select four compounds according to their free energy values for Aβ1-42 in α-helix (Aβ-α), random coil (Aβ-RC) and β-sheet (Aβ-β) conformations. Docking studies revealed that compound 5 had a higher affinity for Aβ-α and Aβ-RC than the other compounds. In vitro, this compound was able to abolish Thioflavin T fluorescence and favored an RC conformation of Aβ1-42 in circular dichroism studies, resulting in the formation of amorphous aggregates as shown by atomic force microscopy. The results obtained from quantum studies allowed us to identify a possible pharmacophore that can be used to design Aβ1-42 aggregation inhibitors. In conclusion, compounds with higher affinity for Aβ-α and Aβ-RC prevented the formation of oligomeric species

Proportions of Aβ_1–42 secondary structures in the absence or presence of 100 μM of the selected compounds.

<p>Proportions of Aβ_1–42 secondary structures in the absence or presence of 100 μM of the selected compounds.</p

AFM analysis after incubating 50 μM Aβ_1–42 alone or in the presence of the selected compounds at 100 μM after 24 h (A to E) or different incubation times (F and G).

<p>Aβ_{<b>1–42</b>} alone (A); Aβ_{<b>1–42</b>} and compound 5 (B); Aβ_{<b>1–42</b>} and compound 8 (C); Aβ_{<b>1–42</b>} and compound 14 (D); Aβ_{<b>1–42</b>} and compound 19 (E). Samples obtained at different incubation times for Aβ_{<b>1–42</b>} alone (F) or with compound 5 (G). Aβ_{<b>1–42</b>} (50 μM in MilliQ water) was incubated at 37°C in a quartz cell in the presence or absence of compounds 5, 8, 14 and 19 (100 μM) and stirred at 250 rpm for 24 h.</p

Comparison of LUMO, HOMO and SOMO (eV) and the electronic energies of the amino acid residues and compounds.

<p><b>(α)</b> After docking studies with <b>Aβ-α</b></p><p><b>(RC)</b> After docking studies with <b>Aβ-RC</b></p><p><b>(β)</b> After docking studies with <b>Aβ-β</b></p><p>Comparison of LUMO, HOMO and SOMO (eV) and the electronic energies of the amino acid residues and compounds.</p

Chemical structures of the inhibitors of Aβ_1–42 fibrillization.

<p>The majority of the compounds shared the presence of amines and/or aromatic rings.</p

Docking results between curcumin, melatonin and ThT with several Aβ_1–42 conformers.

<p>The methodology to obtain the complex is the same as mentioned above for the docking studies with the selected compounds. ΔG values were obtained through docking studies of the ligands with <b>Aβ-α</b> (circles), <b>Aβ-RC</b> (rhombuses) and <b>Aβ-β</b> (squares) (A). The binding modes of curcumin, melatonin and ThT on <b>Aβ-α</b>, <b>Aβ-RC</b>, and <b>Aβ-β</b> (B).</p

Proposed Aβ-α pharmacophore based on the studies with compound 5.

<p>Schematic representation of the polar and nonpolar interactions that favor the interactions with <b>Aβ-α</b> (A); distances between principal chemical groups, the protonated amine (N⁺), the aromatic ring (Ar), and Alkyl substituent (<i>Tert-B</i>) (B). The main interactions involved in the recognition of compound 5 are electrostatic interactions with Glu22 and Asp23, π-π with Phe19 and Phe20 and hydrophobic interactions with Leu17.</p

Chemical structures of the selected compounds used as possible Aβ_1–42 oligomerization inhibitors.

<p>All of the compounds selected contained an amine and/or aromatic ring in their structure. However, not all of the compounds could acquire a positive charge at physiological pH. The compounds are shown with their protonation states based on their pKas.</p

Spatial distribution of SOMO on Aβ-α.

<p>The illustration is based on the mapping of 0.032 isovalues and values onto a total electron density surface contoured at 0.0004 e/au3, which was based on AM1 semi-empirical calculations. The interaction between the LUMOs of compounds 5 and 8 and the SOMO of <b>Aβ-α</b>, in eV (A); <b>Aβ-α</b>–compound 5 complex SOMO (B); and <b>Aβ-α</b>–compound 8 complex SOMO (C) are shown. A map of the electrostatic potentials showing the most positive potential (deepest blue color) and the most negative potential (deepest red color) plotted on a surface with constant electron density (0.02 e/au3). MEP for compound 5 after docking studies with <b>Aβ-α</b> (D); MEP for compound 5 after docking studies with <b>Aβ-β</b> (E); MEP for compound 8 after docking studies with <b>Aβ-α</b> (F); and MEP for compound 8 after docking studies with <b>Aβ-β</b> (G).</p