Structural and thermodynamic folding characterization of triosephosphate isomerases from Trichomonas vaginalis reveals the role of destabilizing mutations following gene duplication

Abstract

We report the structures and thermodynamic analysis of the unfolding of two triosephosphate isomerases (TvTIM1 and TvTIM2) from Trichomonas vaginalis. Both isoforms differ by the character of four amino acids: E/Q 18, I/V 24, I/V 45, and P/A 239. Despite the high sequence and structural similarities between both isoforms, they display substantial differences in their stabilities. TvTIM1 (E18, I24, I45, and P239) is more stable and less dissociable than TvTIM2 (Q18, V24, V45, and A239). We postulate that the identities of residues 24 and 45 are responsible for the differences in monomer stability and dimer dissociability, respectively. The structural difference between both amino acids is one methyl group. In TvTIMs, residue 24 is involved in packing α-helix 1 against α-helix 2 of each monomer and residue 45 is located at the center of the dimer interface forming a "ball and socket" interplay with a hydrophobic cavity. The mutation of valine at position 45 for an alanine in TvTIM2 produces a protein that migrates as a monomer by gel filtration. A comparison with known TIM structures indicates that this kind of interplay is a conserved feature that stabilizes dimeric TIM structures. In addition, TvTIMs are located in the cytoplasm and in the membrane. As TvTIM2 is an easily dissociable dimer, the dual localization of TvTIMs may be related to the acquisition of a moonlighting activity of monomeric TvTIM2. To our knowledge, this is the simplest example of how a single amino acid substitution can provide alternative function to a TIM barrel protein.

The Glycolytic Enzyme Triosephosphate Isomerase of Trichomonas vaginalis Is a Surface-Associated Protein Induced by Glucose That Functions as a Laminin- and Fibronectin-Binding Protein

“…1C). The amino acid sequences of the two TvTIM proteins are 98.4% identical, differing by only four amino acids but showing remarkable differences in physicochemical properties, dimer stability, and enzymatic activity (23,24,44). Thus, it was impossible to differentiate between TvTIM1 and TvTIM2 using an anti-TvTIMr antibody.…”

“…The two proteins were recently identified and characterized as recombinant proteins, and their crystal structure and stability were determined (23,24). TvTIM1 and TvTIM2 are dimeric proteins of 254 amino acids in length.…”

“…Despite a difference of only four amino acids between these two proteins, the difference significantly affects their structural stability. The TvTIM1r dimer is more stable and less dissociable than the TvTIM2r dimer, which is easily dissociable, suggesting that these changes could directly affect protein function (23,24). TvTIMs exhibit dual cellular localizations (in the cytoplasm and on the surface of T. vaginalis) under regular growth conditions, suggesting that these proteins could have alternative novel functions when they are localized to the parasite surface (24).…”

“…The TvTIM1r dimer is more stable and less dissociable than the TvTIM2r dimer, which is easily dissociable, suggesting that these changes could directly affect protein function (23,24). TvTIMs exhibit dual cellular localizations (in the cytoplasm and on the surface of T. vaginalis) under regular growth conditions, suggesting that these proteins could have alternative novel functions when they are localized to the parasite surface (24). Evidence suggests that in addition to their enzymatic activities, these two proteins could also have alternative functions in the parasite.…”

Structural insights from a novel invertebrate triosephosphate isomerase from Litopenaeus vannamei

“…The critical role of TIM is that it serves as a crossroad between glycolysis, lipid metabolism, gluconeogenesis and pentose phosphate pathways. The TIM barrel fold is very conserved among enzymes and the crystal structure of triosephosphate isomerase has been determined in many species such as Gallus gallus [10], Saccharomyces cerevisiae [11], Trypanosoma brucei [12], Escherichia coli [13], Homo sapiens [5], Bacillus stearothermophilus [14], Plasmodium falciparum [15], Trichomonas vaginalis [16] and others.…”