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Neutron structures of Leishmania mexicana triosephosphate isomerase in complex with reaction-intermediate mimics shed light on the proton-shuttling steps.

ABSTRACT: Triosephosphate isomerase (TIM) is a key enzyme in glycolysis that catalyses the interconversion of glyceraldehyde 3-phosphate and dihydroxy-acetone phosphate. This simple reaction involves the shuttling of protons mediated by protolysable side chains. The catalytic power of TIM is thought to stem from its ability to facilitate the deprotonation of a carbon next to a carbonyl group to generate an enediolate intermediate. The enediolate intermediate is believed to be mimicked by the inhibitor 2-phosphoglycolate (PGA) and the subsequent enediol intermediate by phosphoglycolohydroxamate (PGH). Here, neutron structures of Leishmania mexicana TIM have been determined with both inhibitors, and joint neutron/X-ray refinement followed by quantum refinement has been performed. The structures show that in the PGA complex the postulated general base Glu167 is protonated, while in the PGH complex it remains deprotonated. The deuteron is clearly localized on Glu167 in the PGA-TIM structure, suggesting an asymmetric hydrogen bond instead of a low-barrier hydrogen bond. The full picture of the active-site protonation states allowed an investigation of the reaction mechanism using density-functional theory calculations.

SUBMITTER: Kelpsas V

PROVIDER: S-EPMC8256706 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background. Amyloid secondary structure relies on the intermolecular assembly of polypeptide chains through main-chain interaction. According to this, all proteins have the potential to form amyloid structure, nevertheless, in nature only few proteins aggregate into toxic or functional amyloids. Structural characteristics differ greatly among amyloid proteins reported, so it has been difficult to link the fibrillogenic propensity with structural topology. However, there are ubiquitous topologies not represented in the amyloidome that could be considered as amyloid-resistant attributable to structural features, such is the case of TIM barrel topology. Methods. This work was aimed to study the fibrillogenic propensity of human triosephosphate isomerase (HsTPI) as a model of TIM barrels. In order to do so, aggregation of HsTPI was evaluated under native-like and destabilizing conditions. Fibrillogenic regions were identified by bioinformatics approaches, protein fragmentation and peptide aggregation. Results. We identified four fibrillogenic regions in the HsTPI corresponding to the ?3, ?6, ?7 y ?8 of the TIM barrel. From these, the ?3-strand region (residues 59-66) was highly fibrillogenic. In aggregation assays, HsTPI under native-like conditions led to amorphous assemblies while under partially denaturing conditions (urea 3.2 M) formed more structured aggregates. This slightly structured aggregates exhibited residual cross-? structure, as demonstrated by the recognition of the WO1 antibody and ATR-FTIR analysis. Discussion. Despite the fibrillogenic regions present in HsTPI, the enzyme maintained under native-favoring conditions displayed low fibrillogenic propensity. This amyloid-resistance can be attributed to the three-dimensional arrangement of the protein, where ?-strands, susceptible to aggregation, are protected in the core of the molecule. Destabilization of the protein structure may expose inner regions promoting ?-aggregation, as well as the formation of hydrophobic disordered aggregates. Being this last pathway kinetically favored over the thermodynamically more stable fibril aggregation pathway.

Dataset Information

Neutron structures of Leishmania mexicana triosephosphate isomerase in complex with reaction-intermediate mimics shed light on the proton-shuttling steps.

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