Energetically unfavorable protein angles: Exploration of a conserved dihedral angle in triosephosphate isomerase

Abstract

Over the past 3.5 billion years of evolution, enzymes have adopted a myriad of conformations to suit life on earth. However, torsional angles of proteins have settled into limited zones of energetically favorable dihedrals observed in Ramachandran plots. Areas outside said zones are believed to be disallowed to all amino acids, except glycine, due to steric hindrance. Triosephosphate isomerase (TIM), a homodimer with a catalytic rate approaching the diffusion limit, contains an active site lysine residue (K13) with dihedrals within the fourth quadrant (Φ = +51/Ψ = −143). Both the amino acid and the dihedral angles are conserved across all species of TIM and known crystal structures regardless of ligand. Only crystal structures of the engineered monomeric version (1MSS) show accepted β‐sheet dihedral values of Φ = ‐135/Ψ = +170 but experiments show a 1000‐fold loss in activity. Based on these results, we hypothesized that adopting the unfavorable torsion angle for K13 contributes to catalysis. Using both, computational and experimental approaches, four residues that interact with K13 (N11, M14, E97, and Q64) were mutated to alanine. In silico molecular dynamics (MD) simulations were performed using 2JK2 unliganded human TIM as a starting structure. Ramachandran plots, containing K13 dihedral values reveal full or partial loss of disallowed zone angles. N11A showed no detectable catalytic activity and lost the unfavorable K13 dihedral angles across four separate force fields during simulation while all other mutants plus wild type retained activity and retained the conserved K13 dihedral angles.

The Role of Asn11 in Catalysis by Triosephosphate Isomerase

“…Only the N11A variant showed no detectable activity, consistent with the conclusion that the N11–K13 interaction plays an important role in maintaining the unusual K13 Φ dihedral angle. The results of molecular dynamics simulations on these four alanine variants, using PDB ID 2JK2 as the starting structure for Hs TIM, showed that the Φ dihedral angle for K13 was maintained for the M14A, Q65A, and E97A variants but is lost during equilibration of the N11A variant . It was concluded that the change in K13 Φ dihedral angle was responsible for the loss of catalytic activity for the N11A variant.…”

“…We therefore propose that the effect of the N11A substitution on dimer stability is the result of the perturbation of an extended network of active site hydrogen bonds that includes the N11, K13, Q65, E97, and T75′ side chains and the backbone amide from G′76 (Figure A). We propose that a disruption of this hydrogen-bonded network at the N11A variant of Tbb TIM and Lm TIM results in a large change in the preferred Φ dihedral angle for K13, as was previously proposed to occur for the N11A variant of Hs TIM …”

“…The aim of the latter study was to determine the effect of substitution of four side chains (N11, M14, Q65, and E97; using the numbering for Tbb TIM), which interact with the K13 side chain, on the unusual positive Φ dihedral angle for K13. The observed value of Φ = 51ψ = −142° for K13 lies in the fourth quadrant of a Ramachandran plot that is normally reserved for glycine residues . The alanine variants of each of these four side chains were prepared, and the kinetic parameters were determined.…”

“…The N11 side chain amide is positioned to act as a hydrogen bond donor to oxygen-1 of DHAP or GAP (Scheme ), and to the -OH for the T75′ side chain, where N11 and T75′ form an extended network of hydrogen-bonded side chains that includes E97 and K13 (Figure ). It is notable nearly 40 years after the first mutagenesis study on TIM that the catalytic role of the interaction between N11 and substrate was only recently probed by this method in a study that reported that the N11A variant of TIM from Homo sapiens ( Hs TIM) showed no detectable activity toward the isomerization of DHAP . This report appeared during the studies on the N11A variants of Tbb TIM and TIM from Leishmania mexicana ( Lm TIM) that are described in this paper.…”

“…It is notable nearly 40 years after the first mutagenesis study on TIM 19 that the catalytic role of the interaction between N11 and substrate was only recently probed by this method in a study that reported that the N11A variant of TIM from Homo sapiens ( Hs TIM) showed no detectable activity toward the isomerization of DHAP. 42 This report appeared during the studies on the N11A variants of Tbb TIM and TIM from Leishmania mexicana ( Lm TIM) that are described in this paper.…”