Structural Effects of Protein Aging: Terminal Marking by Deamidation in Human Triosephosphate Isomerase

<div><p>Deamidation, the loss of the ammonium group of asparagine and glutamine to form aspartic and glutamic acid, is one of the most commonly occurring post-translational modifications in proteins. Since deamidation rates are encoded in the protein structure, it has been proposed that they can serve as molecular clocks for the timing of biological processes such as protein turnover, development and aging. Despite the importance of this process, there is a lack of detailed structural information explaining the effects of deamidation on the structure of proteins. Here, we studied the effects of deamidation on human triosephosphate isomerase (HsTIM), an enzyme for which deamidation of N15 and N71 has been long recognized as the signal for terminal marking of the protein. Deamidation was mimicked by site directed mutagenesis; thus, three mutants of HsTIM (N15D, N71D and N15D/N71D) were characterized. The results show that the N71D mutant resembles, structurally and functionally, the wild type enzyme. In contrast, the N15D mutant displays all the detrimental effects related to deamidation. The N15D/N71D mutant shows only minor additional effects when compared with the N15D mutation, supporting that deamidation of N71 induces negligible effects. The crystal structures show that, in contrast to the N71D mutant, where minimal alterations are observed, the N15D mutation forms new interactions that perturb the structure of loop 1 and loop 3, both critical components of the catalytic site and the interface of HsTIM. Based on a phylogenetic analysis of TIM sequences, we propose the conservation of this mechanism for mammalian TIMs.</p></div

Structural and Functional Perturbation of <i>Giardia lamblia</i> Triosephosphate Isomerase by Modification of a Non-Catalytic, Non-Conserved Region

<div><p>Background</p><p>We have previously proposed triosephosphate isomerase of <i>Giardia lamblia</i> (GlTIM) as a target for rational drug design against giardiasis, one of the most common parasitic infections in humans. Since the enzyme exists in the parasite and the host, selective inhibition is a major challenge because essential regions that could be considered molecular targets are highly conserved. Previous biochemical evidence showed that chemical modification of the non-conserved non-catalytic cysteine 222 (C222) inactivates specifically GlTIM. The inactivation correlates with the physicochemical properties of the modifying agent: addition of a non-polar, small chemical group at C222 reduces the enzyme activity by one half, whereas negatively charged, large chemical groups cause full inactivation.</p><p>Results</p><p>In this work we used mutagenesis to extend our understanding of the functional and structural effects triggered by modification of C222. To this end, six GlTIM C222 mutants with side chains having diverse physicochemical characteristics were characterized. We found that the polarity, charge and volume of the side chain in the mutant amino acid differentially alter the activity, the affinity, the stability and the structure of the enzyme. The data show that mutagenesis of C222 mimics the effects of chemical modification. The crystallographic structure of C222D GlTIM shows the disruptive effects of introducing a negative charge at position 222: the mutation perturbs loop 7, a region of the enzyme whose interactions with the catalytic loop 6 are essential for TIM stability, ligand binding and catalysis. The amino acid sequence of TIM in phylogenetic diverse groups indicates that C222 and its surrounding residues are poorly conserved, supporting the proposal that this region is a good target for specific drug design.</p><p>Conclusions</p><p>The results demonstrate that it is possible to inhibit species-specifically a ubiquitous, structurally highly conserved enzyme by modification of a non-conserved, non-catalytic residue through long-range perturbation of essential regions.</p></div

Thermostability of WT GlTIM and C222 mutants.

<p>The thermal unfolding of 0.1 mg/ml GlTIM in TED buffer was monitored by recording the change of the circular dichroism signal at 222 nm in a scanning from 25 to 70°C, at a rate of 1°C/min. The fraction of unfolded protein and the Tm values (inset) were calculated as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069031#pone.0069031-EnrquezFlores1" target="_blank">[20]</a>. Experiments were performed by duplicate; in all cases standard errors were less than 5%.</p

The N15D mutation decreases the structural compactness of HsTIM.

<p>The derivatization of cysteine residues in TIM (300 μg/mL) by DTNB (1 mM) was followed spectrophotometrically at 412 nm. The experiment is representative of duplicate experiments qualitatively identical.</p

The two sites of deamidation of HsTIM are found close to each other.

<p>(A) Overall view of the WT HsTIM dimer (PDB code 2JK2) showing the position of N15 and N71; each subunit is depicted in blue and orange. (B) Close-up view of the two deamidating residues. N15 is found in loop 1 and N71 in loop 3.</p

Structural analysis of GlTIM C222D^C.

<p>(A) Structural superposition of the 20 monomers in the crystallographic structure of GlTIM C222D^C; each chain is shown in different color. (B) Close-up of the superposed loop 6 and 7 regions in GlTIM C222D^C, which show the major conformational differences between the different chains; the orientation is the same as in panel A. (C) <i>Per</i> residue Cα RMSD values of the 20 monomers present in the crystallographic structure of GlTIM C222D^C; each chain is shown in a different color.</p

Asymmetric unit of C222D^C.

<p>Each subunit in the asymmetric unit of the GlTIM C222D^C crystal is shown in different color.</p

The dimeric interface is highly perturbed in the structure of the N15D mutant.

<p>Panels A and C correspond to the WT HsTIM, whereas B and D correspond to the mutant N15D. (A) Hydrogen bonds in the interface of the wild-type enzyme that are lost in the N15D mutant (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123379#pone.0123379.s009" target="_blank">S1 Table</a>). The interactions were calculated with the HBplot server (<a href="http://dept.phy.bme.hu/virtuadrug/hbplot/bin/infopage.php" target="_blank">http://dept.phy.bme.hu/virtuadrug/hbplot/bin/infopage.php</a>). (B) There is a new intersubunit hydrogen bond in the N15D mutant that is not present in the wild type enzyme. This interaction is made between the mutated residue 15D and the side chain of S79. (C and D) Surface of a monomer interacting with the neighboring protein subunit. The total contact area per residue decreases from 2486 Ų in the wild type enzyme (C) to 1561 Ų on the N15D mutant (D) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123379#pone.0123379.s010" target="_blank">S2 Table</a>), with the greatest decrease represented by residue M14 (red in C). These panels were drawn according to the results of the Contact Map Analysis server (<a href="http://ligin.weizmann.ac.il/cma/" target="_blank">http://ligin.weizmann.ac.il/cma/</a>), which were plotted on the monomer surface according to the contact area of each residue on the interface on a blue (0 Ų) to red (211.8 Ų for M14) scale basis.</p

Structural divergence of loops 6 and 7 in the crystal structure of GlTIM C222D^C.

<p>(A) Detailed view of the structural differences occurring in loops 6 and 7 in the chain F of C222D^C (red), in comparison with canonical closed (cyan) and open (yellow) states. For the closed state, WT GlTIM crystalized with 2-PG was chosen (4BI7). As GlTIM structure in the open conformation has not been obtained, the closely related structure of <i>T. vaginalis</i> TIM (3QST) was used as representative of the TIM-open state. In panels (B) and (C), the same comparison is shown for GlTIM C222D^C chains E and H, respectively. The substrate analog 2-PG and lateral chains of the YGGS motif are shown as stick models.</p

The N15D mutant is solely responsible for the structural changes induced by deamidation in HsTIM.

<p>(A) The enzyme dimers of the wild type and the N15D mutant do not superpose. Superposition of the dimers of WT HsTIM (cyan) and the N15D mutant (green). The monomers are closer to each other in wild-type TIM when compared to the N15D mutant enzyme. If this superposition is made using a monomer as a reference, the angle of association is changed by 14°, as defined by the DynDom server (<a href="http://fizz.cmp.uea.ac.uk/dyndom/" target="_blank">http://fizz.cmp.uea.ac.uk/dyndom/</a>). (B) The monomeric subunits superpose well. If only the protein monomers are superimposed, the overall RMSD value on Cα is 0.6 Å (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123379#pone.0123379.s005" target="_blank">S5 Fig</a>). There are two main regions that change their overall conformation, loop 1 (in magenta, RMSD of 3 Å) and loop 3 (in red, RMSD of 4 Å). (C) The N71D mutation has no structural effects on the enzyme. A closer look on the region of loop 1 and loop 3 shows that, whereas the N15D mutation (in magenta and red) undergoes a conformational change in both regions, the N71D mutation (in light red) keeps the structure of the WT HsTIM. (D) Residue 15 on the mutant N15D makes two new interactions. A novel intersubunit interaction is made between the side chains of D15 and S79. Also, an intrasubunit interaction is made with the side chain of R17. In the wild-type enzyme, R17 makes intersubunit interactions with the side chains of T70 and N71 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123379#pone.0123379.g009" target="_blank">Fig 9</a>).</p