Perturbation of the Dimer Interface of Triosephosphate Isomerase and its Effect on Trypanosoma cruzi

Most of the enzymes of parasites have their counterpart in the host. Throughout evolution, the three-dimensional architecture of enzymes and their catalytic sites are highly conserved. Thus, identifying molecules that act exclusively on the active sites of the enzymes from parasites is a difficult task. However, it is documented that the majority of enzymes consist of various subunits, and that conservation in the interface of the subunits is lower than in the catalytic site. Indeed, we found that there are significant differences in the interface between the two subunits of triosephosphate isomerase from Homo sapiens and Trypanosoma cruzi (TcTIM), which causes Chagas disease in the American continent. In the search for agents that specifically inhibit TcTIM, we found that 2,2′-dithioaniline (DTDA) is far more effective in inactivating TcTIM than the human enzyme, and that its detrimental effect is due to perturbation of the dimer interface. Remarkably, DTDA prevented the growth of Escherichia coli cells that had TcTIM instead of their own TIM and killed T. cruzi epimastigotes in culture. Thus, this study highlights a new approach base of targeting molecular interfaces of dimers

Alternative Splice Variants in TIM Barrel Proteins from Human Genome Correlate with the Structural and Evolutionary Modularity of this Versatile Protein Fold

<div><p>After the surprisingly low number of genes identified in the human genome, alternative splicing emerged as a major mechanism to generate protein diversity in higher eukaryotes. However, it is still not known if its prevalence along the genome evolution has contributed to the overall functional protein diversity or if it simply reflects splicing noise. The (βα)₈ barrel or TIM barrel is one of the most frequent, versatile, and ancient fold encountered among enzymes. Here, we analyze the structural modifications present in TIM barrel proteins from the human genome product of alternative splicing events. We found that 87% of all splicing events involved deletions; most of these events resulted in protein fragments that corresponded to the (βα)₂, (βα)₄, (βα)₅, (βα)₆, and (βα)₇ subdomains of TIM barrels. Because approximately 7% of all the splicing events involved internal β-strand substitutions, we decided, based on the genomic data, to design β-strand and α-helix substitutions in a well-studied TIM barrel enzyme. The biochemical characterization of one of the chimeric variants suggests that some of the splice variants in the human genome with β-strand substitutions may be evolving novel functions via either the oligomeric state or substrate specificity. We provide results of how the splice variants represent subdomains that correlate with the independently folding and evolving structural units previously reported. This work is the first to observe a link between the structural features of the barrel and a recurrent genetic mechanism. Our results suggest that it is reasonable to expect that a sizeable fraction of splice variants found in the human genome represent structurally viable functional proteins. Our data provide additional support for the hypothesis of the origin of the TIM barrel fold through the assembly of smaller subdomains. We suggest a model of how nature explores new proteins through alternative splicing as a mechanism to diversify the proteins encoded in the human genome.</p></div

Pipeline of bioinformatics analysis to identify the (βα)₈ barrel proteins with splice variants in the human genome.

<p>Summary of our bioinformatics data flow to extract the 135 experimentally confirmed (βα)₈ barrel splice variants in the human genome.</p

Biochemical characterization of a chimeric variant.

<p>The overall standard errors of enzyme kinetic parameters are less than 20%.</p>a<p>The apparent thermal melting temperature (°C).</p>b<p>Data obtained from Ochoa-Leyva et al., 2011 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070582#pone.0070582-OchoaLeyva1" target="_blank">[25]</a>.</p

(βα)₈ subdomains and barrel modifications reported by protein engineering experiments.

<p>NA: Not assigned because the corresponding structure forms the overall barrel domain.</p

Functional annotations of human splice variants.

a<p>Reported by <i>in vitro</i> translation.</p><p>ND: Not determined.</p

Sequence analysis under selective pressure at variable positions in chimera enzymes.

<p>Amino acids are represented by the one code letter. The histograms represent the relative frequencies of the selected versus unselected libraries. The red colored histograms (*) represent those amino acids at the variable positions that had a frequency either significantly higher (2 standard deviations) or lower than expected by chance. The sequence analysis for the α-helix 3 and β-strand 7 from MetR swapped into the TrpF scaffold are shown in A and B, respectively. The variable positions are represented by the NNS codon. The three-dimensional structure of the <i>E. coli</i> enzyme (PDB: 1PII) was used to identify the variable positions. The amino acid numbering of TrpF is according to gene reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070582#pone.0070582-OchoaLeyva1" target="_blank">[25]</a>.</p