An Ancestral Tryptophanyl-tRNA Synthetase Precursor Achieves High Catalytic Rate Enhancement without Ordered Ground-State Tertiary Structures

Abstract

Urzymes—short, active core modules derived from enzyme superfamilies—prepared from the two aminoacyl-tRNA synthetase (aaRS) Classes contain only the modules shared by all related family members. They have been described as models for ancestral forms. Understanding them depends on inferences drawn from the crystal structures of the full-length enzymes. As aaRS Urzymes lack much of the mass of modern aaRS, retaining only a small portion of the hydrophobic cores of the full-length enzymes, it is desirable to characterize their structures. We report preliminary characterization of 15N tryptophanyl-tRNA synthetase Urzyme by heteronuclear single quantum coherence (HSQC) NMR spectroscopy supplemented by circular dichroism, thermal melting, and induced fluorescence of bound dye. The limited dispersion of 1H chemical shifts (0.5 ppm) is inconsistent with a narrow ensemble of well-packed structures in either free or substrate-bound forms, although the number of resonances from the bound state increases, indicating a modest, ligand-dependent gain in structure. Circular dichroism spectroscopy shows the presence of alpha helix and evidence of cold denaturation, and all ligation states induce Sypro Orange fluorescence at ambient temperatures. Although the term “molten globule” is difficult to define precisely, these characteristics are consistent with most such definitions. Active-site titration shows that a majority of molecules retain ~60% of the transition state stabilization free energy observed in modern synthetases. In contrast to the conventional view that enzymes require stable tertiary structures, we conclude that a highly flexible ground-state ensemble can nevertheless bind tightly to the transition state for amino acid activation.

Coding of Class I and II Aminoacyl-tRNA Synthetases

“…It has, however, been possible to prepare isotopically labeled samples of active TrpRS Urzyme. Preliminary 15 N- 1 H HSQC spectra from those samples supported an unexpected, but not unprecedented conclusion—the TrpRS Urzyme is not a folded protein, but has many of the properties expected from a catalytically active molten globule [64]. The HSQC spectrum has a reasonable dispersion of values in the 15 N dimension, but all ~80 peaks are contained between 8–8.5 ppm in the 1 H dimension, which is characteristic of proteins that are not fully folded.…”

“…It can be argued that the effort is fruitless as one cannot run the tape in reverse, that important and relevant witnesses are all extinct, and in particular that there is no way to test hypotheses. Our work [64,40,65,41,55,26,42,32,34,27,7,28,33,29,8,30,36,31,48], and that of others [25,66,52–54,67], tends to rebut this objection. In reality, molecular phylogenies of the Class I and II aaRS have proven to be a rich source of unexpected insights into how translation became possible and robust tools now enable us to investigate, and even recapitulate key events from the past history of life.…”

“…Similarly, it appears likely that the Urzyme level of evolutionary development may utilize tight transition-state binding associated with a large unfavorable negative entropy change as suggested for the TrpRS Urzyme [64] and a chorismate mutase molten globular variant [110]. This possibility, combined with the discussion in §III of the superiority of peptide catalysts suggests strongly that the emergence and selection of catalytic activity itself was vastly more efficient and hence more rapid for peptide, than for ribozymal catalysts.…”

“…The HSQC spectrum has a reasonable dispersion of values in the 15 N dimension, but all ~80 peaks are contained between 8–8.5 ppm in the 1 H dimension, which is characteristic of proteins that are not fully folded. The conclusion that it is probably a molten globule is reinforced by the fact that the temperature dependence of CD spectrum exhibits cold denaturation, and that Thermaflour measurements of thermal melting in the presence of Sypro Orange dye exhibit high fluorescence at all temperatures below ~45 C, above which fluorescence decreases non-cooperatively over a range of ~30 degrees C. Both cold denaturation and high fluorescence over broad temperature ranges in the presence of Sypro Orange are characteristic of limited tertiary structure as in molten globules [64]. …”

Thermodynamic impacts of combinatorial mutagenesis on protein conformational stability: precise, high-throughput measurement by Thermofluor

“…Thus, the modules that remain intact at the highest temperatures are also the most ancient part of the protein which, when excerpted, retain a full range of catalytic activities 24,25,64 . We note the curious consistency between the structural integrity of the TrpRS Urzyme at high temperature and the experimental observation that the Urzyme itself seems to function as a molten globule 65 , whose only possible well-folded state is its complex with the transition state for amino acid activation.…”