Mechanistic Insight into the Reactivation of BCAII Enzyme from Denatured and Molten Globule States by Eukaryotic Ribosomes and Domain V rRNAs

In all life forms, decoding of messenger-RNA into polypeptide chain is accomplished by the ribosome. Several protein chaperones are known to bind at the exit of ribosomal tunnel to ensure proper folding of the nascent chain by inhibiting their premature folding in the densely crowded environment of the cell. However, accumulating evidence suggests that ribosome may play a chaperone role in protein folding events in vitro. Ribosome-mediated folding of denatured proteins by prokaryotic ribosomes has been studied extensively. The RNA-assisted chaperone activity of the prokaryotic ribosome has been attributed to the domain V, a span of 23S rRNA at the intersubunit side of the large subunit encompassing the Peptidyl Transferase Centre. Evidently, this functional property of ribosome is unrelated to the nascent chain protein folding at the exit of the ribosomal tunnel. Here, we seek to scrutinize whether this unique function is conserved in a primitive kinetoplastid group of eukaryotic species Leishmania donovani where the ribosome structure possesses distinct additional features and appears markedly different compared to other higher eukaryotic ribosomes. Bovine Carbonic Anhydrase II (BCAII) enzyme was considered as the model protein. Our results manifest that domain V of the large subunit rRNA of Leishmania ribosomes preserves chaperone activity suggesting that ribosome-mediated protein folding is, indeed, a conserved phenomenon. Further, we aimed to investigate the mechanism underpinning the ribosome-assisted protein reactivation process. Interestingly, the surface plasmon resonance binding analyses exhibit that rRNA guides productive folding by directly interacting with molten globule-like states of the protein. In contrast, native protein shows no notable affinity to the rRNA. Thus, our study not only confirms conserved, RNA-mediated chaperoning role of ribosome but also provides crucial insight into the mechanism of the process

The effect of eukaryotic ribosomes and their domain V rRNA over MG-like states of BCAII.

<p>(<b>A)</b> The <i>S</i>. <i>cerevisiae</i> (RYmg) and <i>L</i>. <i>donovani</i> (RLmg) 80S ribosome-assisted reactivation of molten globule-like (MG-like) BCAII suggests ~60–65% recovery. (<b>B)</b> Domain V RNA of <i>S</i>. <i>cerevisiae</i> (YdVmg) and <i>L</i>. <i>donovani</i> (LdVmg) reactivates 50–55% BCAII from MG-like state. RN represents the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153928#pone.0153928.g001" target="_blank">Fig 1</a> while RS_mg represents self refolding of the MG-like protein in presence of EDTA (~30%). Statistical significance is shown by ** (p < 0.001, one-way ANOVA, N = 5) compared to control. (<b>C-D</b>) Interaction of MG-like state of BCAII protein with the domain V rRNA from <i>L</i>.<i>donovani</i> and <i>S</i>.<i>cerevisiae</i> examined by SPR analysis is shown respectively. Sensograms recorded when varying concentrations of protein (in MG-like state) were passed over the RNA immobilized on the streptavidin chip showing the binding profile of MG-like BCAII with LdV <b>(C)</b> and YdV (<b>E</b>). The interaction pattern suggests significant affinity of the protein in MG-like state towards domain V RNA. Concentration dependent increase in protein (MG-BCAII) binding is shown for LdV (<b>D</b>), and YdV (<b>F</b>).</p

Binding analysis of BCAII in denatured and native states with the eukaryotic rRNA.

<p>Sensogram recorded when varying concentrations of protein (in fully denatured state (<b>A</b>) and native state (<b>B</b>)) were injected over the RNA immobilized on the streptavidin chip. Binding profile of fully denatured BCAII with LdV (<b>A</b>) and YdV (<b>B</b>) shows unusually steep association pattern indicating very fast mode of interaction. The experiment with denatured protein was done in low temperature. No notable concentration dependent increase in binding is observed for native BCAII with either LdV (<b>C</b>) or YdV (<b>D</b>) indicating no significant affinity of the native protein towards RNA.</p

Eukaryotic ribosomal RNA domain V-mediated folding of denatured BCAII.

<p>Secondary structure diagram of 3’end of the LSU-RNA of <i>T</i>. <i>brucei</i> with the domain V (highlighted in yellow) <b>(A)</b> and the same of <i>S</i>. <i>cerevisiae</i> <b>(B)</b>. The peptidyl transferase centre (PTC) in domain V is marked with arrows in (A and B). (<b>C)</b> BCAII reactivation by the domain V RNA of <i>L</i>. <i>donovani</i> (LdV) and <i>S</i>. <i>cerevisiae</i> (YdV) shows ~65% activity recovered. RN and RS represent the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153928#pone.0153928.g001" target="_blank">Fig 1</a>. Statistical significance is shown by ** (p < 0.001, one-way ANOVA, N = 5) compared to control (RN). (<b>D)</b> Time course of reactivation of BCAII by domain V RNA from <i>S</i>. <i>cerevisiae</i> (▪) and <i>L</i>. <i>donovani</i> (•) shows faster reactivation for <i>S</i>. <i>cerevisiae</i>.</p

<i>L</i>. <i>donovani</i> LSUβ RNA-assisted folding of denatured BCAII.

<p>(<b>A)</b> Large subunit rRNA (LSU-RNA) of <i>Trypanosoma brucei</i> (PDB code 4V8M) viewed from intersubunit side with different RNA domains (marked in different colours) shows extra rRNA helices. (<b>B)</b> Alignment of the domain V rRNA sequence of <i>L</i>. <i>donovani</i> with the same from <i>E</i>. <i>coli</i>, <i>S</i>.<i>cerevisiae</i> and <i>human</i>. (<b>C</b>) LSUβ domain of <i>L</i>. <i>donovani</i> shows ~65% reactivation of fully denatured BCAII. RN and RS represent the same as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153928#pone.0153928.g001" target="_blank">Fig 1</a>. Statistical significance is shown by ** (p < 0.001, one-way ANOVA, N = 5) compared to control (RN).</p

Denaturant-induced unfolding of BCAII.

<p>(<b>A)</b> Tryptophan fluorescence of native (orange) and denatured (green) Bovina Carbonic Anhydrase II (BCAII) shows reduction in fluorescence intensity upon denaturation. In the inset a cartoon representation of the crystal structure of BCAII enzyme (PDB code 1V9E) is shown with the tryptophan residues highlighted in red stick. <b>(B)</b> Emission spectra (320–600) of the extrinsic fluorescence of native, fully unfolded (unf+EDTA; unf-EDTA) and molten globule (MG+EDTA;MG-EDTA) BCAII recorded using 8-anilinonaphthalene-1-sulphonic acid (ANS) dye showing considerably higher binding of ANS with molten globule BCAII compared to native or unfolded BCAII. (<b>C)</b> Crystal structure of BCAII enzyme (PDB code 1V9E) having a diameter of ~5nm with its secondary structures highlighted in different colors. Under different conditions, change in the hydrodynamic diameter of native BCA upon denaturation to molten globule-like (~80% population 10nm in 1.5 M GuHCl without EDTA (brown), 100% population 18 nm in 1.5 M GuHCl with EDTA (pink)) and fully unfolded state (~60% population 27 nm in 6 M GuHCl with or without EDTA (blue)) is shown as obtained from dynamic light scattering (DLS) experiments (the experiment was repeated twice for each case, acquiring data twice each time).</p