The evolutionary rewiring of ubiquitination targets has reprogrammed the regulation of carbon assimilation in the pathogenic yeast Candida albicans

Date of Acceptance: 13/11/2012 This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited. Correction for Sandai et al., The Evolutionary Rewiring of Ubiquitination Targets Has Reprogrammed the Regulation of Carbon Assimilation in the Pathogenic Yeast Candida albicans published 20-01-2015 DOI: 10.1128/mBio.02489-14Peer reviewedPublisher PD

3D-modeling of Ssn6 fragments.

<p>(A) Modeling of Δ45: (Left) Cartoon representation of the initial 3D-model used for the explicit MD simulation. (Right) Average structure obtained from the last 10ns of the 100 ns MD trajectory, colored according to estimated atomic B-factors: from blue to red for low and high atomic fluctuations, respectively. (B) Modeling of NTpolyQ_TPR1-3: (Left) Cartoon representation of the initial 3D-model of a NTpolyQ_TPRs2.5 fragment used for the REMD simulations. (Right) (a) Average structures obtained from the last 10ns of the solvated 100 ns MD trajectories of the two REMD models (see “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186363#sec002" target="_blank">Materials & Methods</a>”). Coloring is according to estimated B-factors, as in A. (b), (c) and (d) Average structures obtained from the last 10ns of the second set of solvated 100 ns MD trajectories of the two NTpolyQ_TPR1-3 models colored according to B-factor values for the entire NTpolyQ_TPR1-3, NTpolyQ and TPR domains, respectively, for clarity. Various domains discussed in the text are labeled. Arrows point to susceptible to proteolysis sites of Ssn6, as described in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186363#pone.0186363.ref011" target="_blank">11</a>] (also shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186363#pone.0186363.g001" target="_blank">Fig 1C</a>).</p

Comparison of the Ssn6 NTpolyQ_TPR1-3 models with known TPR structures/interactions from the literature.

<p>(Left) Cartoon representations of the final models shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186363#pone.0186363.g005" target="_blank">Fig 5</a> with a slightly different coloring: the N11 is colored in magenta, for comparison. The known 3D-structures from the literature shown in this figure correspond to the following PDB entries: 1IYG (solution structure of a Fis1-like protein from a mouse cDNA, unpublished); 1ELR (crystal structure of the TPR2 domain of human Hop in complex with an Hsp90 peptide [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186363#pone.0186363.ref018" target="_blank">18</a>]); 3ZFW (crystal structure of the TPR-like domain of KLC2 in complex with a cargo peptide [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186363#pone.0186363.ref050" target="_blank">50</a>]); 2PQR (crystal structure of yeast Fis1p in complex with a fragment of yeast Caf4p [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186363#pone.0186363.ref022" target="_blank">22</a>]); 3GZ1 (crystal structure of the IpgC chaperone from <i>Shigella flexneri</i> in complex with the chaperone binding region of IpgBp [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186363#pone.0186363.ref024" target="_blank">24</a>]) and 3KS2 (crystal structure of a truncated IpgC fragment showing an alternative head-to-head dimerization mode of this chaperone [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186363#pone.0186363.ref025" target="_blank">25</a>]). Molecules participating in dimer formation (obtained from symmetry-related molecules in the corresponding crystal structures) are depicted as ribbon models and indicated as accentuated words. Met10 of Ssn6 and important hydrophobic residues of linear ligand peptides in known TPR-mediated complexes are depicted in sticks and are labeled. Arrows indicate helical regions of the known crystal structures that are spatially similar to the Qx16 and N11 helices in model-1.</p

Ssn6-Tup1 global transcriptional co-repressor: Role of the N-terminal glutamine-rich region of Ssn6

<div><p>The Ssn6-Tup1 complex is a general transcriptional co-repressor formed by the interaction of Ssn6, a tetratricopeptide repeat (TPR) protein, with the Tup1 repressor. We have previously shown that the N-terminal domain of Ssn6 comprising TPRs 1 to 3 is necessary and sufficient for this interaction and that TPR1 plays critical role. In a subsequent study, we provided evidence that in the absence of Tup1, TPR1 is susceptible to proteolysis and that conformational change(s) accompany the Ssn6-Tup1 complex formation. In this study, we address the question whether the N-terminal non-TPR, glutamine-rich tail of Ssn6 (NTpolyQ), plays any role in the Ssn6/Tup1 association. Our biochemical and yeast-two-hybrid data show that truncation/deletion of the NTpolyQ domain of Ssn6 results in its self association and prevents Tup1 interaction. These results combined with <i>in silico</i> modeling data imply a major role of the NTpolyQ tail of Ssn6 in regulating its interaction with Tup1.</p></div

Biochemical characterization of Ssn6 Δ45.

<p>(A) Size exclusion chromatography: Elution profile of Δ45 from the Biogel P100 gel-filtration column relative to the protein standards: bovine serum albumin (<i>BSA)</i>, ovalbumin (<i>OVA)</i>, carbonic anhydrase (<i>CAH)</i> and cytochrom <i>c (CYT</i>). (Inset): The standard log M_w vs K_av curve used to estimate the relative molecular weight of Δ45. Δ45 is indicated by an arrow. (B) Circular dichroism: Far-UV CD spectrum of 20 μM of Δ45 recorded at 10°C in phosphate buffer (pH 8).</p

Details of the two final models of the NTpolyQ_TPR1-3 fragment.

<p>(A) The final models (Top: model-1; Bottom: model-2) in cartoon representation. The N11, Qx16 and TPR regions are colored in cyan, yellow and green, respectively. The TPR domain is also depicted as a surface colored according to (B) electrostatic potentials and (C) Ssn6 mutational data; in red/orange: point mutation sites disrupting/affecting Tup1 interaction [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186363#pone.0186363.ref020" target="_blank">20</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186363#pone.0186363.ref026" target="_blank">26</a>]. Hydrophobic Ssn6 residues are labeled in B. (D) Cartoon representation of the models with details of the mutations colored in C.</p

Two-hybrid assays for Ssn6-Ssn6 and Ssn6-Tup1 interactions.

<p>Two-hybrid assays for Ssn6-Ssn6 and Ssn6-Tup1 interactions.</p

<i>In vivo</i> repression by LexA-Ssn6 and LexA-Tup1 derivatives.

<p><i>In vivo</i> repression by LexA-Ssn6 and LexA-Tup1 derivatives.</p