The non-random clustering of non-synonymous substitutions and its relationship to evolutionary rate

<p>Abstract</p> <p>Background</p> <p>Protein sequences are subject to a mosaic of constraint. Changes to functional domains and buried residues, for example, are more apt to disrupt protein structure and function than are changes to residues participating in loops or exposed to solvent. Regions of constraint on the tertiary structure of a protein often result in loose segmentation of its primary structure into stretches of slowly- and rapidly-evolving amino acids. This clustering can be exploited, and existing methods have done so by relying on local sequence conservation as a signature of selection to help identify functionally important regions within proteins. We invert this paradigm by leveraging the regional nature of protein structure and function to both illuminate and make use of genome-wide patterns of local sequence conservation.</p> <p>Results</p> <p>Our hypothesis is that the regional nature of structural and functional constraints will assert a positive autocorrelation on the evolutionary rates of neighboring sites, which, in a pairwise comparison of orthologous proteins, will manifest itself as the clustering of non-synonymous changes across the amino acid sequence. We introduce a dispersion ratio statistic to test this and related hypotheses. Using genome-wide interspecific comparisons of orthologous protein pairs, we reveal a strong log-linear relationship between the degree of clustering and the intensity of constraint. We further demonstrate how this relationship varies with the evolutionary distance between the species being compared. We provide some evidence that proteins with a history of positive selection deviate from genome-wide trends.</p> <p>Conclusions</p> <p>We find a significant association between the evolutionary rate of a protein and the degree to which non-synonymous changes cluster along its primary sequence. We show that clustering is a non-redundant predictor of evolutionary rate, and we speculate that conflicting signals of clustering and constraint may be indicative of a historical period of relaxed selection.</p

A Novel N-Terminal Domain May Dictate the Glucose Response of Mondo Proteins

<div><p>Glucose is a fundamental energy source for both prokaryotes and eukaryotes. The balance between glucose utilization and storage is integral for proper energy homeostasis, and defects are associated with several diseases, e.g. type II diabetes. In vertebrates, the transcription factor ChREBP is a major component in glucose metabolism, while its ortholog MondoA is involved in glucose uptake. Both MondoA and ChREBP contain five Mondo conserved regions (<em>MCRI-V</em>) that affect their cellular localization and transactivation ability. While phosphorylation has been shown to affect ChREBP function, the mechanisms controlling glucose response of both ChREBP and MondoA remain elusive. By incorporating sequence analysis techniques, structure predictions, and functional annotations, we synthesized data surrounding Mondo family proteins into a cohesive, accurate, and general model involving the <em>MCR</em>s and two additional domains that determine ChREBP and MondoA glucose response. Paramount, we identified a conserved motif within the transactivation region of Mondo family proteins and propose that this motif interacts with the phosphorylated form of glucose. In addition, we discovered a putative nuclear receptor box in non-vertebrate Mondo and vertebrate ChREBP sequences that reveals a potentially novel interaction with nuclear receptors. These interactions are likely involved in altering ChREBP and MondoA conformation to form an active complex and induce transcription of genes involved in glucose metabolism and lipogenesis.</p> </div

Nuclear receptor box conservation.

<p>A LxQLLT motif is largely conserved among animals. Since we could not obtain the full sequence of all sampled species (shown in the species tree), many display alignment gaps, which do not necessarily indicate they lack the putative <i>NRB</i>. However, MondoA in vertebrates exhibits a divergent sequence and lacks the <i>NRB</i>.</p

Mondo conserved regions.

<p>MondoA and ChREBP have five previously defined and uniquely conserved regions, i.e. <i>MCRI-V</i>. These have been grouped into the <i>LID</i> and <i>GRACE</i> regions in ChREBP, and annotated for nuclear export signals (NES1, NES2), α-helix necessary for 14-3-3 binding, and a bipartite nuclear localization signal. These domains, along with newly identified <i>MCR6</i>, are highly conserved among Mondo sequences, with Mondo invariant positions marked with a red ‘X’. Weblogos depicting the particularly conserved sites and regions were created using the full Mondo alignment, with the previously defined MCR regions designated by a red line. We use the red line in MCR6 to accentuate the 12 residues with increased conservation in this region. Amino acids are colored so basic (HKR) residues are blue, acidic (DE) are red, and hydrophobic (AVLIFM) are green. Numbering is according to human ChREBP sequence.</p

<i>MCRII</i> helical wheel.

<p><b>A</b>) MondoA sites 121–138, <b>B</b>) ChREBP sites 81–98. Helical numbering is according to position within <i>MCRII</i> and represented by decreasing circle sizes. Black arrows point to sites indicated as essential for NES and red asterisks mark those necessary for glucose responsive transactivation. Color scheme: blue-basic, pink-acidic, orange-nonpolar, green-polar, uncharged. <b>C</b>) <i>Drosophila</i> sequence. Yellow circles have at least 75% chemical identity among all Mondo sequences.</p

Mondo sequence and structure conservation.

<p><b>A</b>) <b>JS Conservation Score.</b> All Mondo sequences were used to construct an alignment of homologous sites. Black dots represent alignment columns, while sites within domains are colored: red: <i>MCRI-V</i>, orange: Myc box II-like (<i>MCR6</i>), green: nuclear receptor box, blue: basic helix-loop-helix-zipper, cyan: <i>DCD</i>. The dashed line sets the 90% threshold for JS scores <b>B</b>) <b>JS and Entropy Comparison.</b> red: sites with less than 10 residues, black at least 10 residues, where linear regression was performed on the latter with intercept = 0.8745, slope = −1.0803, r² = 0.55467. <b>C</b>) <b>Entropy Distribution.</b> Distribution of entropy values for sites with at least 10 residues <b>D</b>) <b>Domains and Secondary Structure.</b> Consensus secondary structure for ChREBP shown alongside its sequence domains.</p

Mondo and Mlx <i>WMC/DCD</i> alignment.

<p><i>DCD</i> region of Mondo and Mlx sequences from <i>Homo sapiens</i> (Hsap), <i>Rattus norvegicus</i> (Rnor), <i>Drosophila melanogaster</i> (Dmel), <i>Caenorhabditis elegans</i> (Cele), <i>Capitella capitata</i> (Ccap), and <i>Trichoplax adhaerens</i> (Tadh). Red numbering on top corresponds to human ChREBP position, while the bottom represents the Mlx numbering. Sites with >75% identity or chemical similarity are shaded dark and light gray respectively, while the five (four) predicted alpha helices for MondoA and ChREBP (Mlx) are boxed. Mondo invariant positions are marked with a blue ‘X’.</p

MondoA N-terminus structure.

<p>Predicted structure for MondoA:1–490. <b>A</b>) Ribbon structure. <b>B</b>) Filled structure. <i>MCRI</i> is red, <i>MCRII</i> is orange, <i>MCRIII</i> is yellow, <i>MCRIV</i> is green, <i>MCR6</i> is blue, and <i>MCRV</i> is purple. In addition, the first 42 residues potentially targeting MondoA to the OMM are light pink, and putative phosphorylation sites S143 and T187 are magenta, and the serine and threonine residues of <i>MCR6</i> are pale green. Left and right images are rotated 180 degrees.</p

<i>LID</i> and <i>GRACE</i> interaction.

<p><b>A</b>) MondoA (green) and ChREBP (blue) overlay of N-terminal predicted structure. <b>B</b>) Topical view of MondoA:1–490 ribbon structure. <i>MCRV</i> and <i>MCR6</i> are part of the <i>GRACE</i> region, while the <i>LID</i> includes <i>MCRI-IV</i>. <i>MCR</i> domains are colored as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034803#pone-0034803-g008" target="_blank">Figure 8</a>. <b>C</b>) Predicted allosteric affect of G6P binding to <i>MCR6</i>. <i>MCRII</i> and <i>MCRIII</i> release from <i>MCRV</i>, while <i>MCRI</i> and <i>MCRII</i> lock the “open” conformation to separate the <i>LID</i> and <i>GRACE</i> regions and support transactivation.</p