Identifying Structural Domains and Conserved Regions in the Long Non-Coding RNA lncTCF7

Long non-coding RNA (lncRNA) biology is a rapidly growing area of study. Thousands of lncRNAs are implicated as key players in cellular pathways and cancer biology. However, the structure–function relationships of these novel biomolecules are not well understood. Recent structural studies suggest that lncRNAs contain modular structural domains, which play a crucial role in their function. Here, we hypothesized that such structural domains exist in lncTCF7, a conserved lncRNA implicated in the development and progression of several cancers. To understand the structure–function relationship of lncTCF7, we characterized its secondary structure using chemical probing methods. Our model revealed structural domains and conserved regions in lncTCF7. One of the modular domains identified here coincides with a known protein-interacting domain. The model reported herein is, to our knowledge, the first structural model of lncTCF7 and thus will serve to direct future studies that will provide fundamental insights into the function of this lncRNA

Protein function annotation with Structurally Aligned Local Sites of Activity (SALSAs)

<p>Abstract</p> <p>Background</p> <p>The prediction of biochemical function from the 3D structure of a protein has proved to be much more difficult than was originally foreseen. A reliable method to test the likelihood of putative annotations and to predict function from structure would add tremendous value to structural genomics data. We report on a new method, Structurally Aligned Local Sites of Activity (SALSA), for the prediction of biochemical function based on a local structural match at the predicted catalytic or binding site.</p> <p>Results</p> <p>Implementation of the SALSA method is described. For the structural genomics protein PY01515 (PDB ID <ext-link ext-link-id="2aqw" ext-link-type="pdb">2aqw</ext-link>) from <it>Plasmodium yoelii</it>, it is shown that the putative annotation, Orotidine 5'-monophosphate decarboxylase (OMPDC), is most likely correct. SALSA analysis of YP_001304206.1 (PDB ID <ext-link ext-link-id="3h3l" ext-link-type="pdb">3h3l</ext-link>), a putative sugar hydrolase from <it>Parabacteroides distasonis</it>, shows that its active site does not bear close resemblance to any previously characterized member of its superfamily, the Concanavalin A-like lectins/glucanases. It is noted that three residues in the active site of the thermophilic beta-1,4-xylanase from <it>Nonomuraea flexuosa </it>(PDB ID <ext-link ext-link-id="1m4w" ext-link-type="pdb">1m4w</ext-link>), Y78, E87, and E176, overlap with POOL-predicted residues of similar type, Y168, D153, and E232, in YP_001304206.1. The substrate recognition regions of the two proteins are rather different, suggesting that YP_001304206.1 is a new functional type within the superfamily. A structural genomics protein from <it>Mycobacterium avium </it>(PDB ID <ext-link ext-link-id="3q1t" ext-link-type="pdb">3q1t</ext-link>) has been reported to be an enoyl-CoA hydratase (ECH), but SALSA analysis shows a poor match between the predicted residues for the SG protein and those of known ECHs. A better local structural match is obtained with Anabaena beta-diketone hydrolase (ABDH), a known β-diketone hydrolase from <it>Cyanobacterium anabaena </it>(PDB ID <ext-link ext-link-id="2j5s" ext-link-type="pdb">2j5s</ext-link>). This suggests that the reported ECH function of the SG protein is incorrect and that it is more likely a β-diketone hydrolase.</p> <p>Conclusions</p> <p>A local site match provides a more compelling function prediction than that obtainable from a simple 3D structure match. The present method can confirm putative annotations, identify misannotation, and in some cases suggest a more probable annotation.</p

A Tale of Two Isomerases: Compact versus Extended Active Sites in Ketosteroid Isomerase and Phosphoglucose Isomerase

Understanding the catalytic efficiency and specificity of enzymes is a fundamental question of major practical and conceptual importance in biochemistry. Although progress in biochemical and structural studies has enriched our knowledge of enzymes, the role in enzyme catalysis of residues that are not nearest neighbors of the reacting substrate molecule is largely unexplored experimentally. Here computational active site predictors, THEMATICS and POOL, were employed to identify functionally important residues that are not in direct contact with the reacting substrate molecule. These predictions then guided experiments to explore the active sites of two isomerases, <i>Pseudomonas putida</i> ketosteroid isomerase (KSI) and human phosphoglucose isomerase (PGI), as prototypes for very different types of predicted active sites. Both KSI and PGI are members of EC 5.3 and catalyze similar reactions, but they represent significantly different degrees of remote residue participation, as predicted by THEMATICS and POOL. For KSI, a compact active site of mostly first-shell residues is predicted, but for PGI, an extended active site in which residues in the first, second, and third layers around the reacting substrate are predicted. Predicted residues that have not been previously tested experimentally were investigated by site-directed mutagenesis and kinetic analysis. In human PGI, single-point mutations of the predicted second- and third-shell residues K362, H100, E495, D511, H396, and Q388 show significant decreases in catalytic activity relative to that of the wild type. The results of these experiments demonstrate that, as predicted, remote residues are very important in PGI catalysis but make only small contributions to catalysis in KSI