Prediction and Experimental Characterization of nsSNPs Altering Human PDZ-Binding Motifs

<div><p>Single nucleotide polymorphisms (SNPs) are a major contributor to genetic and phenotypic variation within populations. Non-synonymous SNPs (nsSNPs) modify the sequence of proteins and can affect their folding or binding properties. Experimental analysis of all nsSNPs is currently unfeasible and therefore computational predictions of the molecular effect of nsSNPs are helpful to guide experimental investigations. While some nsSNPs can be accurately characterized, for instance if they fall into strongly conserved or well annotated regions, the molecular consequences of many others are more challenging to predict. In particular, nsSNPs affecting less structured, and often less conserved regions, are difficult to characterize. Binding sites that mediate protein-protein or other protein interactions are an important class of functional sites on proteins and can be used to help interpret nsSNPs. Binding sites targeted by the PDZ modular peptide recognition domain have recently been characterized. Here we use this data to show that it is possible to computationally identify nsSNPs in PDZ binding motifs that modify or prevent binding to the proteins containing the motifs. We confirm these predictions by experimentally validating a selected subset with ELISA. Our work also highlights the importance of better characterizing linear motifs in proteins as many of these can be affected by genetic variations.</p></div

nsSNPs can disrupt a PDZ-binding motif, create a new one, or not have an effect.

<p>The two arrows illustrate our algorithm to predict the first two cases. A nsSNP is considered to disrupt a PDZ-binding motif if the wild-type sequence (<i>j</i>) has a score <i>S_ij</i>><i>S_max</i> (green box), while the modified sequence (<i>j′</i>) has a score <i>S_ij′</i><<i>S_min</i> (red box) with at least one PDZ domain (<i>i</i>) (upper arrow). Reversely, nsSNPs can create new PDZ binding motifs if <i>S_ij</i><<i>S_min</i> and <i>S_ij′</i> ><i>S_max</i> (lower arrow). The confidence interval (yellow box) is used to ensure that the scores are different enough between the wild type and the mutant to predict nsSNPs effect on PDZ mediated interactions.</p

nsSNPs disrupting PDZ-binding motifs are only slightly under-represented in human proteins.

<p>The red curve shows the fraction of known nsSNPs predicted to disrupt PDZ-binding motifs for different values of the threshold <i>S_max</i>. The black curve shows the same data for random amino acid substitutions on residues affected by nsSNPs in human C-termini (error bars show standard deviation for 1000 randomization of nsSNPs).</p

PDZ binding is affected by nsSNPs.

<p>(A) Sequence logo representing the peptide binding profile of the first and second PDZ domain of Multiple PDZ Domain Protein (MPDZ) and the PDZ domain of cytosine interacting protein (CYTIP) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094507#pone.0094507-Tonikian1" target="_blank">[19]</a>. The interactions predicted to be disrupted by an nsSNPs are shown above. (B) Saturation binding of MPDZ#1, MPDZ#2 and CYTIP to peptides derived from sequence logos shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094507#pone-0094507-g004" target="_blank">Figure 4A</a> (filled circle) and related peptides containing nsSNPs (open circles). The ELISA signal at 450 nm is plotted vs. the PDZ concentration in mg/ml. Note the C-terminus of PCDHA1 corresponds to the third isoform in UniProt (Q9Y5I3-3).</p

PDZ-binding linear motifs are affected by nsSNPs.

<p>A) Number of nsSNPs that are found in PDZ-binding motif containing C-termini for a wide range of thresholds on PWM scores to define PDZ-binding motifs. Higher threshold values (corresponding to more stringent definitions of PDZ-binding motifs) result in few C-termini being considered as containing a PDZ-binding motif, hence few nsSNPs falling in these motifs. B) Distribution of nsSNPs in PDZ-binding motifs, computed as the ration between the number nsSNPs shown in panel A and the total number of amino acids in PDZ-binding motif containing C-termini. For the highest thresholds (>−6), the number of nsSNPs falling in PDZ-binding motifs is not lower than expected. Then for thresholds between −6 and −9, it becomes slightly lower. For even lower thresholds, we tend to the expected distribution observed for all C-termini (9.9% of positions affected by nsSNPs, dashed line). C) The corresponding P-value assuming a uniform distribution of nsSNPs in all C-terminal segments (binomial test).</p

A Short DNA Aptamer That Recognizes TNFα and Blocks Its Activity <i>in Vitro</i>

Tumor necrosis factor-alpha (TNFα) is a pivotal component of the cytokine network linked to inflammatory diseases. Protein-based, TNFα inhibitors have proven to be clinically valuable. Here, we report the identification of short, single-stranded DNA aptamers that bind specifically to human TNFα. One such 25-base long aptamer, termed VR11, was shown to inhibit TNFα signaling as measured using NF-κB luciferase reporter assays. This aptamer bound specifically to TNFα with a dissociation constant of 7.0 ± 2.1 nM as measured by surface plasmon resonance (SPR) and showed no binding to TNFβ. Aptamer VR11 was also able to prevent TNFα-induced apoptosis as well as reduce nitric oxide (NO) production in cultured cells for up to 24 h. As well, VR11, which contains a GC rich region, did not raise an immune response when injected intraperitoneally into C57BL/6 mice when compared to a CpG oligodeoxynucleotide (ODN) control, a known TLR9 ligand. These studies suggest that VR11 may represent a simpler, synthetic scaffold than antibodies or protein domains upon which to derive nonimmunogenic oligonucleotide-based inhibitors of TNFα

Primary and tertiary structural comparisons between SLT-1 and SLT-2 highlighting the conservation of important ribosomal stalk peptide contact sites.

<p>(A) <i>Left Panel</i> - Surface rendering of the SLT-1 A₁ chain (PDB# 1DM0) depicting the cationic (blue) and hydrophobic (yellow) residues essential for optimal binding to the conserved stalk peptide SDDDMGFGLFD as well as Arg-188 (light blue) which has a modest effect on peptide binding. <i>Right Panel</i> – Structure as shown in the left panel rotated by 140°, highlighting the catalytic residues in green. (B) Three-dimensional stick structures of SLT-1 (left panel), SLT-2 (PDB# 1R4P; middle panel), and the structural alignment of the two toxins (right panel). Cationic residues are labeled in blue and red, while hydrophobic residues are labeled in yellow and orange for SLT-1 and SLT-2 respectively. (C) Primary amino acid sequence alignment of SLT-1 and SLT-2 within residues 158 and 250. Catalytic residues are highlighted in green and cationic and hydrophobic residues in blue and yellow, respectively. Surface and stick renderings and alignments were performed using the The PyMOL Molecular Graphics System (Version 1.3, Schrödinger, LLC), whereas amino acid sequences were aligned using BioEdit software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031191#pone.0031191-Tchorzewski2" target="_blank">[59]</a>.</p

The A₁ chain of SLT-1 harbors a cationic surface composed of a cluster of arginine residues that interact with the ribosomal stalk protein P2 and the conserved C-terminal peptide.

<p>(A) A vector expressing a catalytically inactive variant of the SLT-1 A₁ domain (CIA₁) or one of the arginine-to-alanine point mutants as fusion partners with the GAL4 DNA-BD domain were co-transformed in the yeast strain AH109 with a vector expressing ribosomal protein P2 as a fusion construct to the GAL4-AD. The transformed yeast cells were plated on SD agar −Trp/−Leu. The resulting yeast colonies were grown overnight, and spotted (10 µl) as 10-fold serial dilutions onto SD medium lacking Trp and Leu to select for the presence of each plasmid followed by spotting on SD media lacking Trp, Leu, and His to select for interacting partners leading to colony growth. (B) SPR profiles illustrating the decrease in relative units for the arginine-to-alanine SLT-1 A₁ chain variants in relation to the wild-type A₁ chain, at a concentration of 15 µM, when presented to the immobilized peptide SDDDMGFGLFD. (C) Increasing salt concentrations led to a decrease or loss of binding of wild-type SLT-1 A₁ chain when exposed to the peptide SDDDMGFGLFD. SPR traces were plotted for the wild-type SLT-1 A₁ chain (15 µM) as a function of increasing salt concentrations.</p

The interaction of the A₁ chain of SLT-1 with the ribosomal stalk protein P2 and the C-terminal peptide SDDDMGFGLFD also involves hydrophobic residues within the A₁ chain.

<p>(A) Bait vectors expressing either a catalytically inactive variant of the wild-type SLT-1 A₁ domain (CIA₁) or one of the hydrophobic mutants were co-transformed in the yeast strain AH109 with a prey vector expressing ribosomal protein P2. The transformed yeast cells were plated on SD agar −Trp/−Leu. The resulting yeast colonies were grown overnight, and spotted (10 µl) as 10-fold serial dilutions onto SD medium lacking Trp and Leu to select for the presence of each plasmid followed by spotting on SD media lacking Trp, Leu, and His to select for interacting partners. (B) SPR profiles (plotted at 15 µM) demonstrate that hydrophobic mutants F226A and S235A in the SLT-1 A₁ chain have a minor effect on the binding to the conserved peptide SDDDMGFGLFD and the SLT-1 V191A and L233A A₁ chain mutants cause a drastic decrease in binding. Experiments were performed in triplicate.</p