Evaluating Caveolin Interactions: Do Proteins Interact with the Caveolin Scaffolding Domain through a Widespread Aromatic Residue-Rich Motif?

<div><p>Caveolins are coat proteins of caveolae, small flask-shaped pits of the plasma membranes of most cells. Aside from roles in caveolae formation, caveolins recruit, retain and regulate many caveolae-associated signalling molecules. Caveolin-protein interactions are commonly considered to occur between a ∼20 amino acid region within caveolin, the caveolin scaffolding domain (CSD), and an aromatic-rich caveolin binding motif (CBM) on the binding partner (фXфXXXXф, фXXXXфXXф or фXфXXXXфXXф, where ф is an aromatic and X an unspecified amino acid). The CBM resembles a typical linear motif - a short, simple sequence independently evolved many times in different proteins for a specific function. Here we exploit recent improvements in bioinformatics tools and in our understanding of linear motifs to critically examine the role of CBMs in caveolin interactions. We find that sequences conforming to the CBM occur in 30% of human proteins, but find no evidence for their statistical enrichment in the caveolin interactome. Furthermore, sequence- and structure-based considerations suggest that CBMs do not have characteristics commonly associated with true interaction motifs. Analysis of the relative solvent accessible area of putative CBMs shows that the majority of their aromatic residues are buried within the protein and are thus unlikely to interact directly with caveolin, but may instead be important for protein structural stability. Together, these findings suggest that the canonical CBM may not be a common characteristic of caveolin-target interactions and that interfaces between caveolin and targets may be more structurally diverse than presently appreciated.</p> </div

List of Cav-1-interacting molecules reported as containing CBM-like motifs.

<p>List of Cav-1-interacting molecules reported as containing CBM-like motifs.</p

Cross-eyed stereo view of the context of the CBM of Couet <i>et al</i>[<b>6</b>] seen in the rat Gi1α protein (PDB code 1CIP; [<b>51</b>]).

<p>The β-hairpin structure of the motif is shown as a cartoon, coloured from blue to red, and the aromatic residues drawn as sticks (Phe189 is blue, Phe191 is cyan, Phe196 is yellow and Phe199 is red). The third strand of the three-stranded sheet to which the motif belongs is also shown in pink. The remainder of the protein is shown as lines and surface, the latter coloured green where contributed by side chains of the aromatic residues.</p

Predicted change in folding free energy (ΔΔG) resulting from alanine mutation.

<p>Predictions calculated using PoPMuSiC. ΔΔG values for proteins marked with * were determined from homology models (SWISS MODEL repository) and the PDB code given is that of the template (T) used for the model. Mutations predicted as significantly destabilising (ΔΔG>2.00 kcal/mol) in bold font.</p

SABLE estimates of relative exposed surface area (RSA) of CBM aromatics.

<p>Predictions are given in ranges spanning 10%. Buried residues (RSA<20%) are in bold font.</p

Relative exposed surface area (RSA) of CBM aromatics.

<p>Relative exposed surface area (RSA) of CBM aromatics.</p

Neutrophil elastase does not breakdown oxyhaemoglobin but degrades methaemoglobin.

<p>Neutrophil elastase (2 μM) was incubated at 37°C with 4 μM oxyhaemoglobin (<i>A</i>) or with methaemoglobin (<i>B</i>), in 0.14 M NaCl, 0.1 M Tris-HCl, pH 7.5. The methaemoglobin substrate was formed by incubation of 100 μM oxyhaemoglobin with a 4-fold molar excess of sodium nitrite for 1 h at 37°C. Inset graph in panel <i>A</i>, difference spectra produced by subtraction of the elastase-methaemoglobin spectra from the control methaemoglobin spectra at each time point to show the amounts of methaemoglobin degraded with time (405 nm Soret band). Inset graph in panel <i>B</i>, difference spectra made by subtracting the enzyme incubation spectra from those of the control methaemoglobin spectra at each time period to show the incremental amounts of methaemoglobin degraded.</p

Formation of the HmuY-ferrihaem complex during co-incubation of oxyhaemoglobin with the <i>P. gingivalis</i> HmuY haemophore in the presence of pyocyanin.

<p>HmuY and pyocyanin concentrations were 32 and 50 μM, respectively, whilst that of oxyhaemoglobin was 4 μM (with respect to tetramer). Buffer was 0.14 M NaCl, 0.1 M Tris-HCl, pH 7.5. Arrows indicate the 525 nm Q band plus 558 nm shoulder related to the presence of the HmuY-ferrihaem complex, green line; oxyhaemoglobin at time zero, red line; oxyhaemoglobin plus HmuY, black line.</p

Myeloperoxidase activities (MPO) in homogenates of mouse lungs challenged with <i>P. gingivalis, P. gingivalis</i> plus pyocyanin, or pyocyanin alone.

<p>The MPO activity was expressed as % of control activity (mice inoculated with PBS only). The data points represent the mean ± SD from two independent MPO assays performed for each group (PBS+PCN, <i>P. gingivalis</i>, and <i>P. gingivalis</i>+PCN). The number of animals in the PBS and PBS+PCN groups was 10, while those in the <i>P. gingivalis</i> and <i>P. gingivalis</i>+PCN groups was 8 and 4 (at 24 h), 7 and 3 (at 48 h), and 7 and 0 (at 72 h), respectively. Statistical significance: ***<i>P</i><0.005.</p

Clinical evaluation scores of mice (on a scale of 0 to 3) after intra-tracheal inoculation with 3×10⁹ CFU <i>P. gingivalis</i> W83, or 3×10⁹ CFU <i>P. gingivalis</i> W83 + 40 μg pyocyanin.

<p>Filled bars, mice challenged with <i>P. gingivalis</i> plus pyocyanin. Statistical significance: *<i>P</i><0.01; **<i>P</i><0.05; ***<i>P</i><0.005.</p