A pH-Mediated Topological Switch within the N-Terminal Domain of Human Caveolin-3

Abstract

Caveolins mediate the formation of caveolae, which are small omega-shaped membrane invaginations involved in a variety of cellular processes. There are three caveolin isoforms, the third of which (Cav3) is expressed in smooth and skeletal muscles. Mutations in Cav3 cause a variety of human muscular diseases. In this work, we characterize the secondary structure, dynamics, and topology of the monomeric form of the full-length lipidated protein. Cav3 consists of a series of membrane-embedded or surface-associated helical elements connected by extramembrane connecting loops or disordered domains. Our results also reveal that the N-terminal domain undergoes a large scale pH-mediated topological rearrangement between soluble and membrane-anchored forms. Considering that roughly one-third of pathogenic mutations in Cav3 influence charged residues located in this domain, we hypothesize that this transition is likely to be relevant to the molecular basis of Cav3-linked diseases. These results provide insight into the structure of Cav3 and set the stage for mechanistic investigations of the effects of pathogenic mutations.

Figure 1

Two-dimensional solution NMR spectra of full-length lipidated Cav3 in LPPG micelles. (A) The ¹H ¹⁵N TROSY-HSQC spectrum of lipidated Cav3 in 100 mM imidazole (pH 6.5) containing 1 mM EDTA and 5% LPPG at 318 K and 800 MHz is shown. Peaks corresponding to Asn or Gln side-chain NH₂ groups are connected with a line for reference. The red box highlights the region magnified in the right inset. The tryptophan side-chain peaks are shown in the left inset. The five amide peaks that could not be assigned are marked for reference. (B) The ¹H ¹⁵N HSQC spectra of lipidated Cav3 in 75 mM imidazole containing 25 mM MES, 1 mM EDTA, and 5% LPPG are shown as a function of varying pH. To see this figure in color, go online.

Figure 2

Backbone dynamics of lipidated Cav3 in LPPG micelles. The dynamics of lipidated Cav3 in LPPG micelles at 45°C was probed by solution NMR. The longitudinal relaxation time (T1, A), transverse relaxation time (T2, B), and steady-state ¹H ¹⁵N NOEs (C) were measured using an 800 MHz spectrometer at pH 6.5 and 318 K. Blank regions indicate residues that could not be assigned. Error bars for T1 and T2 reflect the uncertainties associated with the fitting of relaxation data. Error bars for ¹H-¹⁵N NOE values reflect the spectral noise.

Figure 3

Solvent NOEs and paramagnetic relaxation enhancement of backbone amide resonances by paramagnetic probes. (A) An ¹⁵N-edited NOESY-HSQC spectrum of lipidated Cav-3 was acquired and analyzed under conditions of 100 mM imidazole (pH 6.5) containing 1 mM EDTA and 5% LPPG at 45°C. Backbone amide residues that exhibited detectable NOEs with water (blue), LPPG methylene hydrogens (red), or both (green) are indicated in the amino acid sequence. Residues for which NOEs could not be clearly interpreted are shown in black. The ¹H ¹⁵N HSQC spectrum of lipidated Cav3 was recorded in the presence and absence of water soluble (Gd-DTPA) or lipophilic (16-DSA) paramagnets under various conditions. (B) The intensity of backbone amide peaks in the presence of 10 mM Gd-DTPA (black) or 4 mol % 16-DSA (red) relative to those collected in the absence of paramagnetic reagents in 100 mM imidazole (pH 6.5), 1 mM EDTA, and 5% LPPG are shown for each residue. Negative peaks denote resonances for which the ratio could not be accurately determined. (C) The intensity of backbone amide peaks in the presence of 4 mol % 16-DSA relative to those collected in the absence of paramagnetic reagents are shown at pH 5.5 (red) and pH 7.2 (black). Negative bars denote residues for which the ratio could not be accurately determined. To see this figure in color, go online.

Figure 4

Influence of pH on the secondary structure of lipidated Cav3. The effect of pH on the secondary structure of lipidated Cav3 was investigated using CD spectroscopy at 25°C. (A) The far-UV CD spectrum of lipidated Cav3 in 25 mM sodium phosphate containing 0.2% LPPG was recorded with varying pH. The mean residue ellipticity is plotted against the wavelength. (B) The mean residue ellipticity at 222 nm is plotted against the pH. The data were fit with a model derived from the Henderson-Hasselbalch equation (black line), and the apparent pKa of the transition was determined to be 6.35 ± 0.04. To see this figure in color, go online.

Figure 5

pH-mediated conformational transition of the Cav3 N-terminal domain in LPPG micelles and in POPG vesicles. The influence of pH on the fluorescence spectrum of mBBr-labeled V25C Cav3 was assessed in micelles and vesicles. (A) The fluorescence emission spectrum of mBBr-labeled V25C Cav3 in 120 mM NaCl containing 15 mM acetic acid, 15 mM MES, 30mM Tris, 0.5 mM EDTA, and 5% LPPG was collected as a function of pH. The wavelength corresponding to the emission maximum is plotted against the pH. The data were fit with a model derived from the Henderson-Hasselbalch equation (black line), and the apparent pKa of the transition was determined to be 6.8 ± 0.1. (B) mBBr-labeled V25C Cav3 in POPG vesicles was equilibrated in 120 mM NaCl containing 15 mM acetic acid, 15 mM MES, 30 mM Tris, and 0.5 mM EDTA with varying pH before acquisition of the fluorescence emission spectrum. The wavelength corresponding to the emission maximum is plotted against the pH. Closed symbols reflect data for the titration from low to high pH. Open symbols reflect the data for the titration from high to low pH. The data were fit with a model derived from the Henderson-Hasselbalch equation (black line), and the apparent pKa of the transition was determined to be 5.11 ± 0.06.

Figure 6

pH-mediated membrane association of the Cav3 N-terminal domain. (A) Lipidated Cav3 was reconstituted in POPG vesicles and equilibrated in 75 mM imidazole (pH 7.0) containing 25 mM sodium acetate and 0.5 mM EDTA before acquisition of the ¹H ¹⁵N TROSY-HSQC spectrum (left). The pH of the sample was then lowered to pH 4.5 before a second acquisition of the ¹H ¹⁵N TROSY-HSQC spectrum (center). Finally, the pH of the original sample was restored to 7.0 before acquisition of a third ¹H ¹⁵N TROSY-HSQC spectrum (right). (B) The ¹H spectrum is shown for the initial sample in lipid vesicles at pH 7.0 (dark gray), as well as those taken subsequently at pH 4.5 (light gray) and then again at 7.0 (black).

Figure 7

Working model for the organization of monomeric Cav3 in membranes and the nature of its pH-mediated conformational transition. A potential interaction between the acidic caveolin signature sequence and basic residues of the caveolin scaffolding domain is implied in the upper panel. Protonation of acidic residues at low pH may disrupt this interaction and drive association of the N-terminal domain with the membrane interface. It is emphasized that the exact palmitoylation pattern of native Cav3 is not known (the pattern shown here is based on Cav1) and that most of the experiments on which this working model are based were conducted with mutant forms of Cav3—usually chemically modified, and usually using LPPG micelles as the model membranes. To see this figure in color, go online.