Secondary Structure Analysis of a Functional Construct of Caveolin-1 Reveals a Long C-Terminal Helix

Abstract

Caveolin-1 is an integral membrane protein that is the primary component of cell membrane invaginations called caveolae. While caveolin-1 is known to participate in a myriad of vital cellular processes, structural data on caveolin-1 of any kind is severely limited. In order to rectify this dearth, secondary structure analysis of a functional construct of caveolin-1, containing the intact C-terminal domain, was performed using NMR spectroscopy in lyso-myristoylphosphatidylglycerol micelles. Complete backbone assignments of caveolin-1 (residues 62–178) were made, and it was determined that residues 62–79 were dynamic; residues 89–107, 111–128, and 132–175 were helical; and residues 80–88, 108–110, and 129–131 represent unstructured breaks between the helices.

Main Text

Caveolin is the preeminent protein in plasma membrane invaginations called caveolae. In addition to being responsible for the formation of caveolae, caveolin also mediates other vital caveolae-related processes (1). The topology of caveolin is unusual as it is postulated to possess an intramembrane loop structure that places both the N- and C-termini on the cytoplasmic side of the plasma membrane (2). Typically, caveolin-1 is divided into four domains: the N-terminal domain (residues 1–81), the scaffolding domain (residues 82–101), the intramembrane domain (residues 102–134), and the C-terminal domain (residues 135–178).

To date, there is limited structural information on caveolin, and the majority of studies have employed short nonfunctional constructs (2). This has made the formation of a structural consensus difficult as the observed secondary structure appears to be highly dependent on the construct employed. Importantly, there have been no experimental structural studies of the C-terminal domain, either on its own or in the context of the other domains, even though it is vital for many functions of caveolin.

The C-terminal domain is important for movement of caveolin-1 from the Golgi apparatus to the plasma membrane, membrane attachment, the formation of networks of oligomers that are required for the formation of the hallmark striated coat that stabilizes caveolae, and binding signaling molecules such as endothelial nitric oxide synthase, connexin, and Retrovirus NSP4 (1, 3, 4, 5, 6, 7, 8). Additionally, frameshift mutations within the C-terminal domain have been identified in patients with pulmonary arterial hypertension (9).

In this study we probed the secondary structure of a functional (traffics correctly in vivo) construct of caveolin-1, the most ubiquitous of the caveolin isoforms (residues 62–178, Cav1_62–178), which includes the C-terminal domain (3). The advantage of taking this approach is that the effects that the domains have on each other can be accurately characterized to form a more complete picture of caveolin-1 secondary structure. It is important to note, that the secondary structure of caveolin-1 may be the protein’s most important structural feature as it is very much in question as to whether caveolin-1 possesses a significant amount of tertiary structure (unpublished data, S. Plucinsky and K.J. Glover).

Caveolin-1 has three sites of cysteine palmitoylation; however, it has been shown that palmitoylation is not required for proper caveolin-1 trafficking to caveolae (10). In addition, a recent study conducted on caveolin-3 (a close homolog of caveolin-1) showed that the introduction of synthetic palmitoyl groups at the analogous three sites had only minor effects on the protein’s behavior (11). Therefore, in our construct we chose to mutate each of the cysteine residues to serine. Additionally, M111 was mutated to leucine in order to be compatible with protein preparation procedures (see the Supporting Material for details). Clearly, Cav1_62–178 will capture the essence of caveolin-1, and give important insights into caveolin-1’s secondary structure.

Previous studies in our lab have shown that LMPG (lyso-myristoylphosphatidylglycerol) is the most suitable detergent for obtaining high-quality NMR spectra of caveolin-1, and has been used extensively for NMR studies of membrane proteins in general (2, 12, 13). We have obtained complete backbone assignments of Cav1_62–178, and Fig. S1 shows the assigned ¹H-¹⁵N HSQC spectrum.

To analyze the secondary structure, a Cα chemical shift index (CSI) plot was generated (Fig. S2). The plot shows that Cav1_62–178 has significant α-helical character, as evidenced by the presence of stretches of positive ΔCα values. This is corroborated by circular-dichroism spectroscopy data, which shows the characteristic signature of helicity, namely minima at 208 and 222 nm (Fig. S3). In particular, the C-terminal domain, which has not been previously characterized, appears to be highly α-helical. However, there are 27 residues (out of 117, 23%) that could not be attributed to a defined secondary structure using this methodology (shown in red in Fig. S2). The ambiguous residues within the C-terminal domain are isolated between stretches of positive ΔCα (helical) values, and are therefore unlikely to represent breaks in helical structure. This is in contrast to the ambiguous residues that lie outside of the C-terminal domain; they, with the exception of T91, are clustered between helical stretches (e.g., residues 129–134 and 108–110) and in the N-terminal region of the construct. Therefore, as opposed to the C-terminus, the clustering of these ambiguous residues seems to be more indicative of unstructured or dynamic regions. Taken together, the CSI analysis shows the presence of three major helices: residues 87–107, residues 111–129, and residues 135–178.

To clarify the ambiguities observed in the CSI plot and reinforce the secondary structure conclusions, the chemical shifts for the N, NH, CO, Cα, and Cβ were processed using TALOS+ (Table S1) (14). This program is able to accurately determine the secondary structure of polypeptides. The data indicate that residues 62–79 are dynamic, and residues 80–88 are unstructured. In this context, “dynamic” refers to residues with undefined ϕ- and ψ-angles, while unstructured refers to residues that have defined ϕ- and ψ-angles that do not fall within canonical secondary structure motifs (i.e., α-helix or β-sheet). The first major helix (Helix-1) begins at residue 89 and ends at residue 107. Helix-1 is immediately followed by a three-residue break (residues 108–110), and helical character is restored for the second major helix (Helix-2) from residues 111–128. Following the second helix, there is another break (residues 129–131), and the third major helix (Helix-3) begins at residue 132 and continues throughout the entire C-terminus until residue 175, just three residues from the end of the protein. Fig. 1 shows this data pictorially. This TALOS+ analysis clearly agrees with the CSI data (±2 residues at the beginning of Helix-1, ±1 residue at the end of Helix-2, and ±3 residues at the beginning and end of Helix-3) and clarifies that that the C-terminal domain is indeed a single long helix that does not contain any central breaks. In addition, it confirms that the clustering of ambiguous residues observed outside of the C-terminal domain were indeed due to unstructured and/or dynamic regions.

Figure 1.

Cartoon of TALOS+ data for Cav1_62–178. (Zigzag line) Dynamic structure. To see this figure in color, go online.

It should be noted that all of the TALOS+ helical predictions were assigned a value of “good”, meaning that the ϕ- and ψ-values fell within the helical region. Furthermore, the maximum standard deviation for any one residue was a very low 14°. Two residues were assigned as “no prediction” (H79 and V131). However, this ambiguity is not troublesome as the two residues fall outside the helical regions, H79 in the dynamic region and V131 in the second break.

A chemical shift perturbation plot was generated comparing constructs with and without the C-terminal domain (Fig. S4). From this plot, there are two regions showing significant perturbations (residues 80–103 and 129–136). To determine whether a perturbation was significant, the average chemical shift perturbation of all residues was calculated (dashed line in Fig. S4), and perturbations that fell above the average were labeled as significant. While residues 129–136 would be expected to show changes because they are proximal to the construct break point (residue 136), residues 80–103 are not expected, and highlight the importance of utilizing longer multidomain constructs that possess functionality. This insight also supports the important role for the C-terminal domain in the overall structure of caveolin. However, it cannot be ruled out that the perturbations observed for these residues are due in part to disparate protein-detergent effects in the two constructs.

The data shows that aside from residues 62–79, Cav1_62–178 is a primarily α-helical protein composed of three major helices forming a helix-break-helix-break-helix motif. We believe the first break (residues 108–110) may be the location of the putative intramembrane turn that returns the polypeptide chain to the same side of the membrane. The second break, residues 129–131, may allow for a transition from Helix-2 (uniformly hydrophobic) to Helix-3, which is predicted to be amphipathic based on helical wheel analysis (Fig. S5), and would likely rest horizontally on the surface of the membrane (15).

When examining the two break regions (break-1 residues 108–110, and break-2 residues 129–131), it is important to note the sequence similarities; both breaks contain a small side-chain amino acid followed by a β-branched amino acid and a proline. The notable difference is the additional β-branched amino acid in the second break. One important similarity is that both proline residues, 110 in the first break and 132 in the second break, reside at the head of helices; Helix-2 for 110 and Helix-3 for 132. This is quite reasonable as previous studies have shown that proline residues are favorable to initiate helices (16). Therefore it appears that these break regions are critical for preserving and maintaining the proper secondary structure of caveolin-1. When comparing this data to previous studies that employed short nonfunctional constructs, it is clear that the residues at the domain interfaces showed significant changes, and highlights the importance of characterizing the secondary structure of the domains in the context of each other (2).

The determination of the secondary structure of Cav1_62–178 represents a critical step forward in the understanding of caveolin-1 structure. Using a functional construct we now have, for the first time, obtained specific secondary structural data on the C-terminal domain of caveolin-1, and show that it is an amphipathic helix. Additionally, we present detailed secondary structural dataon the longest construct of caveolin-1 to date. These findings represent a significant step forward in the overall structural determination of caveolin-1 and will undoubtedly lead to new insights into this vital protein.

Author Contributions

S.M.P. performed the experiments; S.M.P. and K.J.G. analyzed the data and wrote the article; and K.J.G. designed the experiments.

Editor: Elizabeth Komives.

Supporting Citations

References (17, 18, 19, 20, 21, 22, 23) appear in the Supporting Material.