Abstract

Caveolae are 50–100 nm invaginations found within the plasma membrane of cells. Caveolae are involved in many processes that are essential for homeostasis, most notably endocytosis, mechano-protection, and signal transduction. Within these invaginations, the most important proteins are caveolins, which in addition to participating in the aforementioned processes are structural proteins responsible for caveolae biogenesis. When caveolin is misregulated or mutated, many disease states can arise which include muscular dystrophy, cancers, and heart disease. Unlike most integral membrane proteins, caveolin does not have a transmembrane orientation; instead, it is postulated to adopt an unusual topography where both the N- and C-termini lie on the cytoplasmic side of the membrane, and the hydrophobic span adopts an intramembrane loop conformation. While knowledge concerning the biology of caveolin has progressed apace, fundamental structural information has proven more difficult to obtain. In this mini-review, we curate as well as critically assess the structural data that have been obtained on caveolins to date in order to build a robust and compelling model of the caveolin secondary structure.

Introduction

Caveolae are 50–100 nm wide plasma membrane invaginations that punctuate the plasma membrane of many mammalian cell types [1]. Caveolae are primarily involved in signal transduction, mechano-protection, and endocytosis and have a lipid composition marked by increased levels of sphingomyelin and cholesterol [2]. Caveolins are the most abundant proteins found in caveolae, and it has been demonstrated that they are necessary to promote and stabilize the high degree of membrane curvature found in caveolae [36]. Recently, the cavin family of proteins has been shown to play a critical part in this process as well [7]. The central role that caveolins play in cellular homeostasis is apparent as mutations and misregulation of caveolins are linked to a plethora of human disease states including muscular dystrophy, Alzheimer's disease, heart disease, and cancers [2].

Caveolin is an integral membrane protein, and humans express three isoforms: -1, -2, and -3 (20.5, 16.8, and 17.2 kDa, respectively). Caveolins-1 and -3 share a high degree of sequence identity (52%), while caveolin-2 possesses significantly lower sequence identity with -1 and -3 (29 and 32%, respectively). Caveolins-1 and -2 are co-expressed across many cell types whereas caveolin-3 is specifically located in muscle tissues [8,9]. The expression of caveolin-1 or caveolin-3 is sufficient for caveolae formation; however, caveolin-2 requires the co-expression of caveolin-1 [1012].

The hallmark structural feature of caveolin is a central segment, which is postulated to adopt a loop conformation within the hydrophobic core of the bilayer, resulting in both the N- and C-termini residing on the cytoplasmic side of the plasma membrane and no penetration of the polypeptide chain into the extracellular space [1316]. Traditionally, caveolins are viewed as having four major structural domains: the N-terminal domain (NTD), the scaffolding domain (CSD), the intramembrane domain (IMD), and the C-terminal domain (CTD) [17]. It is important to note that the molecular mass variation between the isoforms arises from differing lengths of the NTD (caveolin-1, 81 residues; caveolin-2, 66 residues; and caveolin-3, 54 residues).

The cellular eminence of caveolins, particularly their distinctive role in curving bilayers, has sparked immense interest in providing a structural model that can explain their biological functions. However, experimentally verifying the structure of caveolin has been a painstaking process due to the intractability of the protein (high degree of insolubility) and has prompted many researchers to take a piecewise approach. The goal of this up-to-date review is to amalgamate the data obtained under different experimental conditions (i.e. various membrane mimics and construct lengths) to produce a coherent and unified thesis on the secondary structure of caveolins.

Caveolin domains

N-Terminal domain

Hydropathy plots predict that the NTD of caveolins is soluble. However, they differ markedly in both length and amino acid composition (caveolins-1 and -2 ∼23% identity, caveolins-1 and -3 ∼39% identity, and caveolins-2 and -3 ∼31% identity; Figure 1). The NTD has been shown to support many critical functions of the caveolin protein; it harbors several important phosphorylation sites and has been implicated in many protein–protein interactions [1822].

Sequence alignment of caveolin isoforms (human).

Figure 1.
Sequence alignment of caveolin isoforms (human).

The colored regions of the sequences denote the originally proposed domain (NTD, purple; CSD, green; IMD, red; CTD, blue). Cysteine residues are bolded. Underline denotes site of palmitoylation. Dotted underline denotes the potential site of palmitoylation.

Figure 1.
Sequence alignment of caveolin isoforms (human).

The colored regions of the sequences denote the originally proposed domain (NTD, purple; CSD, green; IMD, red; CTD, blue). Cysteine residues are bolded. Underline denotes site of palmitoylation. Dotted underline denotes the potential site of palmitoylation.

Much of the experimental data on the structure of the NTD come from circular dichroism spectroscopy (CD) studies on purified N-terminal fragments of various lengths [23,24]. The first study probed the secondary structure of the complete NTD of caveolin-1 in aqueous buffer and yielded a CD spectrum that was an indicative of a mostly random coil conformation [23]. Another study investigated three short segments of the NTD (Cav12–20, Cav119–40, and Cav168–80) in aqueous buffer or in the presence of small unilamellar vesicles (SUVs) [24]. All three peptides were mainly disordered, which is in agreement with the studies for the Cav11–80 fragment. Notably, the presence of SUVs did not result in a significant induction of secondary structure which suggests that these fragments did not interact with the membrane, although the short length of the peptides precludes making any definitive conclusions about the behavior of the NTD as a whole.

In-depth computational studies of caveolin-1 carried out by many researchers all concluded that the NTD was primarily unstructured, but could contain short segments of secondary structure interspersed throughout the domain. Parton et al. identified a single segment, residues 30–50, which could form an amphipathic α-helix that rested on the membrane surface, while Spisni et al. and Ariotti et al. identified many α-helical (Spisni, 30–38, 43–46, 58–61; Ariotti, 30–38, 56–72) and β-strand segments (Spisni, 5–7, 13–14, 48–49, 63–66, 69–72; Ariotti, 12–14, 48–50, and 63–73) [2527]. While the presence of some secondary structure in the NTD was intriguing, these findings needed experimental validation.

A functional caveolin-1 construct that trafficks properly in vivo, Cav162–178, was reconstituted into lysomyristoylphosphatidylglycerol (LMPG) micelles and analyzed by nuclear magnetic resonance spectroscopy (NMR) [28,29]. Chemical shift data revealed that residues 62–80 of the NTD were dynamic and unstructured. This finding was consistent with previous CD studies and the predicted nature of the region by Parton et al. [23,25]. Importantly, this study showed that the presence of the CSD, IMD, and CTD did not impart any structure to the 62–80 region, mitigating the argument that the unstructured nature of the NTD observed by CD was a result of its isolation from the rest of the protein.

NMR studies of a full-length caveolin-3 construct reconstituted into lysopalmitoylphosphatidylglycerol (LPPG) micelles confirmed that the NTD was primarily disordered, and strong solvent-protein Nuclear Overhauser Effect(s) indicated that it was not interacting with the micelle (i.e. soluble) [30]. However, the authors observed by CD, fluorescence spectroscopy, and NMR that a lowering of the pH (∼5) significantly increased the helicity of the NTD (primarily residues 10–24) and caused it to associate with the micelle. While these studies revealed that the NTD has the ability to adopt secondary structure and associate with membranes under acidic conditions, the exact biological implications of this behavior were unclear as caveolin-3 residing in caveolae is unlikely to experience a low pH environment.

Taken together, these studies show that the NTD of caveolins is largely unstructured at physiological pH. However, detailed NMR experiments will need to be employed for residues 1–61 (preferably in the context of the full-length protein) of caveolin-1 to verify whether the secondary structure predicted in silico is actually present. For example, many β-strand segments predicted to be present in the NTD for caveolin-1 by both Spisni et al. and Ariotti et al. are not supported by NMR experiments [26,27,29].

Scaffolding domain

The CSD has been purported to be functionally important to many aspects of caveolin behavior, including associations with numerous signaling proteins [most notably endothelial nitric oxide synthase (eNOS)], cholesterol-binding, and oligomerization [14,15,3135]. The length of the CSD for each isoform is identical (20 amino acids), and the homology is particularly high between caveolins 1 and 3 (caveolins-1 and -2, ∼35% identity; caveolins-1 and -3, ∼75% identity; caveolins-2 and -3, ∼45% identity; Figure 1).

Initial insights into the structure of the CSD were obtained from CD studies which included constructs that contained both the NTD and CSD [23]. Cav11–101 in buffer alone displayed signatures of the α-helical content (∼20%), and since a construct lacking the CSD (Cav11–80) displayed little to no helical character, it was deduced that the helical content was localized to the CSD. Analogous experiments carried out for Cav21–73 and Cav31–74 resulted in the same conclusion; the CSD region contained significant levels of helicity [23].

Computational analyses by Parton et al. and Spisni et al. of caveolin-1 also supported the helical content in the CSD [25,26]. Parton et al. identified residues 81–92 as being helical followed by an unstructured region from residues 92–96. The remaining residues, 97–101, were predicted to be part of a longer α-helix with a terminal residue of F107, which is in the IMD. On the other hand, Spisni et al. reported that the CSD had a β-strand character from residues 84–87 and 91–94 and that only residues 95–101 were α-helical. Ariotti et al. arrived at two conclusions for the CSD: (i) α-helical and β-strand character interspersed in the 83–93 region followed by α-helical character to the end of the CSD and (ii) α-helical over the entire CSD [27].

In an attempt to clarify these computationally based structural insights, many studies, involving all of the three isoforms, further investigated the secondary structure of the CSD utilizing short peptidic fragments ranging in length from 9 to 28 residues [3639]. While these studies did reinforce the general helicity of the CSD (with the exception of a singular study which identified a β-strand segment), they failed to produce a consensus as to the exact location and length of secondary structure elements [40]. This high degree of discordance was clearly attributable to insufficient construct length, as variability in solution conditions (i.e. membrane mimic) alone could not explain the differences. For example, when probing the secondary structure of Cav194–102, Le Lan et al. found that the removal of just one amino acid from the end of the construct resulted in a significant loss of helicity [38].

When a longer construct of caveolin-1, encompassing the full CSD in tandem with the full IMD (residues 82–136), was reconstituted into LMPG micelles, NMR experiments (chemical shift indexing) revealed a helical stretch from residues 87–107 [41]. A complementary study utilizing an even longer construct, residues 62–178, identified the helical stretch similarly as residues 89–107 [29]. NMR studies of full-length caveolin-3 reconstituted into LPPG micelles showed that residues 55–80 (analogous to caveolin-1 residues 82–107) were helical as well [30]. These findings indicated that the degree of helical content observed in the CSD was consistent only when sufficient flanking residues were present. Also, with no break in helicity between the end of the CSD and beginning of the IMD, the postulation that the CSD should be considered as an independently functioning domain was severely weakened.

Intramembrane domain

The IMD is a highly conserved domain among caveolins (caveolins-1 and -2, ∼48% identity; caveolins-1 and -3, ∼70% identity; caveolins-2 and -3, ∼42% identity) and is postulated to form the intramembrane U-conformation that is thought to induce and stabilize the membrane curvature required for caveolae formation (Figure 1) [16]. Thus, the knowledge of the secondary structure of this region is critical for understanding how caveolin interacts with the lipid bilayer.

The secondary structure of the IMD of caveolin-1 was predicted to be composed of two helices separated by an intramembrane turn based on computational modeling by two independent groups [25,26]. A model by Parton et al. indicated that the first helix begins in the CSD at residue 97 and ends at residue 107 followed by a four residue unstructured region, and the second helix spans residues 112–130 [25]. The model by Spisni et al. predicted a similar pattern for the secondary structure; the first helix spanned residues 95–109 until breaking at position 110, and the second helix spanned residues 111–132 [26]. Interestingly, computational analysis by Ariotti et al. also supported a helical IMD for caveolin-1, but did not indicate any potential breaks in helicity [27]. While these predictions were completely reasonable, structural insights from these models necessitated experimental data for validation.

A landmark study by Lee et al. provided the first experimental evidence for the secondary structure of the intact IMD [42]. The construct, which comprised residues 96–136 of caveolin-1 (Cav196–136), was reconstituted into LMPG micelles. CD indicated that the IMD was highly α-helical. Next, NMR was employed and chemical shift indexing revealed that Cav196–136 conformed to a helix–break–helix structure with the first helix starting in the CSD at residue 97 and extending into the IMD to residue 107 (11 residues, Helix-1). It was separated from the second helix spanning IMD residues 111–129 (19 residues, Helix-2) by a three residue (108–110) unstructured region. It is important to note that the experimental data were in good agreement with the computational predictions of Parton et al. and Spisni et al. [25,26]. The break is semi-conserved across caveolin isoforms (caveolin-1, GIP; caveolin-2, AIP; caveolin-3, GVP) and was postulated to be the intramembrane turn. The absolute conservation of the proline suggests that it is the crucial residue for the break as it could arrest the helical structure of Helix-1 (C-terminal), while simultaneously initiating helicity in Helix-2 (N-terminal). The strongest data in support of the break being an intramembrane turn came from Epand et al. who showed in vivo that when P110 was mutated to alanine, the N-terminus of the protein was located on the extracellular side of the plasma membrane [43]. This could reasonably be explained as the loss of the intramembrane turn, but since the C-terminus was not located in the study, one cannot rule out the possibility that both the N- and C-termini were located outside of the cell with the intramembrane turn being retained. Therefore, additional experiments supporting the presence of the intramembrane turn are warranted.

Approximately two years after the first report on the secondary structure of the full IMD, it was followed up with a study that evaluated the secondary and tertiary structure of two incomplete caveolin-1 IMD constructs (101–126 and 93–126) [44]. Aside from the obvious drawback of not having the full IMD, non-native lysine residues were added to the N- and C-termini of the constructs for increased solubility. Expectedly, modest differences were found when comparing the secondary structural features with the 96–136 data, but they were conceivably the consequence of the abbreviated constructs and the unevaluable effects of polylysination. The tertiary structure was also very difficult to rationalize in a bilayer context, particularly with respect to the large number of unstructured residues. Of particular concern was the employment of hexafluoroisopropanol (organic solvent) and sodium dodecyl sulfate micelles as membrane mimics. While reasonable from a secondary structure point of view, these mimics were considered as poor choices for tertiary structure determination due to their extremely harsh (denaturing) characteristics [45]. Although these studies represented a step forward because it was the first time that tertiary structure data were presented for caveolin-1, they will require further experimental validation to determine if critical factors such as the absence of a bilayer or the use of an abridged construct with solubility tagging negatively influenced the reported structure.

To address the question of how the CSD and IMD may influence each other structurally, NMR studies were performed on a Cav182–136 construct in LMPG micelles which contains the complete CSD and IMD [41]. NMR (chemical shift indexing) allowed the determination of the secondary structure for the entire construct. Interestingly, residues 87–107 (21 residues, Helix-1) were in the α-helical conformation, a much longer stretch than what was determined for Cav196–136. Therefore, most of the residues of the CSD are part of Helix-1. Importantly, the longer construct did not impact the location or length of the break or Helix-2 (111–129, 19 residues).

Another NMR study followed up on the work on Cav182–136 by utilizing a longer construct, Cav162–178, reconstituted into LMPG micelles [29]. This construct contained part of the NTD, but the complete CSD, IMD, and CTD [28]. The secondary structure of this construct in the CSD and IMD region was not altered significantly by the inclusion of NTD and CTD portions, mirroring the structural elements determined for Cav182–136. Therefore, Cav182–136 can be considered the minimum length required to display consistent secondary structural trends in the CSD and IMD. It is important to note that the β-strand segment observed by Hoop et al. using a Cav182–109 construct was not observed, again revealing the shortcomings of working with truncated CSD and IMD constructs.

NMR studies of the IMD utilizing a full-length caveolin-3 construct reconstituted into LPPG micelles displayed behavior that was similar to Cav162–178, where the CSD and IMD residues (55–98) were members of a helix-break-helix motif [30]. The length of Helix-1 and Helix-2 for caveolin-3 was 26 and 15 residues, respectively, differing significantly from caveolin-1 (21 and 19 residues). However, some assignments (∼9 residues) were missing in the N-terminal region of Helix-1 precluding strong conclusions about the exact length of the helices. Reminiscent of Cav162–178, the unstructured break between helix contained the triplet of amino acids (GVP).

Taken together, the structural data for the IMD clearly show that it adopts a helix–break–helix structure which is consistent with, but not proving, the presence of an intramembrane turn. The motif is in support of the current model of membrane curvature generation in caveolae; it imparts a ‘wedge-like' shape to the caveolin molecule which perturbs the lipid spacing in the inner and outer leaflets of the plasma membrane asymmetrically and results in curvature generation [46]. In addition, this secondary structural model helps to contextualize experimental findings that have implicated residues in Helix-1 as being involved in binding interactions between caveolin and soluble proteins while at the same time mediating crucial interactions with the bilayer [47,48]. Specifically, the placement of CSD residues within a helix that is partially embedded in the bilayer would limit their location to being membrane proximal, resulting in efficient interactions with acylated proteins (eNOS, G-proteins) and also providing a static geometric feature that is recognizable by a multitude of binding partners [49,50].

C-Terminal domain

The CTD of caveolin-1 has been implicated in many critical caveolin functions including membrane trafficking and oligomerization [17,20,28,5154]. The length of the CTD is similar between isoforms, and the amino acid sequence is only well-conserved between caveolins-1 and -3 (caveolins-1 and -2 ∼23% identity, caveolins-1 and -3 ∼52% identity, and caveolins-2 and -3 ∼20% identity; Figure 1). Caveolin-1 has been shown to be palmitoylated at three cysteine residues located in the CTD (133, 143, and 156), and mutation of these cysteines to serines did not influence the ability of caveolin to traffic to the membrane correctly. Therefore, palmitoylation may have only a limited impact on the caveolin fold [55]. Caveolin-2 has also been shown to be palmitoylated at three cysteine residues, once in the IMD and twice in the CTD (109, 122, and 145) [56]. Caveolin-3 has been shown to be heavily palmitoylated, although the exact sites of palmitoylation are not known, and unclear since caveolin-3 contains 9 cysteine residues spread throughout the entire sequence [57]. Although some have posited that caveolin-3 is palmitoylated at positions 105, 115, and 128 based on alignment with caveolin-1, this assertion has not been demonstrated experimentally [58].

Early insights into the secondary structure of the CTD came from computational studies based on primary sequence analyses of caveolin-1. Parton et al. predicted that the CTD had an amphipathic helix starting at residue 160, and the region containing the palmitoylation sites (133–159) was unstructured [25]. In contrast, Spinsi et al. predicted an amphipathic helix spanning residues 134–167 followed by an unstructured region for the remainder of the protein (168–178) [26]. A later computational investigation performed by Ariotti et al. similarly predicted helicity within the CTD (134–167) with the key difference of observed β-strand propensity in the 170–176 region [27]. Another noteworthy difference between the model of Ariotti et al. and the predictions of others is that there is no break in helicity between the CTD and IMD regions.

This secondary structure of this region was first supported experimentally using an 18 residue peptide corresponding to the end of the CTD that was determined to be 43% helical by CD in the presence of buffer alone or SUVs [24]. The secondary structure of the entire caveolin-1 CTD was investigated by NMR and CD spectroscopy utilizing a Cav162–178 construct reconstituted into LMPG micelles [29]. The results from this study indicated that a three residue unstructured region, 129–131, separates Helix-2 from a long 12-turn α-helix spanning residues 132–175 (Helix-3), in partial agreement with the prediction by Spisni et al. but not supporting the β-strand character in the region suggested by Ariotti et al. [27]. Akin to P110, a conserved proline at position 132 in caveolin-1 appeared to have the role of both a helix breaker and initiator [29]. Furthermore, NMR experiments in which proline 132 was mutated to leucine extended Helix-2 an additional four residues, which supported its role in breaking helicity in the region [59]. With the secondary structure of the CTD known, helical wheel analysis confirmed the amphipathic character predicted by the computational studies [29].

The secondary structure of the CTD of caveolin-3 was probed in the context of the full-length protein in LPPG micelles by NMR [60]. The study also attempted to look at the effect of palmitoylation by attaching an octyl group (via a disulfide bond) to three (C105, C115 and C128) of the four cysteines in the CTD. Due to the lipidation chemistry employed, this necessitated mutation of the remaining seven cysteines to other amino acids. Next, the effect of this modification was probed using a chemical shift perturbation plot. Only minor chemical shift perturbations were observed for the lipidated protein at positions near the thio-octyl attachment sites, indicating that acylation likely had little effect on the secondary structure of caveolin-3 [60]. In a follow-up NMR study, secondary structural elements were partially assigned to a construct containing only two thio-octyl groups (C115 and C128). It was found that the CTD of caveolin-3 did not contain a singular long α-helical stretch, but instead was composed of four short helices (106–113, 117–120, 125–128, and 132–145) separated by unstructured regions [30]. Therefore, the two studies on lipidated caveolin-3, from the same research group no less, appeared to purport very different conclusions; one in which the lipidation causes modest (negligible) effects, and the other where acylation significantly impacts CTD secondary structure. However, within the CTD, there were six missing NMR assignments, which significantly weakened the conclusion of a segmented helical stretch. In addition, the high number of mutations in the construct, the fact that the exact sites of caveolin-3 palmitoylation were not known, and the uncertainty that attaching an octyl group via disulfide bond formation was a good mimic of the actual palmitoyl group inhibited one from making definitive conclusions from these studies. Nonetheless, these studies did show that the CTD of caveolin-3 likely had helical elements.

Beyond secondary structure

In vivo, caveolins are believed to members of large hetero-oligomeric assemblies based on studies using “caveolae” that have been isolated from plasma membranes by detergent extraction [15,62]. If fidelity to the in vivo state of caveolae is accurately preserved in these detergent-extracted complexes, it is plausible that the complex itself could exert an influence on the secondary structure of caveolins [63]. However, precise information on the oligomeric state of the caveolin constructs, under the conditions used to determine the secondary structure, is largely lacking. In a single exception, Fernandez et al. used gel filtration chromatography and analytical ultracentrifugation to show that a construct containing the NTD was likely monomeric, while a construct containing the NTD and CSD showed oligomeric behavior [23]. However, since we now have information that CSD should not be studied independently of the IMD, there is the possibility that the oligomerization observed was due to the truncated construct. Nonetheless, at this point in time, there is simply not enough data to draw any meaningful conclusions on the relationship between oligomeric state and secondary structure.

In recent years, electron microscopy studies have probed the oligomeric structure of caveolin aggregates, and it is interesting to opine on how secondary structure elements may fit into these structures [64,65]. Unfortunately, the structure of a nonameric caveolin-3 aggregate determined in the presence of β-dodecyl-d-maltoside micelles had a resolution (∼17 Å) that was simply too coarse to allow for the positioning of secondary structural elements. In another study, an 80S particle-containing caveolin-1, cavins, and other membrane constituents isolated from HeLa cells using detergent extraction methods revealed that the aggregate was polyhedral in shape, mimicking the caveolar bulb. As was the case for the caveolin-3 nonamer, the low resolution of the cryo-electron microscopy structure made it difficult to confidently place caveolin-1, much less secondary structure elements, within the superstructure's architecture.

Perspective

  • Caveolin is an incredibly important protein that has been woefully under-characterized structurally. It is only now, 27 years after its identification that we are in a position to present a clear picture of its secondary structure. The main source of inconsistencies between published works is construct length (Table 1) and membrane mimic employed (i.e. micelles, vesicles, etc.). Although the data clearly show that construct length can significantly influence the observed secondary structure (particularly the CSD), the role of the membrane mimic appears to be less significant. Perhaps this is not surprising as studies have shown that secondary structure (but not tertiary structure) elements of membrane proteins are remarkably resistant to disruption by detergents [61].

  • We propose that caveolin should be considered as having three functional domains: NTD, IMD, and CTD. The NTD is unstructured, soluble, and does not interact appreciably with the membrane. The IMD has a helix-break-helix motif, is insoluble, and is deeply embedded in the membrane. The C-terminal domain is an amphipathic helix and likely rests on the surface of the membrane. Figure 2 summarizes our conclusions.

  • At this point, the secondary structure of caveolins is fairly clear, although there are areas where more experimentation is warranted. More data on caveolin-2, although it is not surprising that caveolin-2 has received less attention due to its secondary role in caveolae formation [11,12]. More data on palmitoylated constructs, although current evidence suggests that it is unlikely to have a significant effect on secondary structure. Detailed structural data (i.e. NMR) of caveolin-1 residues 1–61 given that the three isoforms diverge significantly in the NTD and that the NTD of caveolin-1 is the longest. In addition, future studies should aim to work with full-length or close to full-length constructs to minimize any truncation artifacts.

Cartoon of caveolin-1 secondary structure based on experimental data.

Figure 2.
Cartoon of caveolin-1 secondary structure based on experimental data.

X represents an amino acid residue and P represents proline.

Figure 2.
Cartoon of caveolin-1 secondary structure based on experimental data.

X represents an amino acid residue and P represents proline.

Table 1
Summary of secondary structure studies
Domain Construct Method Major result References 
NTD Cav11–80 CD NTD has a random coil conformation [23
Cav12–20
Cav119–40
Cav168–80 
CD Mainly disordered structure [24
Cav11–178 CP Amphipathic α-helix (residues 30–50) resting on bilayer surface [25
Cav11–178 CP α-helices (residues 30–38, 43–46, 58–61)
β-strand segments (residues 5–7, 13–14, 48–49, 63–66, 69–72) 
[26
Cav11–178 CP α-helices (residues 30–38, 56–72)
β-strand segments (residues 12–14, 48–50, 63–73) 
[27
Cav162–178 NMR Residues 62–80 dynamic/unstructured [29
Cav31–150 NMR Primarily disordered NTD [30
Cav31–150 FS, CD, NMR pH ∼5 increases helicity of NTD (residues 10–24) [30
CSD Cav11–101 CD α-helical character (∼20%) [23
Cav21–73 CD α-helical character (∼25%) [23
Cav31–74 CD α-helical character (∼25%) [23
Cav11–178 CP α-helix (residues 81–92)
Unstructured (residues 92–96)
Residues 97–101 predicted to be part of longer α-helix 
[25
Cav11–178 CP β-strand (residues 84–87, 91–94)
α-helix (residues 95–101) 
[26
Cav11–178 CP Interspersed β-strand and α-helical character (residues 83–93) followed by α-helical character to end of CSD or
α-helical character for the entire CSD 
[27
Cav182–101 NMR α-helix (residues 84–97) [36
Cav182–101
Cav182–109 
NMR and CD α-helical character only observed in Cav182–109 [37
Cav194–102 NMR Amphipathic α-helix in the polar head group region of DPC micelles [38
Cav369–74
Cav355–74
Cav355–69 
FS, CD, and MS All peptides bound to the bilayer surface [39
Cav182–136 NMR α-helical character (residues 87–107) [41
Cav162–178 NMR α-helical character (residues 89–107) [29
Cav31–150 NMR α-helical character (residues 55–80) [30
IMD Cav11–178 CP α-helix (residues 97–107, 112–130) [25
Cav11–178 CP α-helix (residues 95–109, 111–132) [26
Cav11–178 CP α-helix (entire IMD) [27
Cav196–136 NMR α-helix (residues 97–107, 111–129), helices separated by 3 residue “break’ [42
Cav1103–122 FS and CD N-terminus is extracellular as a result of P110A mutation [43
Cav1101–126
Cav193–126 
NMR Determination of tertiary structure of cav1 peptide fragments [44
Cav182–136 NMR α-helix (residues 87–107, 111–129), helices separated by 3 residue break [41
Cav162–178 NMR Mirrors secondary structure of Cav182–136 [29
Cav31–150 NMR CSD and IMD (residues 55–98) exhibit helix-break-helix motif [30
CTD Cav11–178 CP Unstructured region followed by amphipathic α-helix resting on the bilayer surface starting at residue 160 [25
Cav11–178 CP Long amphipathic α-helix on bilayer surface [26
Cav11–178 CP α-helical character observed in CTD up until residue 168
β-strand (residues 170–176) 
[27
Cav1161–178 CD 43% α-helical character [24
Cav162–178 NMR and CD Unstructured residues 129–131 separate Helix-2 from 12-turn α-helix (residues 132–175) [29
Cav196–136 NMR P132L mutant extends length of Helix-2 by four residues [59
Cav31–150 NMR Palmitoylation provides little to no change in secondary structure [60
Cav31–150 NMR CTD of Cav3 composed of four short α-helices (residues 106–113, 117–120, 125–128, 132–145) [30
Domain Construct Method Major result References 
NTD Cav11–80 CD NTD has a random coil conformation [23
Cav12–20
Cav119–40
Cav168–80 
CD Mainly disordered structure [24
Cav11–178 CP Amphipathic α-helix (residues 30–50) resting on bilayer surface [25
Cav11–178 CP α-helices (residues 30–38, 43–46, 58–61)
β-strand segments (residues 5–7, 13–14, 48–49, 63–66, 69–72) 
[26
Cav11–178 CP α-helices (residues 30–38, 56–72)
β-strand segments (residues 12–14, 48–50, 63–73) 
[27
Cav162–178 NMR Residues 62–80 dynamic/unstructured [29
Cav31–150 NMR Primarily disordered NTD [30
Cav31–150 FS, CD, NMR pH ∼5 increases helicity of NTD (residues 10–24) [30
CSD Cav11–101 CD α-helical character (∼20%) [23
Cav21–73 CD α-helical character (∼25%) [23
Cav31–74 CD α-helical character (∼25%) [23
Cav11–178 CP α-helix (residues 81–92)
Unstructured (residues 92–96)
Residues 97–101 predicted to be part of longer α-helix 
[25
Cav11–178 CP β-strand (residues 84–87, 91–94)
α-helix (residues 95–101) 
[26
Cav11–178 CP Interspersed β-strand and α-helical character (residues 83–93) followed by α-helical character to end of CSD or
α-helical character for the entire CSD 
[27
Cav182–101 NMR α-helix (residues 84–97) [36
Cav182–101
Cav182–109 
NMR and CD α-helical character only observed in Cav182–109 [37
Cav194–102 NMR Amphipathic α-helix in the polar head group region of DPC micelles [38
Cav369–74
Cav355–74
Cav355–69 
FS, CD, and MS All peptides bound to the bilayer surface [39
Cav182–136 NMR α-helical character (residues 87–107) [41
Cav162–178 NMR α-helical character (residues 89–107) [29
Cav31–150 NMR α-helical character (residues 55–80) [30
IMD Cav11–178 CP α-helix (residues 97–107, 112–130) [25
Cav11–178 CP α-helix (residues 95–109, 111–132) [26
Cav11–178 CP α-helix (entire IMD) [27
Cav196–136 NMR α-helix (residues 97–107, 111–129), helices separated by 3 residue “break’ [42
Cav1103–122 FS and CD N-terminus is extracellular as a result of P110A mutation [43
Cav1101–126
Cav193–126 
NMR Determination of tertiary structure of cav1 peptide fragments [44
Cav182–136 NMR α-helix (residues 87–107, 111–129), helices separated by 3 residue break [41
Cav162–178 NMR Mirrors secondary structure of Cav182–136 [29
Cav31–150 NMR CSD and IMD (residues 55–98) exhibit helix-break-helix motif [30
CTD Cav11–178 CP Unstructured region followed by amphipathic α-helix resting on the bilayer surface starting at residue 160 [25
Cav11–178 CP Long amphipathic α-helix on bilayer surface [26
Cav11–178 CP α-helical character observed in CTD up until residue 168
β-strand (residues 170–176) 
[27
Cav1161–178 CD 43% α-helical character [24
Cav162–178 NMR and CD Unstructured residues 129–131 separate Helix-2 from 12-turn α-helix (residues 132–175) [29
Cav196–136 NMR P132L mutant extends length of Helix-2 by four residues [59
Cav31–150 NMR Palmitoylation provides little to no change in secondary structure [60
Cav31–150 NMR CTD of Cav3 composed of four short α-helices (residues 106–113, 117–120, 125–128, 132–145) [30

Abbreviations: CD, circular dichroism spectroscopy; CP, computational (in silico); NMR, nuclear magnetic resonance spectroscopy; FS, fluorescence spectroscopy; MS, mass spectrometry.

Abbreviations

     
  • CD

    circular dichroism spectroscopy

  •  
  • CSD

    scaffolding domain

  •  
  • CTD

    C-terminal domain

  •  
  • DPC

    n-dodecylphosphocholine

  •  
  • eNOS

    endothelial nitric oxide synthase

  •  
  • IMD

    intramembrane domain

  •  
  • LMPG

    lysomyristoylphosphatidylglycerol

  •  
  • LPPG

    lysopalmitoylphosphatidylglycerol

  •  
  • NMR

    nuclear magnetic resonance spectroscopy

  •  
  • NTD

    N-terminal domain

  •  
  • SUV

    small unilamellar vesicle

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript.

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