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Structural and dynamic properties of juxta-membrane segments of caveolin-1 and caveolin-2 at the membrane interface

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Abstract

Caveolins (cav1–3) are essential membrane proteins found in caveolae. The caveolin scaffolding domain of cav-1 includes a short sequence containing a CRAC motif (V94TKYWFYR101) at its C-terminal end. To investigate the role of this motif in the caveolin–membrane interaction at the atomic level, we performed a detailed structural and dynamics characterization of a cav-1(V94-L102) nonapeptide encompassing this motif and including the first residue of cav-1 hydrophobic domain (L102), in dodecylmaltoside (DM) or dodecylphosphocholine (DPC) micelles, as membrane mimics. Cav-1(V94-L102) partitioned better in DPC and in DM/anionic lipid micelles than in DM micelles, as shown by fluorescence titration and CD. NMR data revealed that this peptide folded as an amphipathic helix located in the polar head group region of DPC micelles. The two tyrosine side-chains, flanked by arginine and lysine residues, are situated on one face of this helix, whereas the phenylalanine and tryptophan side-chains are located on the opposite face. Fluorescence studies showed significant Trp subnanosecond rotations, the presence of several rotamers, and a heterogeneous location within the water/micelle interface. NMR studies of the shorter cav-1(V94-R101) peptide and of the homologous sequence of cav-2(I79SKYVMYKF87) allowed the description of the effect of L102 and of the amino acid variations occurring in cav-2 on the structure and localization in DPC micelles. Based on the topological model of caveolins, our results suggest that the cav-1 and cav-2 nonapeptides studied form interfacial α-helix membrane anchors in which the K/RhhhYK/Rh motif, also found in cav-3, may play a significant role.

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Abbreviations

BrDM:

7,8-Dibromododecylmaltoside

BrUM:

10,11-Dibromoundecanoylmaltoside

cmc:

Critical micellar concentration

CD:

Circular dichroism

CRAC:

Cholesterol recognition/interaction amino acid consensus

cav-n :

Caveolin-n with n = 1, 2 or 3

DM:

n-Dodecyl-β-d-maltoside

DMPA:

1,2-Dimyristoyl-sn-glycero-3-phosphate

DMPC:

1,2-Dimyristoyl-sn-glycero-3-phosphocholine

DMPG:

1,2-Dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]

DMPS:

1,2-Dimyristoyl-sn-glycero-3-phospho-l-serine

DPC:

Dodecylphosphocholine

DSS:

Dimethylsilapentane-sulfonic acid

FWHM:

Full width at half maximum

HSQC:

Heteronuclear single quantum correlated spectroscopy

MALDI/TOF:

Matrix-assisted laser desorption ionization time of flight

MEM:

Maximum entropy method

N-MAD:

N-terminal membrane attachment domain

NATA:

N-acetyltryptophanamide

NOESY:

Nuclear Overhauser effect spectroscopy

POPC:

1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine

POPS:

1-Palmitoyl-2-oleoyl-sn-glycero-3-[phospho-l-serine]

POPG:

1-Palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]

TOCSY:

Total correlated spectroscopy

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Acknowledgments

This paper is dedicated to the memory of J-M Neumann, who died during the course of this work.

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Authors and Affiliations

  1. CEA, iBiTecs, SB2SM, 91191, Gif-sur-Yvette, France

    Charlotte Le Lan, Jean Michel Neumann, Béatrice de Foresta & Nadège Jamin

  2. CNRS, URA 2096, 91191, Gif-sur-Yvette, France

    Charlotte Le Lan, Jean Michel Neumann, Béatrice de Foresta & Nadège Jamin

  3. Institut de Biochimie et de Biophysique Moléculaire et Cellulaire, Université Paris-Sud, UMR8619-CNRS, IFR115, 91405, Orsay, France

    Jacques Gallay & Michel Vincent

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  1. Charlotte Le Lan
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  2. Jacques Gallay
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  4. Jean Michel Neumann
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Le Lan, C., Gallay, J., Vincent, M. et al. Structural and dynamic properties of juxta-membrane segments of caveolin-1 and caveolin-2 at the membrane interface. Eur Biophys J 39, 307–325 (2010). https://doi.org/10.1007/s00249-009-0548-4

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