Stepwise activation mechanism of the scramblase nhTMEM16 revealed by cryo-EM

ELife
Valeria KalienkovaCristina Paulino

Abstract

Scramblases catalyze the movement of lipids between both leaflets of a bilayer. Whereas the X-ray structure of the protein nhTMEM16 has previously revealed the architecture of a Ca2+-dependent lipid scramblase, its regulation mechanism has remained elusive. Here, we have used cryo-electron microscopy and functional assays to address this question. Ca2+-bound and Ca2+-free conformations of nhTMEM16 in detergent and lipid nanodiscs illustrate the interactions with its environment and they reveal the conformational changes underlying its activation. In this process, Ca2+ binding induces a stepwise transition of the catalytic subunit cavity, converting a closed cavity that is shielded from the membrane in the absence of ligand, into a polar furrow that becomes accessible to lipid headgroups in the Ca2+-bound state. Additionally, our structures demonstrate how nhTMEM16 distorts the membrane at both entrances of the subunit cavity, thereby decreasing the energy barrier for lipid movement.

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Datasets Mentioned

BETA
EMD-4589
EMD-4593

Methods Mentioned

BETA
X-ray
gel filtration

Software Mentioned

Coot
Phenix
MotionCor2
Relion
PyMOL Molecular Graphics System
ChimeraX
RELION2
WEBMAXC
FOCUS
ctffind4

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