Design of ultra-swollen lipidic mesophases for the crystallization of membrane proteins with large extracellular domains

Alexandru Zabara,Josephine Tse Yin Chong,Chiara Speziale,Brett A Cromer,Calum John Drummond,Raffaele Mezzenga,Isabelle Martiel,Laura Stark

doi:10.1038/s41467-018-02996-5

Abstract

In meso crystallization of membrane proteins from lipidic mesophases is central to protein structural biology but limited to membrane proteins with small extracellular domains (ECDs), comparable to the water channels (3–5 nm) of the mesophase. Here we present a strategy expanding the scope of in meso crystallization to membrane proteins with very large ECDs. We combine monoacylglycerols and phospholipids to design thermodynamically stable ultra-swollen bicontinuous cubic phases of double-gyroid (Ia3d), double-diamond (Pn3m), and double-primitive (Im3m) space groups, with water channels five times larger than traditional lipidic mesophases, and showing re-entrant behavior upon increasing hydration, of sequences Ia3d→Pn3m→Ia3d and Pn3m→Im3m→Pn3m, unknown in lipid self-assembly. We use these mesophases to crystallize membrane proteins with ECDs inaccessible to conventional in meso crystallization, demonstrating the methodology on the Gloeobacter ligand-gated ion channel (GLIC) protein, and show substantial modulation of packing, molecular contacts and activation state of the ensued proteins crystals, illuminating a general strategy in protein structural biology.

Highlights

In meso crystallization of membrane proteins from lipidic mesophases is central to protein structural biology but limited to membrane proteins with small extracellular domains (ECDs), comparable to the water channels (3–5 nm) of the mesophase
Crystallizing membrane proteins is a challenging task, for those with large extracellular domains (ECDs), yet, this provides the foundation for membrane protein structural biology
We exploit the thermodynamic stable nature of these systems to crystallize a representative membrane protein with large ECDs, the Gloeobacter violaceus ligand-gated ion channel (GLIC), otherwise inaccessible to the classical in meso crystallization techniques (Fig. 1), and we show how the crystallization in meso of this protein leads to significantly improved packing of the proteins within the crystals and a differently observed space group compared to all the deposited structures of the same protein obtained by vapor diffusion crystallization, opening a promising strategy for crystallization of challenging membrane proteins

Summary

Introduction

In meso crystallization of membrane proteins from lipidic mesophases is central to protein structural biology but limited to membrane proteins with small extracellular domains (ECDs), comparable to the water channels (3–5 nm) of the mesophase. We combine monoacylglycerols and phospholipids to design thermodynamically stable ultraswollen bicontinuous cubic phases of double-gyroid (Ia3d), double-diamond (Pn3m), and double-primitive (Im3m) space groups, with water channels five times larger than traditional lipidic mesophases, and showing re-entrant behavior upon increasing hydration, of sequences Ia3d→Pn3m→Ia3d and Pn3m→Im3m→Pn3m, unknown in lipid self-assembly We use these mesophases to crystallize membrane proteins with ECDs inaccessible to conventional in meso crystallization, demonstrating the methodology on the Gloeobacter ligand-gated ion channel (GLIC) protein, and show substantial modulation of packing, molecular contacts and activation state of the ensued proteins crystals, illuminating a general strategy in protein structural biology. Among the greatest recent successes of the in meso method, it is noteworthy the resolution of the structure and the understanding of the mechanism of action of the highly relevant class of G-Protein coupled receptors 4–6

Methods

Results

Conclusion