Small-residue packing motifs modulate the structure and function of a minimal de novo membrane protein

Paul Curnow,Paul Verkade,Christopher Williams,Richard B Sessions,Matthew P Crump,Michael R Jones,Lorna R Hodgson,Christopher J Arthur,J L Ross Anderson,Deborah K Shoemark,Richard Stenner,Virginie Dufour,Benjamin J Hardy

doi:10.1038/s41598-020-71585-8

Abstract

Alpha-helical integral membrane proteins contain conserved sequence motifs that are known to be important in helix packing. These motifs are a promising starting point for the construction of artificial proteins, but their potential has not yet been fully explored. Here, we study the impact of introducing a common natural helix packing motif to the transmembrane domain of a genetically-encoded and structurally dynamic de novo membrane protein. The resulting construct is an artificial four-helix bundle with lipophilic regions that are defined only by the amino acids L, G, S, A and W. This minimal proto-protein could be recombinantly expressed by diverse prokaryotic and eukaryotic hosts and was found to co-sediment with cellular membranes. The protein could be extracted and purified in surfactant micelles and was monodisperse and stable in vitro, with sufficient structural definition to support the rapid binding of a heme cofactor. The reduction in conformational diversity imposed by this design also enhances the nascent peroxidase activity of the protein-heme complex. Unexpectedly, strains of Escherichia coli expressing this artificial protein specifically accumulated zinc protoporphyrin IX, a rare cofactor that is not used by natural metalloenzymes. Our results demonstrate that simple sequence motifs can rigidify elementary membrane proteins, and that orthogonal artificial membrane proteins can influence the cofactor repertoire of a living cell. These findings have implications for rational protein design and synthetic biology.

Highlights

Alpha-helical integral membrane proteins contain conserved sequence motifs that are known to be important in helix packing
Most de novo designs of membrane proteins have employed the chemical synthesis of short peptides that can assemble in model lipid bilayers[3,4,7,9,10,11] and biological expression has received less a ttention[5,6,8,12]
The biosynthesis of de novo proteins is an enticing prospect since it could generate large constructs that are inaccessible to chemical synthesis, test the degree of novelty that can be tolerated by living systems and engage with the biochemistry of the living cell

Summary

Introduction

Alpha-helical integral membrane proteins contain conserved sequence motifs that are known to be important in helix packing. Alongside continuing efforts to understand natural membrane proteins, there is an emerging interest in designing artificial membrane proteins from first principles Such de novo proteins can help reveal the fundamental relationships between primary sequence, structure, and function[1,2]. A key contribution to the folding and assembly of these proteins comes from van der Waals interactions between neighbouring transmembrane helices[13] These forces are optimised in natural proteins through a relatively limited number of sequence motifs that minimise the interhelical distance through sidechain packing. It has recently emerged that classical coiled-coil heptads featuring bulky side-chains at the interfacial a and d positions can produce very well-defined tertiary structures through van der Waals forces a lone[7] It remains to be seen whether motifs incorporating small sidechains can be integrated into de novo designs. A key challenge is that helical interfaces based around small residues can lack the favourable steric and energetic effects that arise from the interdigitation of larger groups

Methods

Results

Conclusion