New approach for membrane protein reconstitution into peptidiscs and basis for their adaptability to different proteins

Abstract

Previously we introduced peptidiscs as an alternative to detergents to stabilize membrane proteins in solution (Carlson et al., 2018). Here, we present ‘on-gradient’ reconstitution, a new gentle approach for the reconstitution of labile membrane-protein complexes, and used it to reconstitute Rhodobacter sphaeroides reaction center complexes, demonstrating that peptidiscs can adapt to transmembrane domains of very different sizes and shapes. Using the conventional ‘on-bead’ approach, we reconstituted Escherichia coli proteins MsbA and MscS and find that peptidiscs stabilize them in their native conformation and allow for high-resolution structure determination by cryo-electron microscopy. The structures reveal that peptidisc peptides can arrange around transmembrane proteins differently, thus revealing the structural basis for why peptidiscs can stabilize such a large variety of membrane proteins. Together, our results establish the gentle and easy-to-use peptidiscs as a potentially universal alternative to detergents as a means to stabilize membrane proteins in solution for structural and functional studies.

Cryo-EM structure of an elusive pre-transport intermediate of the multidrug ABC transporter BmrCD reveals modes of asymmetric drug binding

“…Table 2). In comparison to other prokaryotic exporters of known structure 2,6,[18][19][20][21][22][23][24] and related members of the ABCB 3,13,25 and ABCC 4,5 families, BmrCD geometry is most similar in architecture to the heterodimeric exporter TM287/288 asymmetrically bound to AMP-PNP in one NBS (PDB ID: 4q4a 22 ; RMSD 3.135 Å) (Supp. Fig.…”

“…Mutants selected are of a subset of residues observed interacting with Hoechst-33342 in the BmrCD-QQ H/ATP cryo-EM structure (see Figure 4). Supplemental Table 1 19 , 6bpp 20 , 6uz2 21 , 3b5y 23 , and 3b5z 23 , TM2987/288 PDB ID: 2hyd 24 , BmrA PDB ID: 6r81 (unpublished); McjD PDB ID: 4pl0 6 ) as well as from the ABCB (or Pgp) and ABCC families . The noncanonical NBDs, or the first NBD (half 1) in homomeric or single polypeptide transporters, are colored cyan, whereas the consensus NBDs, or the second NBD in homomeric or single polypeptide transporter, are colored orange.…”

Changes in Membrane Protein Structural Biology

“…The reconstitution can be achieved by "on-column", "in-gel" or "on-bead" approaches and MPs later purified by tag-based affinity chromatography [105]. An alternative "on-gradient" approach can be used to reconstitute a detergent solubilised MP [149]. MPs reconstituted into peptidiscs have been solved by cryo-EM and include the MscS ion channel and MsbA transporter [149].…”

“…An alternative "on-gradient" approach can be used to reconstitute a detergent solubilised MP [149]. MPs reconstituted into peptidiscs have been solved by cryo-EM and include the MscS ion channel and MsbA transporter [149].…”

A molecular mechanism for the generation of ligand-dependent differential outputs by the epidermal growth factor receptor

“…We purified full-length human EGFR expressed in human HEK293S GnTIcell line 17 , and reconstituted the complex of EGFR with EGF into four membrane-mimetic environments: detergent micelles, lipid nanodiscs 18 , amphipols 19 , and peptidiscs 20,21 (Extended Data Fig. 1).…”