Structural and Functional Adaptability of Sucrose and Lactose Permeases from Escherichia coli to the Membrane Lipid Composition

Abstract

The lipid environment in which membrane proteins are embedded can influence their structure and function. Lipid–protein interactions and lipid-induced conformational changes necessary for protein function remain intractable in vivo using high-resolution techniques. Using Escherichia coli strains in which the normal phospholipid composition can be altered or foreign lipids can be introduced, we established the importance of membrane lipid composition for the proper folding, assembly, and function of E. coli lactose (LacY) and sucrose (CscB) permeases. However, the molecular mechanism underlying the lipid dependence for active transport remains unknown. Herein, we demonstrate that the structure and function of CscB and LacY can be modulated by the composition of the lipid environment. Using a combination of assays (transport activity of the substrate, protein topology, folding, and assembly into the membrane), we found that alterations in the membrane lipid composition lead to lipid-dependent structural changes in CscB and LacY. These changes affect the orientation of residues involved in LacY proton translocation and impact the rates of protonation and deprotonation of E325 by affecting the arrangement of transmembrane domains in the vicinity of the R302-E325 charge pair. Furthermore, the structural changes caused by changes in membrane lipid composition can be altered by a single-point mutation, highlighting the adaptability of these transporters to their environment. Altogether, our results demonstrate that direct interactions between a protein and its lipid environment uniquely contribute to membrane protein organization and function. Because members of the major facilitator superfamily present with well-conserved functional architecture, we anticipate that our findings can be extrapolated to other membrane protein transporters.

Minimal Out-of-Equilibrium Metabolism for Synthetic Cells: A Membrane Perspective

“…Membrane proteins have evolved to operate in a biological membrane and often exhibit dependencies on specific lipids for (optimal) functionality. Therefore, other vesicle-forming compounds, such as single chain-amphiphiles and block copolymers, generally do not or poorly support membrane protein function, with a few notable exceptions. − Membrane transport proteins that undergo large conformational changes in the translocation step typically make essential interactions with, e.g., lipid head groups or require specific hydrophobic chains. − Thus, if metabolism and energy conservation are to rely on multiple membrane proteins for selective communication with the external environment (Box ), natural lipids are key building blocks to achieve compartmentalization for life-like entities. In addition, lipids can be synthesized using biosynthetic routes, which enable the required genetic encoding for a synthetic cell (see Section ).…”

“…Selecting a lipid bilayer composition consisting of a minimal set of defined phospholipids that enable the functionality of a wide variety of membrane proteins is essential in building a synthetic cell (Figure ). Phospholipid bilayers of differing complexity have been used to reconstitute purified membrane proteins. − Complex mixtures extracted from natural sources, such as polar lipid extracts (e.g., from E. coli , soy, etc. ), are also used, as their varied composition provides a close-to-native environment that meets the structure/activity requirements of many proteins.…”

Eugene P. Kennedy’s Legacy: Defining Bacterial Phospholipid Pathways and Function

“…However, pssA null strains require mmolar levels of lactose as an energy source and all the amino acids. These requirements are due to mis-folding, as discussed in more detail later, of secondary transporters, which couple substrate accumulation to the proton electrochemical potential, for probably all amino acids and as well as lactose (DeChavigny et al, 1991;Zhang et al, 2003Zhang et al, , 2005Hariharan et al, 2018;Vitrac et al, 2020). It had been well known that PE is required for reconstitution of energy dependent uphill transport of substrate by the secondary transporter lactose permease (LacY) in proteoliposomes (Newman and Wilson, 1980;Newman et al, 1981).…”

“…Several other secondary transporters also undergo topological inversions in the absence of PE or show local structural changes that affect their function ( Zhang et al, 2003 , 2005 ; Hariharan et al, 2018 ; Vitrac et al, 2011 , 2020 ). Others using different approaches, have reported similar effects of lipid environment and EMD charge on MP structural organization ( Bowie, 2006 , 2013 ; Hickey and Buhr, 2011 ; Tunuguntla et al, 2013 ; McIlwain et al, 2015 ).…”