**1. Introduction**

Biological membranes form barriers separating cellular or organellar contents from the external environment. These are comprised of complex mixtures of polar lipids and membrane proteins. The long-standing Fluid-Mosaic model described the structure of the membrane as a bilayer of freely laterally diffusing polar lipids forming a highly hydrophobic core and acting as solvent for the membrane proteins [1]. Whilst some features of this model still hold true, it is becoming increasingly clear that the membrane is more organised than this model suggested [2], with the presence of discrete domains or rafts in the plasma membrane acting as signalling hubs [3]. A further key feature of biological membranes is their asymmetry, with the individual monolayers that make up the bilayer having distinct lipid compositions and associated functional implications [4]. It is also becoming increasingly evident that the membrane lipids do not just act as a solvent for membrane proteins but have critical roles in their structure and function [5]. Indeed, it seems in many cases that the functional unit is a complex of membrane protein and associated lipids. An interesting study using the rhomboid protease GlpG as a model protein suggested that the presence of cavities and pockets on the external membrane-facing surfaces of a protein induce instability key for the membrane protein conformational changes. Lipid interactions in these regions do not limit the conformational flexibility of the protein but do reduce the instability associated with the presence of the cavities [6].

Until recently it has been challenging to obtain definitive information on the precise nature and role of the interactions between individual membrane proteins and membrane lipids. This has been due in part to the limitations in technologies and the fact that membrane proteins are typically solubilised from the membrane for structural and other

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**Citation:** Renard, K.; Byrne, B. Insights into the Role of Membrane Lipids in the Structure, Function and Regulation of Integral Membrane Proteins. *Int. J. Mol. Sci.* **2021**, *22*, 9026. https://doi.org/10.3390/ ijms22169026

Academic Editor: Masoud Jelokhani-Niaraki

Received: 20 July 2021 Accepted: 17 August 2021 Published: 21 August 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

biophysical analysis, a process using detergents that is designed to remove most if not all the interacting lipids. This has led to some controversy regarding some membrane protein structures [7]. Despite this, it has been possible to explore many important membrane protein–lipid interactions. In this review, we summarise recent advances in our understanding of the roles of lipids in a range of membrane proteins and membrane protein complexes (Table 1).

**Table 1.** Summary of the lipid interactions covered in this manuscript.



**Table 1.** *Cont.*

#### **2. Lipids and Respiratory Complexes**

High-resolution structural studies have provided a number of insights into the role that tightly associated lipids play in the structure of membrane proteins. In virtually all cases the proteins are extracted from the membrane using detergent. However, in many cases, tightly associated lipids remain in complex with the protein even after detergent extraction and purification. If the lipids are sufficiently ordered, then they can be observed in X-ray crystal structures. One early example of lipids being clearly visible was in the crystal structure of Formate Dehydrogenase-N (Fdh-N) [50]. Each protomer of Fdh-N contains three subunits with the γ-subunit and a single transmembrane (TM) domain of the βsubunit forming the integral membrane region. FdhN crystallised as a physiological trimer with the interactions between the individual membrane domains mediated by molecules of cardiolipin (CL) forming essential interactions between the integral membrane regions of the individual protomers and clearly stabilising the oligomer. The integral membrane regions of the individual protomers have little role in this interaction, with the lipid almost entirely responsible for mediating trimer formation within the membrane. Although Fdh-N functions as a monomer, it is highly likely that trimer formation is critical for stability and thus the CL molecules are essential for formation of the physiological oligomeric state of the protein [50].

More recently, and in an exciting development, researchers have exploited a detergentfree approach using styrene maleic acid copolymer (SMA) to extract the Alternative Complex III (ACIII) from the bacterium *Flavobacterium johnsoniae* to produce SMA lipid particles

(SMALPs) containing the ACIII along with native lipids [58]. The SMA approach punches holes in the membrane and surrounds the lipid and protein particles rather than disrupting the hydrophobic interactions between the membrane protein and the membrane lipids as detergents do [59]. One very appealing consequence of this mode of extraction from the membrane is that hitherto uncharacterised membrane complexes can be isolated. The researchers in this case effectively isolated a super complex of ACIII and cytochrome c oxidase. In addition, electron density assigned to 11 phospholipid (PL) molecules was also discernible in the structure in two key regions. The first region is between two of the ACIII subunits, suggesting a role for the PLs in the stability of the protein, and the second region flanks a triacylated cysteine residue in the ActB subunit, close to the site of menaquinol entry into the protein, suggesting a role for the PLs in the function of the protein [58]. Reports of lipids binding to other respiratory complexes are covered in an earlier review [60]. These include the yeas<sup>t</sup> bc1 complex with an initial crystal structure obtained in complex with five closely associated lipid molecules [61]. Interestingly, in this case the researchers were able to alter the amount of bound lipid by changing the purification protocol, limiting the amount of time the protein spent on an ion exchange chromatography column. Protein produced with this optimised purification protocol was more active and yielded a crystal structure with an additional bound lipid [62].

A cryo-EM structure revealed lipid bound to the complete F-type ATPase from sheep [63]. Two lipid molecules are bound into the c-ring, part of the integral membrane, Fo domain, central to proton translocation. The lipids are bound into both the matrix and the intramembrane space sides of the c-ring. The e subunit of Fo, which forms part of the hook apparatus, interacts with the lipid, possibly a lysolipid, bound to the intra-membrane side of the c-ring via the C-terminal Lys residue. This lipid-mediated connection between different regions of the Fo domain is thought to increase the stability of the complex but is also likely to play a key role in ATPase function. Confirmational changes in the protein would cause movement of subunit e away from the c-ring, removing the associated lipid. It is suggested that this is an early step in full opening of the proton translocation channel [63].

#### **3. Secondary Active Transporters**
