Membrane proteins account for 20% to 30% of all gene products in most genomes, and they are the targets of 50% of modern drugs.[1] Proteins embedded in or traversing the lipid bilayer mediate exchange of information and materials across membrane barriers. They are architecturally and functionally diverse. Single-pass transmembrane proteins have functional extracellular and/or intracellular domains connected by a single membrane-spanning helix. By contrast, integral membrane proteins typically have much of their mass embedded within the lipid bilayer, with multiple membrane-spanning segments connected by cytoplasmic and extracellular loops. Historically, integral membrane proteins have been difficult to study at a structural level. However, innovations in membrane protein crystallization and protein engineering have made such studies more tractable, allowing elucidation of many important structures at near-atomic resolution. Most membrane-embedded proteins are predominately helical, although β-strand membrane proteins also occur. Diverse ion channels and GPCRs are integral membrane proteins. One of the largest and most complicated membrane protein complexes characterized to date is that of mitochondrial complex I. This huge proton-pumping machine features 82 transmembrane helices, accounting for approximately half of its molecular mass.[2]

GPCRs are the largest family of membrane proteins—more than 800 have been identified in the human genome. GPCRs mediate fundamental signal transduction processes, touching virtually every aspect of human physiology, from vision, taste, and smell to cardio vascular, endocrine, immunologic, and reproductive functions. Not surprisingly, they represent an important class of drug target. The conserved domain structure of GPCRs includes seven transmembrane helices that pack together across the lipid bilayer. They form a ligand-binding cleft that opens to the extracellular space. The cleft can vary dramatically in size and shape in different GPCRs because some receptors recognize small molecules (e.g., the β2-adrenergic receptor) whereas others have protein ligands (e.g., chemokine receptors).

Structural studies of the β2-adrenergic receptor have revealed its mechanism of transmembrane signal transduction via the heterotrimeric G protein Gαs βγ (Fig.1A).[3] Binding of agonist in the extracellular-facing cleft induces key conformational changes in the cytoplasmic region, in particular a large movement of the sixth transmembrane helix and an extension of the cytoplasmic end of the fifth transmembrane helix (TM5). These alterations promote binding to the Gα-subunit of Gαs βγ. In binding Gα, the agonist-bound receptor functions as a guanine-nucleotide exchange factor (GEF), inducing exchange of guanosine diphosphate (GDP) for guanosine triphosphate (GTP). GTP-bound Gα dissociates from the βγ heterodimer to activate adenyl cyclase, while the free βγ component signals to Ca2+ channels. The inactive and active β2-adrenergic receptor structures are illustrated in Fig. 1A. Currently, at least 70 unique GPCR structures are available, providing substantial impact in drug development.[4]

Fig1. MEMBRANE PROTEIN STRUCTURES. (A) Structures of the β2 adrenergic receptor (β2 AR) in the inactive (left) and active (right) conformations. Like all G protein–coupled receptors, β2 AR contains seven transmembrane helices. The inactive structure was determined with the antagonist carazolol bound in the ligand-binding site, while the active structure was determined in complex with the tight-binding agonist BI-167107 (ligand are shown as yellow spheres). Agonist binding induces conformational changes on the cytoplasmic face of the receptor, including reorientation of the sixth transmembrane helix (TM6) and lengthening of TM5, which in turn promote binding of the heterotrimeric Gαβγ complex. Interactions with the receptor are mediated by the Gαs subunit (green). This interaction induces exchange of guanosine diphosphate for guanosine triphosphate in the Gαs subunit, and promotes dissociation of βγ complex (shown in cyan and magenta for β and γ, respectively). Illustration is drawn from PDB entries 2RH1 and 3SN6 for the inactive and active structures, respectively. T4 lysozyme fusion partners and a nanobody that was engineered to facilitate crystallization are not illustrated. (B) Structure of a bacterial homolog of the human vitamin K epoxide reductase (VKOR). VKOR is expected to bind vitamin K in a pocket homologous to that formed by four conserved transmembrane helices (green) that is occupied by a ubiquinone (magenta) in this structure. Warfarin inhibits VKOR by displacing vitamin K from this pocket. See text for further details. Illustration drawn from PDB entry 3KP9. PDB, Protein Data Bank.

Membrane proteins can also fulfill catalytic roles, and they operate not only in the plasma membrane but in every lipid membrane in the cell. One example of interest is vitamin K epoxide reductase (VKOR), the target of the anticoagulant drug warfarin. VKOR resides in the endoplasmic reticulum (ER) membrane and catalyzes a key step in the vitamin K cycle—regeneration of vita min K hydroquinone. This compound is a cofactor for the enzyme that converts glutamic acid residues in the N-termini of vitamin K dependent clotting factors to γ-carboxy glutamate (Gla) residues. This PTM is required for interaction of these proteins with Ca2+ and thus for their function in coagulation. Although the structure of the human enzyme has not been elucidated, the structure of a homologous bacterial protein has been described.[5] In this crystal structure (Fig. 1B), the bacterial homolog of VKOR is naturally fused to a thioredoxin (Trx)-like domain, which supplies reducing equivalents. The core of VKOR structure is a four-helix bundle embedded in the membrane (transmembrane helices TM1-TM4, shown in green) with a TM5 linked to the Trx-like domain on the extracellular surface (topologically equivalent to the luminal side of ER-resident mammalian VKOR). The ubiquinone compound has its quinone ring located near the membrane surface with its isoprenyl tail intercalated into the V-shaped cleft between TM2 and TM3. All the enzymatically important residues are on or close to the extracellular side of the membrane, adjacent to the Trx-like domain, providing a plausible path for electron transfer.[5] Interestingly, mapping of mutations that confer resistance to warfarin onto the bacterial structure reveals striking clustering around the ligand-binding pocket, confirming that it is also the site of warfarin binding and that warfarin exerts its inhibitory effect by displacing vitamin K.[5]

References

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[1] Overington JP, Al-Lazikani B, Hopkins AL. How many drug targets are there? Nat Rev Drug Discov. 2006;5:993–996. https://doi.org/10.1038/ nrd2199.

 [2] Zickermann V, Wirth C, Nasiri H, et al. Structural biology. Mechanistic insight from the crystal structure of mitochondrial complex I. Science. 2015;347:44–49. https://doi.org/10.1126/science.1259859.

[3] Rasmussen SG, DeVree BT, Zou Y, et al. Crystal structure of the β2 adrenergic receptor-Gs protein complex. Nature. 2011;477:549–555. https://doi.org/10.1038/nature10361.

[4] Congreve M, de Graaf C, Swain NA, Tate CG. Impact of GPCR structures on drug discovery. Cell. 2020;181:81–91. https://doi.org/10.1016/j.cell. 2020.03.003.

[5] Li W, Schulman S, Dutton RJ, et al. Structure of a bacterial homologue of vitamin K epoxide reductase. Nature. 2010;463:507–512. https://doi. org/10.1038/nature08720.

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