10.1: Membrane and Membrane Proteins

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Learning Goals (ChatGPT o3-mini, 2/2/25)

1. Explain the Functional Role of Membrane Bilayers:
   • Describe how membrane bilayers not only serve as physical barriers separating cellular compartments but also provide platforms for signal transduction and molecular trafficking.

2. Differentiate Types of Membrane Proteins:
   • Compare and contrast peripheral (extrinsic) and integral (intrinsic) membrane proteins, including the concepts of monotopic, bitopic (single-pass), and polytopic (multi-pass) proteins, and explain their respective modes of association with the lipid bilayer.

3. Describe Mechanisms of Membrane Protein Localization:
   • Explain how various post-translational modifications (e.g., N-myristoylation, S-palmitoylation, prenylation, and glycosylphosphatidylinositol (GPI) anchoring) target proteins to membranes and influence their function and interactions.

4. Analyze Protein-Lipid Interactions:
   • Understand how peripheral proteins such as MMP-12 and prothrombin interact with lipid bilayers via electrostatic interactions (e.g., Ca²⁺-mediated binding of GLA domains) and hydrophobic patches, and how these interactions affect their catalytic activity.

5. Explore Structural Classes of Integral Membrane Proteins:
   • Describe the structural features of transmembrane proteins, including alpha-helical polytopic proteins (such as GPCRs and kinases) and beta-barrel proteins (such as OMF and VDAC), and discuss how their structure supports their function in transport and signal transduction.

6. Examine Membrane Protein Models and Mimetics:
   • Evaluate the different experimental systems used to study membrane proteins, including detergent micelles, bicelles, nanodiscs, and SMALPs, and understand how these systems mimic the native lipid bilayer environment.

7. Discuss Protein Complexes Involved in Cell Adhesion and Signaling:
   • Analyze the roles of adhesive proteins such as cadherins and dystroglycan in maintaining cell–cell interactions and connecting the extracellular matrix to the cytoskeleton, as well as their implications in disease when mutated.

8. Understand the Role of Membrane Proteins in Signal Transduction:
   • Explain how transmembrane receptors, including GPCRs and receptor tyrosine kinases, transduce extracellular signals into intracellular responses through conformational changes and downstream signaling cascades.

9. Interpret Structural Data from Molecular Models:
   • Utilize interactive 3D molecular visualization tools (e.g., iCn3D) to analyze the architecture of membrane proteins and their interactions with lipids, highlighting how structural insights can inform understanding of protein function and pharmacological targeting.

10. Relate Membrane Protein Function to Therapeutic Applications:
    • Discuss how targeting membrane-associated enzymes and transporters (e.g., P-glycoprotein, Ras proteins) can influence disease outcomes, particularly in cancer and other signal transduction disorders, and explore strategies for drug design based on membrane protein structures.

These learning goals will help you integrate theoretical knowledge with practical examples, providing a comprehensive understanding of how membrane proteins contribute to cellular function and how their structural features are harnessed for biological regulation and therapeutic intervention.

Introduction

One easily understandable function of membrane bilayers is to separate the inside and outside of the cell or intracellular organelles. Yet, as we mentioned before, such barriers can not be so rigid and impenetrable that they prevent the movement of materials across the membrane. Also, all cells must sense and respond to their environment through signal transduction. We have already discussed lipid molecules involved in signaling. Now, let's turn our attention to proteins associated with the membrane and confer added functionalities. Figure \(\PageIndex{1}\) reviews some of the features of membranes we've discussed before and shows a simple bilayer (top) to the complicated membrane/cell wall of bacteria.

Figure \(\PageIndex{1}\): Membrane from simple to complex

Types of Membrane Proteins

Although we presented this image earlier, Figure \(\PageIndex{2}\) reviews the details that should now be clearer. In this section, we will explore membrane proteins in more detail.

Figure \(\PageIndex{2}\): A cell membrane with peripheral and integral membrane proteins. https://commons.wikimedia.org/wiki/F...Components.jpg. This file is licensed under the Creative Commons Attribution 4.0 International license.

Proteins can be loosely associated with the membrane (peripheral or extrinsic), embed deeply, or pass through the membrane and become a transmembrane (also called integral or intrinsic) protein. Sometimes, they pass through using a single alpha helix, while others pass through multiple times (for example, seven times in G-protein coupled receptors). They can also be classified based on the number of membrane leaflets they cross, as shown in Figure \(\PageIndex{3}\).

Figure \(\PageIndex{3}\): Peripheral and biotopic, monotopic, and polytopic integral membrane proteins

Peripheral Proteins

The proteins interact with a membrane through protein-lipid head group interactions but might slightly penetrate the membrane. Those that do would be classified as monotopic peripheral. Peripheral proteins are generally easy to remove from a membrane in vitro by changing solution ion concentration, as the interactions are often of an ion-ion nature. The first model below shows a matrix Metalloproteinase (MPP) 12 binding to a lipid bilayer. This protein is involved in inflammation, wound healing, arthritis, cardiovascular disease, and remodeling of neural synapses, suggesting a broad role in recovery from cell and tissue aberrations. Macrophages secrete MMP-12, so it is considered a water-soluble (aqueous) protein. It travels to viral cells and appears to display activity not in aqueous solution but near membranes, implying activation of the enzyme through binding to the bilayer.

Studies show the catalytic domain of MMP 12 can bind bilayers through both α- and β- secondary structure regions of the protein. Figure \(\PageIndex{4}\) shows an interactive iCn3D model of the protein and its interaction with the membrane through the alpha-helical region. Once bound to the membranes, catalytic activity increases.

Figure \(\PageIndex{4}\): Catalytic domain of peripheral membrane Protein MMP 12 Interacting with a bilayer. (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...wyF6WvRMx9JP6A

Other MMPs localize to membranes in other ways. MMP-7 interacts with heparan sulfate proteoglycans (CD44) and lipid rafts. Others bind transmembrane proteins like integrins.

Other examples of peripheral protein include many precursor forms of protein clotting factors. Clotting is initiated when the serine protease thrombin cleaves fibrinogen to form fibrin, which self-associates to form a fibrin clot or when thrombin activates platelet receptors. The soluble precursor of thrombin, prothrombin, a zymogen, is activated on membrane binding through interactions with several proteins assembled on a negatively charged phospholipid (like phosphatidylserine) bilayer in the prothrombinase complex. How does the precursor zymogen interact with the membrane? It requires calcium ions, which bind to a series of gamma-carboxylated glutamic acid (GLA) residues on the zymogen. The enzyme that carboxylates the zymogen depends on Vitamin K.

Figure \(\PageIndex{5}\) shows an interactive iCn3D model of bovine prothrombin Fragment 1 (N terminal) bound to a bilayer through its GLA domain (1NL2).

Figure \(\PageIndex{5}\): Bovine prothrombin Fragment 1 (N terminal) bound to a bilayer through its GLA domain (1NL2). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...SG6qByvpAsvwe6

The Gla sidechains are shown in CPK-colored sticks and interact with Ca²⁺ ions (gray spheres). Click on this link to see a zoomed view of just the calcium ions and Gla sidechains: https://structure.ncbi.nlm.nih.gov/i...xydtj8a4WHgdb7

The Gla domain, in the absence of calcium ions, is disordered. On binding, an ordered linear alignment of bound calcium ions is formed, stabilizing the ordered structure of the Gla domain and allowing interaction with the membrane. Three nonpolar amino acid side chains, Phe 5, Leu 6, and Val 9, are now clustered and exposed, allowing penetration of this hydrophobic patch part-way into the membrane. They are represented in cyan spacefill just underneath the surface of the red dots in the model above (the red dots are dummy atoms that represent the outer bilayer leaflet). Given this penetration, this protein domain would then be considered monotopic.

What is not shown in the model is the role of negatively charged phosphatidyl serine. Studies have shown that the head group of serine in lysophosphatidylserine (which has only one acyl group) provides additional ion-ion interactions with the Ca²⁺ ions that bind Gla residues 17 and 21. Arg 10 and Arg 16 also interact with the phosphatidyl serine head group. Phosphatidylcholine could also spatially fit into the active site, but electrostatic interactions would prevent it. Why?

Lipid-Anchored Proteins

We have studied lipids, proteins, and carbohydrates. Although phospholipids can spontaneously form bilayers, the actual structure of biological membranes is made much more complicated by adding protein and carbohydrate substituents to the membrane. Soluble proteins can be made to insert into bilayers by the addition of nonpolar attachments. Localization to a membrane changes the functional expression of the protein. Several examples of such attachments are described below.

Fatty acid linkers

Two common covalent modifications of proteins are N-myristoylation (attached myristic acid - 14:0 - through an amide link) and S-palmitoylation (attached palmitic acid - 16:0 - through a thioester link with a Cys).

Myristoylation is usually a cotranslational modification in eukaryotic and viral proteins after cleavage of the N-terminal methionine. Figure \(\PageIndex{6}\) shows an image of the serine/threonine phosphatase 2C (1A6Q) with its N-terminal glycine myristoylated. It should be obvious how this post-translationally modified protein interacts with a membrane.

Figure \(\PageIndex{6}\): The serine/threonine phosphatase 2C (1A6Q) with its N-terminal glycine myrisoylated

This modification is a key part of initiating immune system signal transduction pathways. The modification is catalyzed by N-myristoyltransferase (NMT) using myristoyl-coenzyme as the fatty acid acyl donor. This activates the protein's function partly by reducing the dimensionality of substrate diffusion to the protein to the 2D surface of the membrane instead of a 3D search in the cytoplasm. NMT acylates protein at this consensus sequence: G¹X²X³X⁴S/T⁵X⁶R⁷R⁸.

Likewise, many signaling proteins are palmitoylated, leading to protein recruitment to membranes. Small G proteins like Ras, Rho, and the alpha subunit of heterotrimeric G proteins are often palmitoylated. This modification can also be found in transmembrane proteins where localization is not an issue (see example xx below). In such circumstances, the modification might help target the proteins to rafts within the membrane. Palmitic acid is saturated, and attaching it to a protein might target the modified protein to more ordered membrane regions with cholesterol and sphingolipids within rafts.

Isoprenoids linkers:

The isoprenoids farnesyl (15C) or geranylgeranyl (20C)are added to a CAAX carboxy-terminal sequence in a target protein like RAS, where C is Cys, A is aliphatic, and X is any amino acid, which helps target proteins to the membrane. The enzymes used for these modifications are farnesyltransferase (FTase) and protein geranlygeranyltransferase I (GGTase I), respectively. This and the other modifications have the potential to do more than target proteins to the membrane. The modification can also modulate protein-ligand interactions and protein stability. Ras, a key signaling protein, is a target of prenylation.

Ras and other small G proteins are involved in many human cancers. As the G protein Ras has somewhat of a billiard ball surface with obvious sites to target drugs that would affect its aberrant function in cancers, efforts have been made to target the prenyltransferases necessary to target it to the membrane.

In humans, there are three genes in the Ras family, H-Ras and N-Ras, whose gene products localize to both plasma and Golgi membranes, and K-Ras, which localizes predominantly to the plasma membrane. These and other G proteins bind GTP and possess GTPase activity. The GTP-bound form is active, while the GDP form is inactive. Point mutations that attenuate or prevent GTP cleavage leave the protein continually activated, contributing to oncogenesis.

KRas has two predominant isoforms, 4A, the canonical form (also called 2A), and 4B (also called 2B), that arise from alternative splicing of the primary RNA transcript. The C-terminal protein sequences of isoform 4A and 4B differ significantly.

Isoform 4A: QYRLKKISKEEKTPGCVKIKKCIIM

Isoform 4B: KHKEKMSKDGKKKKKKSKTKCVIM

The farnesylation motif site containing the modified Cys are highlighted in yellow above. The same cysteine is also often carboxymethylated. The Cys six residues from the farnesylated Cys in isoform 4A are also often palmitoylated

Figure \(\PageIndex{7}\) shows isoform KRas 4B bound to a membrane bilayer through its farnesylated tail. (PDB file provided by Alemayehu (Alex) Gorfe. Viney Nair and Andrew McCammon). The tail is essential for its function at the plasma membrane, where KRAS-mediated signaling events occur. Phosphodiesterase-δ (PDEδ) binds to KRAS4b and is important in targeting it to cellular membranes. Note that the farnesyl attachments only penetrate part of the upper leaflet.

Figure \(\PageIndex{7}\): Isoform KRas 4B bound to a membrane bilayer through its farnesylated tail

Glycosyl-phosphatidylinositol linkers

Soluble cytosolic proteins can attach to membranes normally by adding glycosylphosphatidylinositol (GPI). The attachment usually contains a conserved tetrasaccharide core of three mannoses (Man) and one unacetylated glucosamine (GlcN) linked to the carboxy terminus of the protein. The GPI can be further modified with extra galactoses and mannoses and additions to the PI group, which secures the protein in the membrane. Figure \(\PageIndex{8}\) shows the common backbone for GPI anchors. Note the additions of the phosphoethanolamines to the core polysaccharide.

Figure \(\PageIndex{8}\): Common backbone of GPI anchors

GPIs in eukaryotic cells link many surface antigens, adhesion molecules, and hydrolases to the membrane. GPIs from Plasmodium falciparum, the malarial parasite that kills about two million people yearly, appear to act as a toxin and are the most common CHO modification of the parasite protein. Mice immunized against the GPI sequence, NH₂-CH₂-CH₂-PO₄-Man (α1-2) 6Man (α1-2) Man (α1-6) Man (α1-4) GlcNH2 (α1-6) myo-inositol-1,2-cyclic-phosphate, were substantially protected from malarial symptoms and death after they were exposed to the actual parasite.

Figure \(\PageIndex{9}\) shows a cross-section of a membrane (with cholesterol, PE, SM) containing the glycosylated form of the human complement regulatory protein CD59 protein (1cdr) with a GPI anchor attached at its C-terminus. Note that the middle part of the anchor (glycan) holds the protein well above the top of the lipid bilayer. The soluble protein is also glycosylated. The protein binds to complement proteins C8 and/or C9, which are effector immune proteins that assemble on the surface of a cell undergoing lysis.

Figure \(\PageIndex{9}\): Cross section of a membrane containing the glycosylated form of the human complement regulatory protein CD59 protein (1cdr) with a GPI anchor attached at its C-terminus

The GPI anchor is shown in spacefill. Note that it only extends halfway into the bilayer, as expected from the size of the fatty acids attached to the phosphatidyl inositol. The glycan part of the GPI is shown in spacefill between the lipid and its protein attachment site. The protein is also glycosylated in the extracellular domain.

Something new!

An unexpected type of glycosylated molecule has been found in the outer leaflet of mammalian cells - a glycosylated RNA, as shown in Figure \(\PageIndex{10}\). This adds RNA to the lipids and proteins as a target for glycosylation. These surface glycoRNAs interact with antibodies against ds-RNA and the Siglec lectin family. They are found in cells in vivo and cultured cells in vitro.

Figure \(\PageIndex{10}\): A glycoRNA - a small noncoding RNAs with sialylated glycans. Park. https://doi.org/10.14348/molcells.2021.0178
www.molcells.org. Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.

Flynn et al., Small RNAs are modified with N-glycans and displayed on the surface of living cells, Cell (2021), https://doi.org/10.1016/j.cell.2021.04.023

Transmembrane (Integral) Proteins

These proteins pass through the membrane either one in one pass, usually with a single alpha-helix or many membrane-spanning helices. For example, G protein-coupled receptors, often called serpentine receptors, cross the membrane seven times. There are three types based on the number of types the protein crosses the membrane and the type of secondary structure used in crossing: biotopic (single pass), alpha-helical polytopic, and beta-barrel. These proteins are found in all types of membranes and have many types of functions, from receptors, receptor ligands, structural, adhesion, transport, gene regulation, and transport.

Transmembrane Biotopic - Single Pass Proteins

The are four types of single-pass transmembrane proteins:

• Type I: N-terminal outside of the cell (extracellular) and the precursor signal sequence on the N-terminus, which is a localization sequence, is removed
• Type II: N-terminal intracellular and with the transmembrane domain close to the N-terminus
• Type III: N-terminus extracellular and no signal sequence in precursor protein
• Type IV: N-terminus intracellular and the transmembrane domain close to the C-terminus

The transmembrane domain of single-pass integral membrane proteins consists of a single alpha-helix with nonpolar side chains extending outward from the helical axis, interacting with the nonpolar lipid parts of the membrane. These nonpolar sides are more stable in nonpolar environments.

To study such proteins in a less complex environment, membranes are often "dissolved" in nonpolar, single-chain amphiphilic detergents. These single-chain amphiphiles form micelles without membrane proteins but form mixed micelles in which the nonpolar part of the protein is surrounded in the detergent micelle by the nonpolar acyl chains of the detergent.

Figure \(\PageIndex{11}\) (top) below shows just the transmembrane and juxtamembrane (next to the membrane) domains of the single pass Notch protein, which is critical in many signal transduction pathways.

Figure \(\PageIndex{11}\): Transmembrane and juxtamembrane domains of the single pass Notch protein in a bilayer and an octylglucoside micelle

The top images in the figure above show different ways to represent the protein in the bilayer, with the right-hand image showing a cross-section through the membrane to show better how the protein passes through the bilayer.

The bottom images in the figure above show the protein after excess detergent, in this case, octylglucoside, is added to the protein-containing bilayer.

 

Studying membrane proteins in lipid aggregates - NMR applications

Recent Updates: 9/26/24

Various discrete lipid aggregates are used to conduct NMR studies of membrane proteins in a more realistic biological environment.  Examples are shown in the Figure \(\PageIndex{12A}\) below. 

Figure \(\PageIndex{12A}\): Schematic diagram of different membrane mimetic systems used in NMR studies: detergent micelle, bicelle, nanodisc, liposome, amphipols, salipro, and styrene–malic acid co-polymer lipid particles (SMALPS). The membrane protein is represented as a blue cartoon block. Yeh, V.; Goode, A.; Bonev, B.B. Membrane Protein Structure Determination and Characterization by Solution and Solid-State NMR. Biology 2020, 9, 396. https://doi.org/10.3390/biology9110396.  Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).  For an alternative image see https://www.cell.com/chem/fulltext/S...2822%2900092-4. 

An alternative description and use for general membrane protein purification and studies include a general polymer form and virus-like particles, as shown in Figure \(\PageIndex{12B}\) below. 

Figure \(\PageIndex{12B}\): Alternative descriptions and uses in membrane protein solubilization, purification, and characterization.  Creative BioLabs.  MAGICTM MEMBRANE PROTEIN SERVICES & PRODUCTS.  With permission (10/20/24)

Micelles:  We've studied these in Chapter 10.2:  Lipid Aggregates and the above descriptions. They form from the self-assembly of single-chain amphiphiles (detergents, such as octylglucoside and [mono]dodecylphosphocholine) as well as double-chain amphiphiles with short alkyl chains such as hexanol-phosphatidylcholine (DHPC also abbreviated C6DH).

Bicelle:  Bicelles are made of phospholipids (such as dimyristoyl phosphatidylcholine - DMPC) that can form a bilayer and a detergent (a single chain amphiphile like that can form a micelle.  The detergent fills in the hydrophobic "edges" of the bilayer part to form the final structure. Bicelle size varies with a Q value, the [phospholipid]/[detergent] ratio.  When Q=0 (high detergent), a micelle forms.

A phospholipid with short acyl chains such as dihexyl phosphatidylcholine (DHPC or C6DH) can replace the detergent. The structures of these two phospholipids (DMPC and DHPC) are shown in Figure \(\PageIndex{13}\) below.

Figure \(\PageIndex{13}\):  Structures of dimyristoyl phosphatidylcholine (DMPC) and dihexyl phosphatidylcholine (DHPC or C6DH)

Figure \(\PageIndex{14}\) below represents a cross-section view of a bicelle made from the above lipids.  The central white/gray chains represent the alkyl chains of the DMPC phospholipid bilayer.  The cyan spheres represent the carbon atoms of the smaller DHPC "detergent" that fill the edges of the central bilayer.  

Figure \(\PageIndex{14}\): Cross-sectional view of a bicelle made with DMPC (white carbons) and DHPC (cyan carbons) which are seen around the periphery.

Figure \(\PageIndex{15}\) below shows an iCn3D model of the same bicelle.  The black lines represent the alkyl chains of the central DMPC bilayers.  The cyan sticks represent the alkyl chains of the DHPCs that fill the edges.  Other atoms are shown in their own CPK colors.

Figure \(\PageIndex{15}\): Bicelle

Open iCn3D and follow these prompts to get an interactive model of this bicelle:  

• Download this file to your computer
• in iCn3D, File, Open File, and then openiCn3D PNG Image

Nanodiscs:  Nanodiscs are like bicelles that use two copies of an amphiphilic protein (membrane scaffold protein (MSP) from high-density lipoprotein (HDL) instead of a detergent or small double-chain amphiphile to protect the exposed hydrophobic edges of a small section of a bilayer.  Figure \(\PageIndex{16}\) shows a cross-sectional view of a nanodisc composed of DMPC and two copies of the protein MSP.

Figure \(\PageIndex{16}\): Cross-sectional view of a nanodisc made with DMPC (white carbons) and the membrane scaffold protein (MSP, red helices). Carbon atoms are shown in cyan.  Image made with Pymol.

Figure \(\PageIndex{17}\) below shows an iCn3D model of the same nanodisc.  The cyan atoms represent the carbons in the alkyl chains of the DMPCs.  Other atoms are shown in their own CPK colors.  The two helical amphiphilic proteins are shown in cartoon form in red.

Figure \(\PageIndex{17}\):  A nanodisc with DMPC and the protein MSP.

Open iCn3D and follow these prompts to get an interactive model of this nanodisc:  

• Download this file to your computer
• in iCn3D, File, Open File, and then openiCn3D PNG Image

Follow these instructions to view the hydrophobicity of the two alpha helices.

With the full model in the iCn3D window:

• Analysis, Defined Sets, choose Proteins
• View, View Selection,
• Style, SideChains, Sticks
• Color, Hydrophobicity, Normalized

Rotate the structure to see a top-down view and you will observe that the hydrophobic side chains are all pointed inward in a fashion that would stabilize the exposed nonpolar alkyl chains of the phospholipids.

SMALPS (styrene–malic acid co-polymer lipid particles):  These are nanodiscs wrapped with this amphiphilic polymer instead of an amphiphilic protein.  The structure of SMALPS is shown in Figure \(\PageIndex{18}\) below.

Figure \(\PageIndex{18}\)

Figure \(\PageIndex{19}\) below shows an iCn3D model of SMALPS.  Rotate the image to confirm its that it is amphiphilic.

Figure \(\PageIndex{19}\):  SMALPS (styrene–malic acid co-polymer lipid particles)

Here are some examples of bitopic single-pass transmembrane proteins

Cadherins

All structures need support and connections. At the macro level, the skeleton supports the mass and organization of organs and tissues in whole organisms. Within an organ, how can cells hold together? How do they adhere to each other? Certainly not through outer leaflet lipid contacts, as the outer surface of the leaflet is typically charged. The extracellular matrix does provide some of the glue that holds cells together. At a more detailed level, transmembrane proteins are involved. One class of adhesive proteins is cadherins, a calcium-dependent cell adhesion molecule. There are over 100 human cadherins. They are mostly ditopic, single transmembrane pass proteins. Their cytoplasmic domains interact with proteins like catenin, which then bind to the interior cytoskeletal network composed of actin and other proteins. This provides a way for the intracellular region to regulate the extracellular interactions of the cell.

The extracellular domain is composed of five repeating "cadherin" domains, each around 110 amino acids, that can fold independently. Calcium ions bind at the domain interfaces. A cadherin can interact with other cadherin domains on other cadherins on other cells, leading to cell adhesion. Essentially, the receptor cadherin on one cell binds to the ligand cadherin on the other. As metastatic tumor cells lose their adhering feature and leave the primary tumor site, you would expect mutations in cadherins are often involved. They may also be involved in cell sorting during morphogenesis, regulation of tight and gap junctions, and in the control of intercellular spacing.

Figure \(\PageIndex{20}\) shows a "constructed" image of cadherin-1 (1L3W) interacting with cytoplasmic β-catenin (1I7X) through a modeled transmembrane helix (amino acids QIPAILGILGGILALLILILLLLLFLRR, amino acids 706-731). No full-length structure of cadherin in a membrane is available.

Figure \(\PageIndex{20}\): A constructed" image of cadherin-1 (1L3W) interacting with cytoplasmic β-catenin (1I7X) through a modeled transmembrane helix

Membrane Protein Kinases

Kinases are enzymes that phosphorylate substrates. Hexokinase is a protein enzyme that catalyzes the phosphorylation of a hexose substrate, such as glucose. A protein kinase is a protein enzyme that phosphorylates a protein substrate. That protein could be another copy of itself or another protein. We will see in Chapter 12 that many protein kinases are involved in cell signaling. Many tyrosine protein kinases are bitopic single-pass integral membrane proteins that become active when binding a ligand. Typically, on binding an extracellular ligand, two monomeric copies of the kinase form a dimer in the membrane, activating a tyrosine kinase cytoplasmic domain, which typically phosphorylates (using ATP as a substrate), the other member of the dimer in an "autophosphorylation" reaction. Sometimes, the dimers are held together by disulfide bonds.

Figure \(\PageIndex{21}\) shows a "constructed" image of the human dimeric insulin receptor.

Figure \(\PageIndex{21}\): aA "constructed" image of the human dimeric insulin receptor

One of the monomers is shown in gray. The other monomer is shown in colors corresponding to the domain organization of the protein. Each extracellular dimer (6PXV) has two insulins bound (yellow spacefill). The intracellular domains (1IR3) are activated on insulin binding. No full-length structure of the full insulin receptor in a membrane is available.

Almost half of all helical membrane proteins in humans are bitopic, compared to 20-25% in prokaryotes. Humans have 10-20 fold more bitopic proteins than E.Coli. There appear to be about 196 bitopic proteins in E. coli (located in the inner membrane ) and 70 in M. jannaschii (Archea in plasma membrane). In humans, 57% are in the plasma membrane, with the rest distributed between the Golgi, ER, nuclear, mitochondrial, and chloroplast membranes. In single-celled yeast, only 8% are in the plasma membrane.

Beta-Dystroglycan

This protein is another bitopic protein with a single alpha-helix membrane domain. Dystroglycan is a dimer of alpha and beta subunits. Alpha-dystroglycan is a peripheral protein that binds beta-dystroglycan, a transmembrane protein. Alpha dystroglycan also binds Lassa virus and lymphocytic choriomeningitis virus glycoprotein, serving as viral receptors. It also binds the protein dystrophin, which is missing in Duchenne muscular dystrophy and affects 1 out of 5000 live male births. As an integral transmembrane protein, beta-dystroglycan connects the extracellular matrix to the cytoskeleton through dystrophin. Alpha- and beta-dystroglycan share the same gene, which codes one long protein proteolyzed post-translationally to form the alpha (N-terminal end) and beta subunits (C-terminal end).

Figure \(\PageIndex{22}\) shows a schematic outline of dystrophin and the dystrophin-associated glycoprotein complex (DAGC).

Figure \(\PageIndex{22}\): Schematic outline of dystrophin and the dystrophin-associated glycoprotein complex (DAGC). Dystrophin contains N-terminal (NT), middle rod, cysteine-rich (CR), and C-terminal (CT) domains. The middle rod domain is composed of 24 spectrin-like repeats (numerical numbers in the cartoon, positively charged repeats are marked in white color) and four hinges (H1, H2, H3, and H4). Dystrophin has two actin-binding domains at NT and repeats 11-15, respectively. Repeats 1-3 interact with the negatively charged lipid bilayer. Repeats 16 and 17 form the neuronal nitric oxide synthase (nNOS)-binding domain. Dystrophin interacts with microtubules through repeats 20-23. Part of H4 and the CR domain binds to the β-subunit of dystroglycan (βDG). The CT domain of dystrophin interacts with syntrophin (Syn) and dystrobrevin (Dbr). Dystrophin links cytoskeleton components (actin and microtubule) to laminin in the extracellular matrix. Sarcoglycans and sarcospan do not interact with dystrophin directly, but they strengthen the entire DAGC, which consists of dystrophin, DG, sarcoglycans, sarcospan, Syn, Dbr, and nNOS.Disease Models & Mechanisms (2015) doi:10.1242/dmm.018424vailable via license: CC BY 3.0

Transmembrane - Alpha-helical polytopic

There are so many intriguing examples of these proteins. We'll illustrate just two.

Rhodopsin-like receptors and pumps

These proteins are involved in cell signaling and are the target of most pharmaceutic drugs. G protein-coupled receptors (GPCRs) are incredibly important, and we will discuss them extensively in the capstone chapter (28.2: At the cell membrane- receptors and receptor enzymes) for Vol 1.  

GPCRs are cell receptors that span the membrane seven times in a serpentine fashion. They bind ligands (neurotransmitters, hormones, etc.) in the extracellular or internal membrane domains (the latter for hydrophobic ligands) and, through propagated conformational changes, alter the cytoplasmic domain where they functionally interact with a heterotrimeric G protein.

Figure \(\PageIndex{23}\) shows an interactive iCn3D model of the human cannabinoid receptor with bound cholesterol and Δ ⁹ -tetrahydrocannabinol (Δ⁹ -THC) in spacefill (5xra). The red dummy atoms represent the outer leaflet, and the blue the inner.

Figure \(\PageIndex{23}\): Human cannabinoid receptor with bound cholesterol and Δ ⁹ -tetrahydrocannabinol (Δ⁹ -THC). (5xra). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...PaTFrkGFgKeP2A

Δ ⁹ -THC is a partial agonist and tunes the response of the receptor. The active site is conformationally somewhat flexible or plastic. Other ligands bound to it act as antagonists instead of agonists and must do so by eliciting nonactive conformations.

ABC Transporter

Figure \(\PageIndex{24}\) shows an interactive iCn3D model of the P-glycoprotein multidrug resistance transporter protein (6nf1).

Figure \(\PageIndex{24}\): P-glycoprotein multidrug resistance transporter protein (6nf1). (Copyright; author via source). Click the image for a popup or use this external link:https://structure.ncbi.nlm.nih.gov/i...zzXduCZjhCMaw9

The spacefill ligands represent Zosuquidar, which binds with high affinity to P-glycoprotein and inhibits its activity, making it a cancer agent as it prevents chemotherapeutic drugs that have entered the cell from being pumped out. The protein chain interacting with it on the cytoplasmic face is an antibody fragment that stabilizes the P-glycoprotein so crystals can form.

Transmembrane Beta-barrel Fold

We will focus on two of these proteins.

Outer Membrane Factor (OMF) - Gram-negative bacteria

Figure \(\PageIndex{25}\) shows an interactive iCn3D model of a beta-barrel transmembrane protein OPRM - Outer Membrane Factor (4y1k) from Pseudomonas aeruginosa that acts as a pore.  The transmembrane part of the protein consists of a beta-barrel fold (BBF). The protein also has a palmitoyl fatty acid in thioester linkage to Cys 1 of the protein for extra but unneeded anchorage.

Figure \(\PageIndex{25}\): Beta-barrel transmembrane protein OPRM - Outer Membrane Factor (4y1k) from Pseudomonas aeruginosa (4y1k). (Copyright; author via source). Click the image for a popup or use this external link:https://structure.ncbi.nlm.nih.gov/i...xrAuW1oFtY5FL7

Use this link for another view: https://structure.ncbi.nlm.nih.gov/i...FtY5FL7&t=4Y1K (OPM) in iCn3D

This protein is part of a large complex of proteins that spans both the inner and outer membranes of Gram-negative (examples, E. Coli and Pseudomonas aeruginosa) bacterial cell walls. Unfortunately for humans, this protein complex pumps out toxins (to the bacteria) like antibiotics, which makes bacteria resistant to these drugs. The OPRM acts as the outer passageway or duct for the pumped molecules. The Bacterial Outer Membrane Factor (OMF) protein differs in sequence, but they all form the beta-barrel duct. The E. Coli version of OMF has a triacylated lipid modification of the N-terminus. The N terminal lipid modification might be necessary for the initial attachment of the protein to a membrane before the insertion of the beta-barrel. As such, the enzymes involved in tail attachment could be targets for new antibiotics.

Voltage-dependent anion channel (VDAC) - mouse

This protein, which also contains a beta-barrel fold, regulates the movement of molecules between the cytoplasm and the interior of the mitochondria across the outer mitochondrial membrane. VDAC also serves as a docking site or scaffold for assembling molecules into a complex that regulates mitochondrial function. The protein's conformation and function are regulated by changes in the transmembrane potential, which we will explore in the following sections. Hence, the protein and its function are voltage-dependent. Figure \(\PageIndex{26}\) shows an interactive iCn3D model of mouse VDAC with a beta-barrel fold formed by 19 beta-strands (3emn).

Figure \(\PageIndex{26}\): Mouse voltage-dependent anion channel (VDAC) (3emn). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...WxUqWaGPxgHH79 

Note the N-terminal alpha-helix resident inside the channel opening. This helical section moves on changes in membrane potential, gating open and regulating the flow of metabolites and ions across the membrane through the pore. The conductance is high at a low transmembrane potential (10 mV) as the channel is the open state. When the potential increases to 30 mV (either + or -), conductance drops as the protein forms the closed state.

An alternative beta-barrel fold is found in the protein enzyme Triose phosphate IsoMerase (TIM) which we will see in Chapter x. That fold has eight α-helices connecting 8 parallel β-strands that form the barrel. The TIM beta-barrel fold (TBF) is found in up to 10% of protein enzymes.  The transmembrane pore proteins above superficially resemble the TIM barrels, but they have loops, not helices, connecting the β-strands, which are antiparallel, not parallel as in TIM. They represent a different fold called the beta-barrel fold (BBF).

Membrane proteins containing the beta-barrel fold (BBF) can now be designed de novo (see Chapter 4.14:Predicting Structure from Sequence and Sequence from Structure/Function) to create nanopores of predefined sizes.  These could serve as designed sensors.  The links below will take you to iCn3D models containing designed proteins with TIM barrel folds (TBF).

• 8UZL: Designed Transmembrane beta-barrel- TMB10_163 (10-stranded)
• 9FDG: Solution structure of a de novo designed 12-stranded transmembrane beta-barrel in LDAO micelles

Now that you understand the structure of membrane proteins, let's explore a key function of a subset of integral membrane proteins in the next chapter section: the movement of molecules/ions across the membrane.

Summary

This chapter provides a comprehensive overview of membrane proteins and their vital roles in cellular membranes. While the primary function of a lipid bilayer is to act as a semi-permeable barrier that distinguishes the inside from the outside of a cell or organelle, the incorporation of proteins adds layers of functionality, including signal transduction, adhesion, transport, and enzymatic activity.

Key topics include:

• Types of Membrane Proteins:
  The chapter categorizes proteins based on their association with the membrane. Peripheral proteins, which interact with the membrane through transient, non-covalent bonds (e.g., via lipid head groups or through binding to other membrane proteins), are contrasted with integral (transmembrane) proteins that span the bilayer. These integral proteins are further classified as:

  • Bitopic (Single-Pass) Proteins:
    These include various types (I–IV) depending on the orientation of their domains relative to the membrane. Examples, such as prothrombin’s GLA domain and the Notch protein, illustrate how even single-pass proteins can play crucial roles in signaling and cellular regulation.
  • Polytopic (Multi-Pass) Proteins:
    These proteins, including G protein-coupled receptors (GPCRs) and ion channels, typically traverse the membrane multiple times (e.g., seven transmembrane domains in GPCRs), mediating a wide array of cellular functions.
  • Beta-Barrel Proteins:
    Found primarily in the outer membranes of Gram-negative bacteria and in mitochondrial membranes (e.g., VDAC), these proteins form pore-like structures that facilitate the transport of molecules.

• Protein Modifications and Membrane Targeting:
  The chapter describes how covalent modifications such as fatty acid attachment (myristoylation, palmitoylation), isoprenoid addition (farnesylation, geranylgeranylation), and glycosylphosphatidylinositol (GPI) anchoring serve to target proteins to the membrane. These modifications not only anchor proteins to specific membrane regions but also influence their interactions and functional states.

• Membrane Dynamics and Protein Interactions:
  The dynamic nature of membranes is underscored by the lateral and transverse mobility of lipids and proteins, essential for processes such as vesicle trafficking, membrane fusion, and signal transduction. Detailed examples, including the interaction of matrix metalloproteinases (MMPs) and clotting factors with lipid bilayers, illustrate how protein binding can enhance catalytic activity or trigger signaling cascades.

• Experimental Approaches for Studying Membrane Proteins:
  Various membrane mimetics—such as detergent micelles, bicelles, nanodiscs, and SMALPs—are introduced as tools to study membrane proteins in environments that mimic native membranes. These systems facilitate structural and functional analyses using techniques like NMR, electron microscopy, and molecular dynamics simulations.

• Functional Relevance in Health and Disease:
  Finally, the chapter connects the structural and functional aspects of membrane proteins to their roles in disease. Examples include the involvement of cadherins in cell adhesion, the critical function of GPCRs in signal transduction (with implications in pharmacology), and the role of transporters like P-glycoprotein in drug resistance.

In summary, this chapter weaves together the principles of membrane biochemistry, highlighting how the unique architecture of lipid bilayers, combined with diverse protein modifications and dynamic interactions, underlies the complex functionalities required for cellular communication, adhesion, transport, and signal transduction. Understanding these interrelationships is crucial for unraveling cellular processes and developing targeted therapeutic interventions.