Ann M. Dixon

Membrane protein folding: beyond the two stage model

The folding of α‐helical membrane proteins has previously been described using the two stage model, in which the membrane insertion of independently stable α‐helices is followed by their mutual interactions within the membrane to give higher order folding and oligomerization. Given recent advances in our understanding of membrane protein structure it has become apparent that in some cases the model may not fully represent the folding process. Here we present a three stage model which gives considerations to ligand binding, folding of extramembranous loops, insertion of peripheral domains and the formation of quaternary structure.

Evidence for role of transmembrane helix–helix interactions in the assembly of the Class II major histocompatibility complex

The Major Histocompatibility Complex Class II (Class II MHC) and invariant chain (Ii) proteins are key initiators of an immune response to invading pathogens. Following biosynthesis, three MHCalpha/beta hetero-dimers associate with an Ii homotrimer to form a nine-chain protein complex. Only as part of this complex are the MHC molecules exported to the cell surface to trigger an immune response. Previous reports implicate the transmembrane (TM) domains of all three proteins in correct assembly, ligand binding and function of Class II MHC. Building on our previous work that revealed the Ii TM domain may contribute significantly to correct assembly of the full-length protein, we have used a variety of genetic, biophysical and computational methods to investigate the role of the TM domains in stabilizing MHCalpha/beta heterodimers. Using the in vivo GALLEX assay, we find that the TM domains of both proteins form strong homo- and hetero-oligomers in natural membranes that are stabilized by GXXXG motifs within the sequence. Förster resonance energy transfer (FRET) measurements, using fluorescently-tagged peptides derived from the TM domains of each protein, were then employed to confirm the presence of TM helix-helix hetero-interactions in detergent micelles, as well as the stoichiometry of these interactions. Our results are summarized in a revised model of Class II MHC-Ii complex formation that illustrates key protein-protein contacts. This work provides the first evidence that the TM domains of the Class II MHC molecules are capable of significant protein-protein interactions that may help to stabilize or even initiate formation of the MHC-Ii complex.

Strong oligomerization behavior of PDGFβ receptor transmembrane domain and its regulation by the juxtamembrane regions

The platelet-derived growth factor beta-receptor (PDGFbetaR) represents an important subclass of receptor tyrosine kinase (RTK) thought to be activated by ligand-induced dimerization. Interestingly, the receptor is also activated by the bovine papillomavirus E5 oncoprotein, an interaction involving the transmembrane domains of both proteins and resulting in constitutive downstream signalling. This unique mode of activation along with emerging data for other RTKs raises important questions about the role of the PDGFbetaR transmembrane domain in signalling. To address this, we have investigated the murine PDGFbetaR transmembrane and juxtamembrane domains. We show for the first time the strong oligomerization behavior of PDGFbetaR transmembrane domain, forming dimers and trimers in natural membranes and detergents; and that these self-interactions are mediated by a leucine-zipper-like motif. The juxtamembrane regions are found to regulate these helix-helix interactions and select specifically for dimer formation. These data provide evidence that PDGFbetaR is able to form ligand-independent dimers, supporting similar observations in a number of other RTKs. A point mutant in the PDGFbetaR juxtamembrane domain previously shown to cause receptor activation was studied and yielded no change in oligomerization or folding, suggesting (in-line with observations of the c-Kit receptor) that it may moderate interactions with other regions of PDGFbetaR.

The brown adipocyte protein CIDEA promotes lipid droplet fusion via a phosphatidic acid-binding amphipathic helix.

Maintenance of energy homeostasis depends on the highly regulated storage and release of triacylglycerol primarily in adipose tissue, and excessive storage is a feature of common metabolic disorders. CIDEA is a lipid droplet (LD)-protein enriched in brown adipocytes promoting the enlargement of LDs, which are dynamic, ubiquitous organelles specialized for storing neutral lipids. We demonstrate an essential role in this process for an amphipathic helix in CIDEA, which facilitates embedding in the LD phospholipid monolayer and binds phosphatidic acid (PA). LD pairs are docked by CIDEA trans-complexes through contributions of the N-terminal domain and a C-terminal dimerization region. These complexes, enriched at the LD–LD contact site, interact with the cone-shaped phospholipid PA and likely increase phospholipid barrier permeability, promoting LD fusion by transference of lipids. This physiological process is essential in adipocyte differentiation as well as serving to facilitate the tight coupling of lipolysis and lipogenesis in activated brown fat. DOI: http://dx.doi.org/10.7554/eLife.07485.001

In vitro dimerization of the bovine papillomavirus E5 protein transmembrane domain

The E5 protein from bovine papillomavirus is a type II membrane protein and the product of the smallest known oncogene. E5 causes cell transformation by binding and activating the platelet-derived growth factor beta receptor (PDGFbetaR). In order to productively interact with the receptor, it is thought that E5 binds as a dimer. However, wild-type E5 and various mutants have also been shown to form trimers, tetramers, and even higher order oligomers. The residues in E5 that drive and stabilize a dimeric state are also still in question. At present, two different models for the E5 dimer exist in the literature, one symmetric and one asymmetric. There is universal agreement, however, that the transmembrane (TM) domain plays a vital role in stabilizing the functional oligomer; indeed, mutation of various TM domain residues can abolish E5 function. In order to better resolve the role of the E5 TM domain in function, we have undertaken the first quantitative in vitro characterization of the E5 TM domain in detergent micelles and liposomes. Circular and linear dichroism analyses verify that the TM domain adopts a stable alpha-helical structure and is able to partition efficiently across lipid bilayers. SDS-PAGE and analytical ultracentrifugation demonstrate for the first time that the TM domain of E5 forms a strong dimer with a standard state free energy of dissociation of 5.0 kcal mol (-1). We have used our new results to interpret existing models of E5 dimer formation and provide a direct link between TM helix interactions and E5 function.

Self-association of Transmembrane Domain 2 (TM2), but Not TM1, in Carnitine Palmitoyltransferase 1A : ROLE OF GXXXG(A) MOTIFS

Carnitine palmitoyltransferase 1 (CPT1) controls the rate of entry of long-chain fatty acids into the mitochondrial matrix for β-oxidation and has been reported to exist as an oligomer. We have investigated the in vivo oligomerization of full-length rat CPT1A (rCPT1A) along with those of the N-terminal truncation/deletion mutants Δ(1–82), Δ(1–18), and Δ(19–30) expressed in yeast mitochondria. The data indicate that in liver mitochondria in vivo CPT1A exists as a hexamer but that during preparation and storage of mitochondria the order of oligomerization is rapidly reduced to the trimer, such that a mixture of hexamer and trimer is observed in isolated mitochondria in vitro. Mutants bearing deletions of different segments of the N terminus (including the more N-terminal of the two transmembrane domains) have the same pattern of oligomerization when expressed in yeast mitochondria. The self-association of the individual rCPT1A transmembrane (TM) domains (TM1, TM2) was also studied using the TOXCAT assay (which measures TM self-association in the Escherichia coli inner membrane). There was minimal self-association of the sequence corresponding to TM1 but significant self-association of TM2 in TOXCAT. Chemical cross-linking and analytical ultracentrifugation of a TM2-derived synthetic peptide showed oligomerization with a similar trimer/hexamer equilibrium to that observed for native rCPT1A in isolated mitochondria. Therefore, there was a correlation between the oligomerization behavior of TM2 peptide and that of the full-length protein. In silico molecular modeling of rCPT1A TM2 highlighted the favorable orientation of GXXXG and GXXXA motifs in the formation of the TM2 hexamer.

Helical membrane peptides to modulate cell function

In recent years there has been an abundance of research into the potential of helical peptides to influence cell function. These peptides have been used to achieve a variety of different outcomes from cell repair to cell death, depending upon the peptide sequence and the nature of its interactions with cell membranes and membrane proteins. In this critical review, we summarise several mechanisms by which helical peptides, acting as either transporters, inhibitors, agonists or antibiotics, can have significant effects on cell membranes and can radically affect the internal mechanisms of the cell. The various approaches to peptide design are discussed, including the role of naturally-occurring proteins in the design of these helical peptides and current breakthroughs in the use of non-natural (and therefore more stable) peptide scaffolds. Most importantly, the current successful applications of these peptides, and their potential uses in the field of medicine, are reviewed (131 references).

Contributions of the Transmembrane Domain and a Key Acidic Motif to Assembly and Function of the TatA Complex

The twin-arginine translocase (Tat) pathway transports folded proteins across bacterial and thylakoid membranes. In Escherichia coli, a membrane-bound TatA complex, which oligomerizes to form complexes of less than 100 to more than 500 kDa, is considered essential for translocation. We have studied the contributions of various TatA domains to the assembly and function of this heterogeneous TatA complex. The TOXCAT assay was used to analyze the potential contribution of the TatA transmembrane (TM) domain. We observed relatively weak interactions between TatA TM domains, suggesting that the TM domain is not the sole driving force behind oligomerization. A potential hydrogen-bonding role for a TM domain glutamine was also investigated, and it was found that mutation blocks transport at low expression levels, while assembly is unaffected at higher expression levels. Analysis of truncated TatA proteins instead highlighted an acidic motif directly following the TatA amphipathic helix. Mutating these negatively charged residues to apolar uncharged residues completely blocks activity, even at high levels of TatA, and appears to disrupt ordered complex formation.

Artificial Transmembrane Oncoproteins Smaller than the Bovine Papillomavirus E5 Protein Redefine Sequence Requirements for Activation of the Platelet-Derived Growth Factor β Receptor

ABSTRACT The bovine papillomavirus E5 protein (BPV E5) is a 44-amino-acid homodimeric transmembrane protein that binds directly to the transmembrane domain of the platelet-derived growth factor (PDGF) β receptor and induces ligand-independent receptor activation. Three specific features of BPV E5 are considered important for its ability to activate the PDGF β receptor and transform mouse fibroblasts: a pair of C-terminal cysteines, a transmembrane glutamine, and a juxtamembrane aspartic acid. By using a new genetic technique to screen libraries expressing artificial transmembrane proteins for activators of the PDGF β receptor, we isolated much smaller proteins, from 32 to 36 residues, that lack all three of these features yet still dimerize noncovalently, specifically activate the PDGF β receptor via its transmembrane domain, and transform cells efficiently. The primary amino acid sequence of BPV E5 is virtually unrecognizable in some of these proteins, which share as few as seven consecutive amino acids with the viral protein. Thus, small artificial proteins that bear little resemblance to a viral oncoprotein can nevertheless productively interact with the same cellular target. We speculate that similar cellular proteins may exist but have been overlooked due to their small size and hydrophobicity.

Differential Transmembrane Domain GXXXG Motif Pairing Impacts Major Histocompatibility Complex (MHC) Class II Structure

Background: Major histocompatibility complex class II molecules are structurally and functionally heterogeneous. Results: Combined mutagenesis and structural studies establish a role for pairing between conserved transmembrane (TM) GXXXG dimerization motifs in determining class II conformation. Conclusion: Differential pairing of highly conserved TM domain dimerization motifs contributes to class II structure and function. Significance: Global conformation contributes to the function of peptide-class II complexes. Major histocompatibility complex (MHC) class II molecules exhibit conformational heterogeneity, which influences their ability to stimulate CD4 T cells and drive immune responses. Previous studies suggest a role for the transmembrane domain of the class II αβ heterodimer in determining molecular structure and function. Our previous studies identified an MHC class II conformer that is marked by the Ia.2 epitope. These Ia.2+ class II conformers are lipid raft-associated and able to drive both tyrosine kinase signaling and efficient antigen presentation to CD4 T cells. Here, we establish that the Ia.2+ I-Ak conformer is formed early in the class II biosynthetic pathway and that differential pairing of highly conserved transmembrane domain GXXXG dimerization motifs is responsible for formation of Ia.2+ versus Ia.2− I-Ak class II conformers and controlling lipid raft partitioning. These findings provide a molecular explanation for the formation of two distinct MHC class II conformers that differ in their inherent ability to signal and drive robust T cell activation, providing new insight into the role of MHC class II in regulating antigen-presenting cell-T cell interactions critical to the initiation and control of multiple aspects of the immune response.