Receptor tyrosine kinases bind ligands such as cytokines, hormones, and growth factors and regulate key cellular processes, including cell division. They are also implicated in the development of many types of cancer. One such example is the Neu receptor tyrosine kinase found in rats (homologous to the human ErbB2 protein), which can undergo a valine to glutamic acid (V664E) mutation at the center of its α-helical transmembrane domain. This substitution results in receptor activation and oncogenesis. The molecular basis of this dramatic change in behavior upon introduction of the V664E mutation has been difficult to pin down, with conflicting results reported in the literature. Here we report the first quantitative, thermodynamic analysis of dimerization and biophysical characterization of the rat Neu transmembrane domain and several mutants in a range of chemical environments. These data have allowed us to identify the effects of the V664E mutation in the isolated TM domain with respect to protein–protein and protein–lipid interactions, membrane insertion, and secondary structure. We also report the results from a 100 ns atomistic molecular dynamics simulation of the Neu transmembrane domain in a model membrane bilayer (dipalmitoylphosphatidylcholine). The results from simulation and experiment are in close agreement and suggest that, in the model systems investigated, the V664E mutation leads to a weakening of the TM dimer and a change in sequence-dependent interactions. These results are contrary to recent results obtained in mammalian membranes, and the implications of this are discussed.

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).

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 MHCα/β 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 MHCα/β 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.

Membrane-spanning epidermal growth factor receptor ErbB2 is of key importance in cell division, in which a dimeric complex of the protein is responsible for tyrosine kinase activation following ligand binding. The rat homologue of this receptor (Neu) is prone to a valine to glutamic acid mutation in the transmembrane domain (TM), resulting in permanent activation and oncogenesis. In this study, the TM domains of Neu and the corresponding oncogenic mutant Neu*, which contains a V to E mutation at position 664 in the TM domain, have been analyzed to improve our understanding of the structural effects of the oncogenic V664E mutation. Building on previous work, we have focused here on understanding the sequence dependence of TM helix−helix interactions and any differences in behavior upon introduction of the V664E mutation. Using a variety of biochemical and biophysical methods, we find that the rat Neu TM domain forms strong oligomers and, similar to previous observations for the human ErbB2 TM domain, the oncogenic mutation results in a reduced level of self-association. Our data also strongly indicate that the proto-oncogenic Neu TM domain can adopt multiple (at least two) oligomeric conformations in the membrane, possibly corresponding to the active and inactive forms of the receptor, and can “switch” between the two. Further, the oncogenic Neu* mutant appears to inhibit this “conformational switching” of TM dimers, as we observe that dimerization of the Neu* TM domain in the Escherichia coli inner membrane strongly favors a single conformation stabilized by an IXXXV motif (I659-XXX-V663) originally identified by site-specific infrared spectroscopic studies.

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 (PDGFβR). 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 α-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.

•Carboxypeptidase G2 is of therapeutic interest in cancer and autoimmune treatment.•Characterization of key features of CPG2 has been hampered by low protein yields.•We report the first high-yield bacterial expression of soluble and active CPG2.•We also report the first study of the isolated catalytic and dimerization domains.•The results led to design of a highly truncated enzyme which retains 62% activity.Due to its applications in the treatment of cancer and autoimmune diseases, the 42 kDa zinc-dependent metalloenzyme carboxypeptidase G2 (CPG2) is of great therapeutic interest. An X-ray crystal structure of unliganded CPG2 reported in 1997 revealed the domain architecture and informed early rational drug design efforts, however further efforts at co-crystallization of CPG2 with ligands, substrates or inhibitors have not been reported. Thus key features of CPG2 such as the location of the active site, the presence of additional ligand-binding sites, stability, oligomeric state, and the molecular basis of activity remain largely unknown, with the current working understanding of CPG2 activity based primarily on computational modelling. To facilitate renewed efforts in CPG2 structural biology, we report the first high-yield (250 mg L−1) recombinant expression (and purification) of soluble and active CPG2 using the Escherichia coli expression system. We used this protocol to produce full-length enzyme, as well as protein fragments corresponding to the individual catalytic and dimerization domains, and the activity and stability of each construct was characterised. We adapted our protocol to allow for uniform incorporation of NMR labels (13C, 15N and 2H) and present preliminary solution-state NMR spectra of high quality. Taken together, our results offer a route for production and solution-state characterization that supports renewed effort in CPG2 structural biology as well as design of significantly truncated CPG2 proteins, which retain activity while yielding (potentially) improved immunogenicity.

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.

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).