Computational analysis of membrane proteins: genomic occurrence, structure prediction and helix interactions

Lehnert, Ursula; Xia, Yu; Royce, Thomas; Goh, Chern-Sing; Liu, Yang; Senes, Alessandro; Yu, Haiyuan; Zhang, Zhao Lei; Engelman, Donald M.; Gerstein, Mark

doi:10.1017/s003358350400397x

Cited by 62 publications

(45 citation statements)

References 218 publications

(215 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To randomize the TM domain, we used a degenerate oligonucleotide in which positions corresponding to codons 12-30 were synthesized with a mixture of nucleotides such that approximately 82% of the amino acids encoded by this segment were hydrophobic. This composition closely matches that of naturally-occurring TM domains (1). In addition, position 33 was randomized to encode six different hydrophilic amino acids because a hydrophilic amino acid is required at this position in the E5 protein (19).…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Construction and genetic selection of small transmembrane proteins that activate the human erythropoietin receptor

Cammett¹,

Jun²,

Cohen³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

This work describes a genetic approach to isolate small, artificial transmembrane (TM) proteins with biological activity. The bovine papillomavirus E5 protein is a dimeric, 44-amino acid TM protein that transforms cells by specifically binding and activating the platelet-derived growth factor β receptor (PDGFβR). We used the E5 protein as a scaffold to construct a retrovirus library expressing ∼500; 000 unique 44-amino acid proteins with randomized TM domains. We screened this library to select small, dimeric TM proteins that were structurally unrelated to erythropoietin (EPO), but specifically activated the human EPO receptor (hEPOR). These proteins did not activate the murine EPOR or the PDGFβR. Genetic studies with one of these activators suggested that it interacted with the TM domain of the hEPOR. Furthermore, this TM activator supported erythroid differentiation of primary human hematopoietic progenitor cells in vitro in the absence of EPO. Thus, we have changed the specificity of a protein so that it no longer recognizes its natural target but, instead, modulates an entirely different protein. This represents a novel strategy to isolate small artificial proteins that affect diverse membrane proteins. We suggest the word "traptamer" for these transmembrane aptamers.bovine papilloma virus | E5 protein | expression libraries | protein engineering | traptamers I t is thought that up to 30% of all proteins span cell membranes (1). Most membrane-spanning protein segments adopt an α-helical conformation that is largely independent of their amino acid sequence and minimally influenced by extramembraneous protein domains. Although transmembrane (TM) domains were once thought to be rather nonspecific protein segments whose primary function was to anchor proteins in membranes, they can participate in highly-specific, inter-and intramolecular side-by-side interactions that control important biological processes (2).The 44-amino acid bovine papillomavirus type 1 (BPV) E5 protein is essentially an isolated TM domain. It contains a hydrophobic central segment and exists in the intracellular membranes of transformed cells as a type II TM homodimer stabilized by disulfide bonds in its carboxy-terminus (3-5). The E5 dimer binds to the TM domains of two molecules of the platelet-derived growthfactor β receptor (PDGFβR) tyrosine kinase, resulting in receptor dimerization and activation, leading to cell transformation (6-10). The E5 protein does not bind to other TM domains or to certain PDGFβR TM mutants (11).Based on our analysis of the E5 protein, we proposed that it might be possible to construct small, artificial TM proteins that regulate a variety of viral and cellular TM proteins in trans (11, 12). To identify small proteins with different TM sequences that transform cells, we constructed expression libraries in which the TM domain of the E5 protein was replaced with randomized sequences of primarily hydrophobic amino acids and isolated clones from these libraries that induced focus formation or growth-factor independe...

show abstract

Section: Resultsmentioning

confidence: 99%

“…I t is thought that up to 30% of all proteins span cell membranes (1). Most membrane-spanning protein segments adopt an α-helical conformation that is largely independent of their amino acid sequence and minimally influenced by extramembraneous protein domains.…”

mentioning

confidence: 99%

Construction and genetic selection of small transmembrane proteins that activate the human erythropoietin receptor

Cammett¹,

Jun²,

Cohen³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

show abstract

“…Here, we have followed their example and constructed extremely small transmembrane proteins that modulate a much larger cellular target, thereby exerting a tremendous effect on cell behavior. Because up to 30% of all cellular proteins are thought to have membrane-spanning helices (25), a substantial fraction of the biochemical functions of cells could be modulated by similar mechanisms. It seems likely that cells express similar, small transmembrane …”

Section: Discussionmentioning

confidence: 99%

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

Talbert-Slagle¹,

Marlatt²,

Barrera

et al. 2009

J Virol

Self Cite

View full text Add to dashboard Cite

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) ␤ receptor and induces ligand-independent receptor activation. Three specific features of BPV E5 are considered important for its ability to activate the PDGF ␤ 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 ␤ 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 ␤ 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.Viruses are tiny relative to the cells they infect. The largest animal viruses encode at most a few hundred genes, in contrast to the tens of thousands of genes expressed by the host cell. To overcome the constraints imposed by their small size, viruses use multiple or even overlapping reading frames and alternative splicing to produce more than one protein from single transcripts, build capsids from repeating subunits, harness cellular mechanisms to produce viral products, and express extremely small proteins. Several of these small viral proteins are membrane anchored, including the 44-residue SH protein of parainfluenza virus 5, which blocks apoptosis of infected cells (16), and the 96-residue M2 protein of influenza A, which forms an ion channel in endosomal membranes of infected cells and is required for infectivity (reviewed in reference 38). One of the best-characterized small viral transmembrane proteins is the E5 protein encoded by bovine papillomavirus type 1 (BPV E5). BPV E5 contains only 44 amino acids and is thus essentially an isolated transmembrane domain (Fig. 1) (reviewed in reference 47). It is the primary translation product from a small open reading frame and is sufficient to cause tumorigenic cell transformation, making it the smallest known autonomous oncoprotein.BPV E5 induces cell transformation by specifically activating a much larger cellular target, the platelet-derived growth factor (PDGF) ␤ receptor tyrosine kinase (8,14,30,35). The PDGF ␤ receptor, a single-span transmembrane protein of more than 1,000 amino acids, is normally activated by the binding of its dimeric ligand, PDGF, to the extracellular domain of the receptor. In contrast, a dimer of the BPV E5 protein binds to the transmembrane doma...

show abstract

“…roteins that span cellular membranes are thought to comprise up to 30% of the proteome (1). The membrane-spanning segments of most of these transmembrane proteins adopt an ␣-helical conformation and can undergo highly specific, lateral interactions to form oligomeric protein complexes or properly folded multipass transmembrane proteins (2,3).…”

mentioning

confidence: 99%

Compensatory Mutants of the Bovine Papillomavirus E5 Protein and the Platelet-Derived Growth Factor β Receptor Reveal a Complex Direct Transmembrane Interaction

Edwards

Xie

Bowers

et al. 2013

J Virol

View full text Add to dashboard Cite

eThe 44-amino-acid E5 protein of bovine papillomavirus is a dimeric transmembrane protein that exists in a stable complex with the platelet-derived growth factor (PDGF) ␤ receptor, causing receptor activation and cell transformation. The transmembrane domain of the PDGF ␤ receptor is required for complex formation, but it is not known if the two proteins contact one another directly. Here, we studied a PDGF ␤ receptor mutant containing a leucine-to-isoleucine substitution in its transmembrane domain, which prevents complex formation with the wild-type E5 protein in mouse BaF3 cells and inhibits receptor activation by the E5 protein. We selected E5 mutants containing either a small deletion or multiple substitution mutations that restored binding to the mutant PDGF ␤ receptor, resulting in receptor activation and growth factor independence. These E5 mutants displayed lower activity with PDGF ␤ receptor mutants containing other transmembrane substitutions in the vicinity of the original mutation, and one of them cooperated with a receptor mutant containing a distal mutation in the juxtamembrane domain. These results provide strong genetic evidence that the transmembrane domains of the E5 protein and the PDGF ␤ receptor contact one another directly. They also demonstrate that different mutations in the E5 protein allow it to tolerate the same mutation in the PDGF ␤ receptor transmembrane domain and that a mutation in the E5 protein can allow it to tolerate different mutations in the PDGF ␤ receptor. Thus, the rules governing direct interactions between transmembrane helices are complex and not restricted to local interactions. Proteins that span cellular membranes are thought to comprise up to 30% of the proteome (1). The membrane-spanning segments of most of these transmembrane proteins adopt an ␣-helical conformation and can undergo highly specific, lateral interactions to form oligomeric protein complexes or properly folded multipass transmembrane proteins (2, 3). These helical interactions are often essential for biological activity, but the structural basis of the vast majority of transmembrane interactions is not understood because of difficulties in obtaining high-resolution structures of transmembrane helical bundles. Mutational analysis showed that homodimeric helix-helix interactions can be determined by highly specific interactions between amino acid side chains (4). Formation of heteromeric transmembrane complexes can be mediated by interactions between hydrophilic amino acid side chains (5-7), but the structural basis for the specificity of heteromeric transmembrane interactions has not been studied in detail. In our laboratory, we have developed genetic methods to analyze heteromeric transmembrane interactions in mammalian cells and showed that artificial transmembrane proteins can undergo specific functional interactions with native transmembrane proteins (8-12).The 44-amino-acid E5 oncoprotein of bovine papillomavirus type 1 (BPV) is essentially an isolated transmembrane domain that forms a homodime...

show abstract

Computational analysis of membrane proteins: genomic occurrence, structure prediction and helix interactions

Cited by 62 publications

References 218 publications

Construction and genetic selection of small transmembrane proteins that activate the human erythropoietin receptor

Construction and genetic selection of small transmembrane proteins that activate the human erythropoietin receptor

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

Compensatory Mutants of the Bovine Papillomavirus E5 Protein and the Platelet-Derived Growth Factor β Receptor Reveal a Complex Direct Transmembrane Interaction

Contact Info

Product

Resources

About