Packing contacts can mediate highly specific interactions between artificial transmembrane proteins and the PDGFbeta receptor.

We used proteins with randomized transmembrane (TM) domains to explore the role of hydrophobic amino acids in mediating specific interactions between transmembrane helices. The 44-aa bovine papillomavirus E5 protein, which binds to the TM domain of the PDGFbeta receptor (PDGFbetaR) was used as a scaffold to construct a library encoding small dimeric proteins with randomized, strictly hydrophobic TM domains, and proteins were selected that induced focus formation in mouse C127 cells by activating the PDGFbetaR. Analysis of these proteins identified a motif of two hydrophobic residues that, when inserted into a 17-residue polyleucine TM domain, generated a protein that activated the PDGFbetaR and transformed cells. In addition, we identified transforming proteins that activated the wild-type PDGFbetaR but did not activate a series of PDGFbetaR TM point mutants that were efficiently activated by the E5 protein, indicating that these proteins were more specific than the E5 protein. Our results implied that multiple van der Waals interactions distributed along the entire length of the TM domains were required for productive interaction between the PDGFbetaR and some small proteins lacking hydrophilic TM residues. Our results also suggested that excluding hydrophilic residues from small TM proteins and peptides is a strategy to increase the specificity of heteromeric TM helix-helix interactions.

Modulation of cell function by small transmembrane proteins modeled on the bovine papillomavirus E5 protein.

Viruses have been subjected to intense study because of their medical importance and because they can provide fundamental insights into normal and pathological cellular processes. Indeed, much of our knowledge about basic cellular biology and biochemistry was acquired through the study of viruses, and some of medicine's greatest triumphs and challenges involve viruses. Since viruses have evolved to exploit important cell processes, they can provide tools and approaches to manipulate cell function. The small transmembrane E5 protein of bovine papillomavirus type 1 transforms cells by a unique mechanism involving ligand-independent activation of the platelet-derived growth factor beta receptor. Experiments summarized in this review suggest that it may be possible to use the E5 protein as a model to design an entirely new class of small, modular transmembrane proteins with novel biological activities.

Conserved locus-specific silencing functions of Schizosaccharomyces pombe sir2+.

In Schizosaccharomyces pombe, three genes, sir2(+), hst2(+), and hst4(+), encode members of the Sir2 family of conserved NAD(+)-dependent protein deacetylases. The S. pombe sir2(+) gene encodes a nuclear protein that is not essential for viability or for resistance to treatment with UV or a microtubule-destabilizing agent. However, sir2(+) is essential for full transcriptional silencing of centromeres, telomeres, and the cryptic mating-type loci. Chromatin immunoprecipitation results suggest that the Sir2 protein acts directly at these chromosomal regions. Enrichment of Sir2p at silenced regions does not require the HP1 homolog Swi6p; instead, Swi6-GFP localization to telomeres depends in part on Sir2p. The phenotype of sir2 swi6 double mutants supports a model whereby Sir2p functions prior to Swi6p at telomeres and the silent mating-type loci. However, Sir2p does not appear to be essential for the localization of Swi6p to centromeric foci. Cross-complementation experiments showed that the Saccharomyces cerevisiae SIR2 gene can function in place of S. pombe sir2(+), suggesting overlapping deacetylation substrates in both species. These results also suggest that, despite differences in most of the other molecules required, the two distantly related yeast species share a mechanism for targeting Sir2p homologs to silent chromatin.

Specific locations of hydrophilic amino acids in constructed transmembrane ligands of the platelet-derived growth factor beta receptor.

The 44 amino acid E5 transmembrane protein is the primary oncogene product of bovine papillomavirus. Homodimers of the E5 protein activate the cellular PDGF beta receptor tyrosine kinase by binding to its transmembrane domain and inducing receptor dimerization, resulting in cellular transformation. To investigate the role of transmembrane hydrophilic amino acids in receptor activation, we constructed a library of dimeric small transmembrane proteins in which 16 transmembrane amino acids of the E5 protein were replaced with random, predominantly hydrophobic amino acids. A low level of hydrophilic amino acids was encoded at each of the randomized positions, including position 17, which is an essential glutamine in the wild-type E5 protein. Library proteins that induced transformation in mouse C127 cells stably bound and activated the PDGF beta receptor. Strikingly, 35% of the transforming clones had a hydrophilic amino acid at position 17, highlighting the importance of this position in activation of the PDGF beta receptor. Hydrophilic amino acids in other transforming proteins were found adjacent to position 17 or at position 14 or 21, which are in the E5 homodimer interface. Approximately 22% of the transforming proteins lacked hydrophilic amino acids. The hydrophilic amino acids in the transforming clones appear to be important for driving homodimerization, binding to the PDGF beta receptor, or both. Interestingly, several of the library proteins bound and activated PDGF beta receptor transmembrane mutants that were not activated by the wild-type E5 protein. These experiments identified transmembrane proteins that activate the PDGF beta receptor and revealed the importance of hydrophilic amino acids at specific positions in the transmembrane sequence. Our identification of transformation-competent transmembrane proteins with altered specificity suggests that this approach may allow the creation and identification of transmembrane proteins that modulate the activity of a variety of receptor tyrosine kinases.

Selection and characterization of small random transmembrane proteins that bind and activate the platelet-derived growth factor beta receptor.

Growth factor receptors are typically activated by the binding of soluble ligands to the extracellular domain of the receptor, but certain viral transmembrane proteins can induce growth factor receptor activation by binding to the receptor transmembrane domain. For example, homodimers of the transmembrane 44-amino acid bovine papillomavirus E5 protein bind the transmembrane region of the PDGF beta receptor tyrosine kinase, causing receptor dimerization, phosphorylation, and cell transformation. To determine whether it is possible to select novel biologically active transmembrane proteins that can activate growth factor receptors, we constructed and identified small proteins with random hydrophobic transmembrane domains that can bind and activate the PDGF beta receptor. Remarkably, cell transformation was induced by approximately 10% of the clones in a library in which 15 transmembrane amino acid residues of the E5 protein were replaced with random hydrophobic sequences. The transformation-competent transmembrane proteins formed dimers and stably bound and activated the PDGF beta receptor. Genetic studies demonstrated that the biological activity of the transformation-competent proteins depended on specific interactions with the transmembrane domain of the PDGF beta receptor. A consensus sequence distinct from the wild-type E5 sequence was identified that restored transforming activity to a non-transforming poly-leucine transmembrane sequence, indicating that divergent transmembrane sequence motifs can activate the PDGF beta receptor. Molecular modeling suggested that diverse transforming sequences shared similar protein structure, including the same homodimer interface as the wild-type E5 protein. These experiments have identified novel proteins with transmembrane sequences distinct from the E5 protein that can activate the PDGF beta receptor and transform cells. More generally, this approach may allow the creation and identification of small proteins that modulate the activity of a variety of cellular transmembrane proteins.