The BPV-1 E5 protein, the 16 kDa membrane pore-forming protein and the PDGF receptor exist in a complex that is dependent on hydrophobic transmembrane interactions.

The E5 oncoprotein of bovine papillomavirus type 1 is a 44 amino acid, highly hydrophobic protein that induces the stable transformation of immortalized murine fibroblasts, presumably through its activation of growth factor receptors. Previous studies have shown that the E5 protein complexes with the 16 kDa (16k) pore-forming protein of vacuolar H(+)-ATPases. This integral membrane protein is essential for the acidification and function of subcellular compartments that process growth factor receptors. Using an SV40 expression system in COS cells, we analyzed whether the E5-16k complexes bind additional cellular proteins, including growth factor receptors. These studies demonstrate that E5 binds to both the 16k protein and the PDGF receptor and that this tri-component complex can be isolated with antibodies specific for each protein. Importantly, the 16k protein bound to the PDGF receptor in the absence of E5, suggesting that E5 binds to the PDGF receptor via its interaction with the 16k protein. An E5 mutant lacking the hydrophilic carboxyl-terminal 14 amino acids retained binding to both 16k and the PDGF receptor, indicating that E5 binds to these proteins through its hydrophobic, membrane-associating domain. These studies reveal that hydrophobic, intramembrane interactions govern the association of E5, 16k and the PDGF receptor, suggesting a ligand-independent mechanism for receptor activation and a potential link between receptor signal transduction pathways and membrane pore activity.     

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.     

E5 oncoprotein transmembrane mutants dissociate fibroblast transforming activity from 16-kilodalton protein binding and platelet-derived growth factor receptor binding and phosphorylation.

The E5 oncoprotein of bovine papillomavirus type 1 is a 44-amino-acid, hydrophobic polypeptide which localizes predominantly in Golgi membranes and appears to transform cells through the activation of tyrosine kinase growth factor receptors. In fibroblasts, E5 interacts with both the 16-kilodalton vacuolar ATPase subunit and the platelet-derived growth factor receptor (PDGF-R) via its hydrophobic transmembrane domain and induces autophosphorylation of the receptor. To further analyze the correlation between E5 biological activity and its ability to bind these cellular proteins, a series of nine E5 transmembrane mutants was evaluated. In 32D mouse hematopoietic cells, there was an incomplete correlation between the abilities of the E5 mutant proteins to associate the PDGF-R and to transform cells. However, all transforming E5 mutant proteins induced PDGF-R tyrosine phosphorylation. In NIH 3T3 and C127 mouse fibroblasts, both transforming and nontransforming E5 mutant proteins were defective for PDGF-R binding. In addition, while most of the transforming E5 proteins induced PDGF-R phosphorylation, one hypertransforming mutant (serine 17) neither bound nor induced receptor autophosphorylation. These findings support the hypothesis that the transformation of fibroblasts by E5 transmembrane mutants can involve alternative cellular targets or potentially independent activities of the E5 protein. In addition, these results underscore the critical role of the transmembrane domain in mediating E5 biological activities.     

The central hydrophobic domain of the bovine papillomavirus E5 transforming protein can be functionally replaced by many hydrophobic amino acid sequences containing a glutamine.

The 44-amino-acid E5 transforming protein of bovine papillomavirus can induce growth transformation of cultured rodent fibroblast cell lines. Previous studies revealed that efficient transformation of mouse C127 cells by the E5 protein required a central core of hydrophobic amino acids and several specific carboxyl-terminal amino acids. Although a randomly derived sequence of hydrophobic amino acids could functionally replace the wild-type hydrophobic core, most such sequences could not. We show here that the conserved glutamine at position 17 in the hydrophobic domain is also important for transformation and that insertion of the glutamine can rescue the transforming activity of many but not all otherwise defective mutants containing random hydrophobic sequences. However, a class of mutants was identified that transform efficiently even in the absence of glutamine, demonstrating that the presence of this amino acid is not absolutely required for efficient transformation. E5 proteins containing the glutamine appear to display increased homodimer formation compared with mutant proteins lacking the glutamine, but this amino acid has no apparent effect on protein stability.     

The E5 oncoprotein of bovine papillomavirus binds to a 16 kd cellular protein.

The E5 oncoprotein of bovine papillomavirus type 1 is the smallest known viral transforming protein. It is a 44 amino acid polypeptide asymmetrically oriented in Golgi and plasma membranes which appears to modify (either directly or indirectly) the internalization and phosphorylation of at least two growth factor receptors: EGF and CSF-1. To identify cellular proteins associated with E5, we have constructed two E5 fusion proteins, each of which contains a well-characterized epitope at the E5 amino terminus. These E5-epitope fusion proteins are biologically active, localize normally to cellular membranes and form dimers. Both monoclonal and polyclonal antibodies against the inserted epitopes specifically co-precipitate E5 and an associated 16 kd cellular protein. A transformation-defective E5 mutant containing a substitution within the hydrophobic portion of E5 is defective in its ability to bind the 16 kd protein. These findings suggest a role for E5/16 kd binding in the process of cellular transformation.     

The human T-cell leukemia/lymphotropic virus type I p12I protein cooperates with the E5 oncoprotein of bovine papillomavirus in cell transformation and binds the 16-kilodalton subunit of the vacuolar H+ ATPase.

The human T-cell leukemia/lymphotropic virus type I (HTLV-I) induces T-cell leukemia and transforms human T cells in vitro. A recently identified protein with a molecular weight of 12,000 (12K) (p12I), encoded by single- and double-spliced mRNAs transcribed from the 3' end of the HTLV-I genome, has been shown to localize in the perinuclear compartment and in the cellular endomembranes. The p12I protein exhibits significant amino acid sequence similarity to the E5 oncoprotein of bovine papillomavirus type 1 (BPV-1). Both proteins are very hydrophobic, contain a glutamine residue in the middle of a potential transmembrane region(s), and are localized in similar cellular compartments. Because of these observations, we investigated whether the p12I resemblance to E5 correlated with a similarity in their biological behavior. We expressed the p12I protein to evaluate its ability to functionally cooperate with the BPV-1 E5 oncoprotein and to bind to a cellular target of the E5 protein, the 16K component of the vacuolar H+ ATPase. Cotransfection of the mouse C127 cell line with the p12I and E5 cDNAs showed that although p12I alone could not induce focus formation, it strongly potentiated the transforming activity of E5. In addition, the p12I protein bound to the 16K protein as efficiently as the E5 protein. These findings might provide new insight for potential mechanisms of HTLV-I transformation and suggest that p12I and E5 represent an example of convergent evolution between RNA and DNA viruses.     

Transglutaminase 2 inhibits Rb binding of human papillomavirus E7 by incorporating polyamine

Transglutaminase 2 (TGase 2) is one of a family of enzymes that catalyze protein modification through the incorporation of polyamines into substrates or the formation of protein crosslinks. However, the physiological roles of TGase 2 are largely unknown. To elucidate the functions of TGase 2, we have searched for its interacting proteins. Here we show that TGase 2 interacts with E7 oncoprotein of human papillomavirus type 18 (HPV18) in vitro and in vivo. TGase 2 incorporates polyamines into a conserved glutamine residue in the zinc-binding domain of HPV18 E7 protein. This modification mediates the inhibition of E7's Rb binding ability. In contrast, TGase 2 does not affect HPV16 E7, due to absence of a glutamine residue at this polyamination site. Using E7 mutants, we demonstrate that TGase 2-dependent inhibition of HPV E7 function correlates with the presence of the polyamination site. Our results indicate that TGase 2 is an important cellular interfering factor and define a novel host-virus interaction, suggesting that the inability of TGase 2 to inactivate HPV16 E7 could explain the high prevalence of HPV16 in cervical cancer.     

Mutagenesis of bacterial elongation factor Tu at lysine 136. A conserved amino acid in GTP regulatory proteins

We have studied the effects of specific amino acid replacements in EF-Tu upon the protein's interactions with guanine nucleotides and elongation factor Ts (EFTs). We found that alterations at the lysine residue of the Asn-Lys-Cys-Asp sequence, the guanine ring-binding sequence, differentially affect the protein's ability to bind guanine nucleotides. Wild type EF-Tu (Lys-136) binds GDP and GTP much more tightly than do many of the altered proteins. Replacing lysine by arginine lowers the protein's affinity for GDP by about 20-fold relative to the change in its affinity for EF-Ts. Substitutions at residue 136 by glutamine (K136Q) and glutamic acid (K136E) further lower the protein relative affinity for GDP by factors of about 4 and 10, respectively. In contrast, replacement of the residue by isoleucine (K136I) eliminates guanine nucleotide binding as well as EF-Ts binding. Apparently, the distortion of this loop by substitution at residue 136 of a bulky hydrophobic residue can hamper the binding for both substrates or disrupt the folding of the protein. All altered proteins except EF-Tu(K136I) are able to bind tRNA(Phe); however, they require much higher concentrations of GTP than wild type EF-Tu. In minimal media, Escherichia coli cells harboring plasmids encoding EF-Tu(K136E) or EF-Tu(K136Q) suffer growth retardation relative to cells bearing the same plasmid encoding wild type EF-Tu. Co-transformation of these cells with a compatible plasmid bearing the EF-Ts gene reverses this growth problem. The growth retardation effect of some of the altered proteins can be explained by their sequestering EF-Ts. These results indicate that EF-Ts is essential to the growth of E. coli and suggest a technique for studying EF-Ts mutants as well as for identifying other guanine nucleotide exchange enzymes.     

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.     

The v-sis protein retains biological activity as a type II membrane protein when anchored by various signal-anchor domains, including the hydrophobic domain of the bovine papilloma virus E5 oncoprotein

Membrane-anchored forms of the v-sis oncoprotein have been previously described which are oriented as type I transmembrane proteins and which efficiently induce autocrine transformation. Several examples of naturally occurring membrane-anchored growth factors have been identified, but all exhibit a type I orientation. In this work, we wished to construct and characterize membrane-anchored growth factors with a type II orientation. These experiments were designed to determine whether type II membrane-anchored growth factors would in fact exhibit biological activity. Additionally, we wished to determine whether the hydrophobic domain of the E5 oncoprotein of bovine papilloma virus (BPV) can function as a signal-anchor domain to direct type II membrane insertion. Type II derivatives of the v-sis oncoprotein were constructed, with the NH2 terminus intracellular and the COOH terminus extracellular, by substituting the NH2 terminal signal sequence with the signal-anchor domain of a known type II membrane protein. The signal-anchor domains of neuraminidase (NA), asialoglycoprotein receptor (ASGPR) and transferrin receptor (TR) all yielded biologically active type II derivatives of the v-sis oncoprotein. Although transforming all of the type II signal/anchor-sis proteins exhibited a very short half-life. The short half-life exhibited by the signal/anchor-sis constructs suggests that, in some cases, cellular transformation may result from the synthesis of growth factors so labile that they activate undetectable autocrine loops. The E5 oncoprotein encoded by BPV exhibits amino acid sequence similarity with PDGF, activates the PDGF beta-receptor, and thus resembles a miniature membrane-anchored growth factor with a putative type II orientation. The hydrophobic domain of the E5 oncoprotein, when substituted in place of the signal sequence of v-sis, was indistinguishable compared with the signal-anchor domains of NA, TR, and ASGPR, demonstrating its ability to function as a signal-anchor domain. NIH 3T3 cells transformed by the signal/anchor-sis constructs exhibited morphological reversion upon treatment with suramin, indicating a requirement for ligand/receptor interactions in a suramin- sensitive compartment, most likely the cell surface. In contrast, NIH 3T3 cells transformed by the E5 oncoprotein did not exhibit morphological reversion in response to suramin.     

Functional analysis at the Cys706 residue of the retinoblastoma protein.

A missense mutation at cysteine 706, resulting in a retinoblastoma (RB) protein defective in phosphorylation and oncoprotein binding, has been isolated from a human tumor cell line. Since this residue is conserved in murine RB and in the related p107 protein, we studied the activity of in vitro mutants flanking this position. These experiments demonstrated that the thiol atom at codon 706 does not possess intrinsic functional activity as small polar or nonpolar residues could substitute at either codons 706 or 707, while bulkier R-group changes in these positions interfered with in vitro oncoprotein binding or in vivo protein phosphorylation. A series of missense mutants in an adjacent leucine repeat domain also demonstrated a loss of oncoprotein binding that was proportional to the magnitude of amino acid substitutions. To determine whether the cysteine 706 --> phenylalanine RB mutant retained any protein binding activity, we examined its ability to precipitate MYC, which was recently identified as a potential RB-associated protein. These experiments demonstrated that the mutant RB product is capable of binding in vitro to c-myc and L-myc proteins with comparable affinity as wild-type RB. These findings raise questions about the functional role of the RB:MYC interactions and emphasize important differences in the binding patterns between MYC and the other RB-associated proteins.     

Mutational analysis of human papillomavirus type 11 E5a oncoprotein.

In this study, we investigated the structural basis of human papillomavirus type 11 (HPV-11) E5a transforming activity at the amino acid level. The effects of insertion, deletion , and substitution mutations on teh E5a transforming activity were determined by the assay of anchorage-independent growth. In the conserved Cys-X-Cys structure, substitution of Ser for Cys-73 resulted in indistinguishable transforming activity, whereas substitution of Ser for Cys-75 or Ser for both Cys-73 and Cys-75 retained 50 and 42% transformation, respectively. This suggests that Cys at position 75 may be important for transformation. Charge and structural changes at teh COOH termini of several mutants impaired transformation significantly, but those at the middle region did so only mildly. In addition, the 16,000-molecular-weight pore-forming protein (16K protein) is known to associate with BPV-1, HPV-6, and HPV-16 E5 proteins. In this study, we investigated the correlation between E5a-16K binding affinity and the transforming activity of E5a by the use of 11 E5a mutants. Results show that E5a and these 11 E5a mutants could bind to the 16K protein when these proteins were coexpressed in COS cells, suggesting that simple binding of the 16K protein by E5a may not be sufficient for cell transformation.     

The conserved C-terminal domain of the bovine papillomavirus E5 oncoprotein can associate with an alpha-adaptin-like molecule: a possible link between growth factor receptors and viral transformation.

The bovine papillomavirus E5 gene encodes an oncoprotein that can independently transform rodent fibroblasts. This small 44-amino-acid protein is thought to function through the activation of growth factor receptors. E5 activation of the epidermal growth factor receptor results in an increase in the number of activated receptors at the cell surface. This finding suggests that E5 may act by inhibiting the normal down regulation of activated epidermal growth factor receptor via coated pit-mediated endocytosis. We have constructed a fusion protein consisting of glutathione S-transferase and the conserved C-terminal domain of E5 (GST-E5) in order to identify E5-associated cellular proteins that may be involved in its transforming activity. We have identified a 125-kDa cellular protein with a strong associated serine kinase activity that specifically associated with GST-E5 in the reduced form but not with GST-E5 fusions that contained changes in several conserved amino acids. Microsequence and biochemical analyses suggest that p125 is a novel member of the alpha-adaptin family. Since alpha-adaptins have previously been shown to be involved in coated pit-mediated cell surface receptor endocytosis and down regulation, these results suggest that p125 may be an alpha-adaptin-like molecule involved in growth factor receptor down regulation and that E5 may act by inhibiting its activity.     

Role of Glutamine 17 of the Bovine Papillomavirus E5 Protein in Platelet-Derived Growth Factor β Receptor Activation and Cell Transformation

The bovine papillomavirus E5 protein is a small, homodimeric transmembrane protein that forms a stable complex with the cellular platelet-derived growth factor (PDGF) β receptor through transmembrane and juxtamembrane interactions, resulting in receptor activation and cell transformation. Glutamine 17 in the transmembrane domain of the 44-amino-acid E5 protein is critical for complex formation and receptor activation, and we previously proposed that glutamine 17 forms a hydrogen bond with threonine 513 of the PDGF β receptor. We have constructed and analyzed mutant E5 proteins containing all possible amino acids at position 17 and examined the ability of these proteins to transform C127 fibroblasts, which express endogenous PDGF β receptor. Although several position 17 mutants were able to transform cells, mutants containing amino acids with side groups that were unable to participate in hydrogen bonding interactions did not form a stable complex with the PDGF β receptor or transform cells, in agreement with the proposed interaction between position 17 of the E5 protein and threonine 513 of the receptor. The nature of the residue at position 17 also affected the ability of the E5 proteins to dimerize. Overall, there was an excellent correlation between the ability of the various E5 mutant proteins to bind the PDGF β receptor, lead to receptor tyrosine phosphorylation, and transform cells. Similar results were obtained in Ba/F3 hematopoietic cells expressing exogenous PDGF β receptor. In addition, treatment of E5-transformed cells with a specific inhibitor of the PDGF receptor tyrosine kinase reversed the transformed phenotype. These results confirm the central importance of the PDGF β receptor in mediating E5 transformation and highlight the critical role of the residue at position 17 of the E5 protein in the productive interaction with the PDGF β receptor. On the basis of molecular modeling analysis and the known chemical properties of the amino acids, we suggest a structural basis for the role of the residue at position 17 in E5 dimerization and in complex formation between the E5 protein and the PDGF β receptor.     

Membrane topography of ColE1 gene products: the hydrophobic anchor of the colicin E1 channel is a helical hairpin.

The paucity of crystallographic data on the structure of intrinsic membrane proteins necessitates the development of additional techniques to probe their structures. The colicin E1 ion channel domain contains one prominent hydrophobic region near its COOH terminus that has been proposed to be an anchor for the assembly of the channel. Saturation site-directed mutagenesis of the hydrophobic anchor region of the colicin E1 ion channel was used to probe whether it spanned the bilayer once or twice. A nonpolar amino acid was replaced by a charged residue in 29 mutations made at 26 positions in the channel domain. Substitution of the charged amino acid at all positions except those in the center of the hydrophobic region and the periphery of the hydrophobic region caused a large decrease in the cytotoxicity of the purified mutant colicin E1 protein. This result implies that the hydrophobic domain spans the membrane bilayer twice in a helical hairpin loop, with the center of this domain residing in an aqueous or polar phase. The lengths of the trans-membrane helices appear to be approximately 18 and 16 residues. The absence of significant changes in ion selectivity in five of nine mutants indicated that these mutations did not cause a large change in the channel structure. The ion selectivity changes in four mutants and those previously documented for the flanking Lys residues imply that the hydrophobic hairpin is part of the channel lumen. Water may "abhor" the hydrophobic side of the channel, explaining the small effects of residue charge changes on ion selectivity.     

Phosphotyrosine binding domains of Shc and insulin receptor substrate 1 recognize the NPXpY motif in a thermodynamically distinct manner

Phosphotyrosine binding (PTB) domains of the adaptor protein Shc and insulin receptor substrate (IRS-1) interact with a distinct set of activated and tyrosine-phosphorylated cytokine and growth factor receptors and play important roles in mediating mitogenic signal transduction. By using the technique of isothermal titration calorimetry, we have studied the thermodynamics of binding of the Shc and IRS-1 PTB domains to tyrosine-phosphorylated NPXY-containing peptides derived from known receptor binding sites. The results showed that relative contributions of enthalpy and entropy to the free energy of binding are dependent on specific phosphopeptides. Binding of the Shc PTB domain to tyrosine-phosphorylated peptides from TrkA, epidermal growth factor, ErbB3, and insulin receptors is achieved via an overall entropy-driven reaction. On the other hand, recognition of the phosphopeptides of insulin and interleukin-4 receptors by the IRS-1 PTB domain is predominantly an enthalpy-driven process. Mutagenesis and amino acid substitution experiments showed that in addition to the tyrosine-phosphorylated NPXY motif, the PTB domains of Shc and IRS-1 prefer a large hydrophobic residue at pY-5 and a small hydrophobic residue at pY-1, respectively (where pY is phosphotyrosine). These results agree with the calculated solvent accessibility of these two key peptide residues in the PTB domain/peptide structures and support the notion that the PTB domains of Shc and IRS-1 employ functionally distinct mechanisms to recognize tyrosine-phosphorylated receptors.     

The E5 oncoprotein of human papillomavirus type 16 inhibits the acidification of endosomes in human keratinocytes.

The human papillomavirus type 16 E5 oncoprotein possesses mitogenic activity that acts synergistically with epidermal growth factor (EGF) in human keratinocytes and inhibits the degradation of the EGF receptor in endosomal compartments after ligand-stimulated endocytosis. One potential explanation for these observations is that E5 inhibits the acidification of endosomes. This may be mediated through the 16-kDa component of the vacuolar proton-ATPase, since animal and human papillomavirus E5 proteins bind this subunit protein. Using a ratio-imaging technique to determine endosomal pH, we found that the acidification of endosomes in E5-expressing keratinocytes was delayed at least fourfold compared with normal human keratinocytes and endosomes in some cells never completely acidified. Furthermore, E5 expression increased the resistance of keratinocytes to protein synthesis inhibition by diphtheria toxin, a process dependent on efficient endosomal acidification. Finally, artificially inhibiting endosomal acidification with chloroquine during the endocytosis of EGF receptors in keratinocytes demonstrated many of the same effects as the expression of human papillomavirus type 16 E5, including prolonged retention of undegraded EGF receptors in intracellular vesicles.     

Identification of amino acids in the transmembrane and juxtamembrane domains of the platelet-derived growth factor receptor required for productive interaction with the bovine papillomavirus E5 protein.

The bovine papillomavirus E5 protein forms a stable complex with the cellular platelet-derived growth factor (PDGF) beta receptor, resulting in receptor activation and cell transformation. Amino acids in both the putative transmembrane domain and extracytoplasmic carboxyl-terminal domain of the E5 protein appear important for PDGF receptor binding and activation. Previous analysis indicated that the transmembrane domain of the receptor was also required for complex formation and receptor activation. Here we analyzed receptor chimeras and point mutants to identify specific amino acids in the PDGF beta receptor required for productive interaction with the E5 protein. These receptor mutants were analyzed in murine Ba/F3 cells, which do not express endogenous receptor. Our results confirmed the importance of the transmembrane domain of the receptor for complex formation, receptor tyrosine phosphorylation, and mitogenic signaling in response to the E5 protein and established that the threonine residue in this domain is required for these activities. In addition, a positive charge in the extracellular juxtamembrane domain of the receptor was required for E5 interaction and signaling, whereas replacement of the wild-type lysine with either a neutral or acidic amino acid inhibited E5-induced receptor activation and transformation. All of the receptor mutants defective for activation by the E5 protein responded to acute treatment with PDGF and to stable expression of v-Sis, a form of PDGF. The required juxtamembrane lysine and transmembrane threonine are predicted to align precisely on the same face of an alpha helix packed in a left-handed coiled-coil geometry. These results establish that the E5 protein and v-Sis recognize distinct binding sites on the PDGF beta receptor and further clarify the nature of the interaction between the viral transforming protein and its cellular target.     

Ferredoxin-NADP+ reductase uses the same site for the interaction with ferredoxin and flavodoxin

The enzyme ferredoxin-NADP(+) reductase (FNR) forms a 1 : 1 complex with ferredoxin (Fd) or flavodoxin (Fld) that is stabilised by both electrostatic and hydrophobic interactions. The electrostatic interactions occur between acidic residues of the electron transfer (ET) protein and basic residues on the FNR surface. In the present study, several charge-reversal mutants of FNR have been prepared at the proposed site of interaction of the ET protein: R16E, K72E, K75E, K138E, R264E, K290E and K294E. All of these mutants have been assayed for reactivity with Fd and Fld using steady-state and stopped-flow kinetics. Their abilities for complex formation with the ET proteins have also been tested. The data presented here indicate that the mutated residues situated within the FNR FAD-binding domain are more important for achieving maximal ET rates, either with Fd or Fld, than those situated within the NADP(+)-binding domain, and that both ET proteins occupy the same region for the interaction with the reductase. In addition, each individual residue does not appear to participate to the same extent in the different processes with Fd and Fld.     

Molecular Determinants of PAM2 Recognition by the MLLE Domain of Poly(A)-Binding Protein

MLLE (previously known as PABC) is a peptide-binding domain that is found in poly(A)-binding protein (PABP) and EDD (E3 isolated by differential display), a HECT E3 ubiquitin ligase also known as HYD (hyperplastic discs tumor suppressor) or UBR5. The MLLE domain from PABP recruits various regulatory proteins and translation factors to poly(A) mRNAs through binding of a conserved 12 amino acid peptide motif called PAM2 (for PABP-interacting motif 2). Here, we determined crystal structures of the MLLE domain from PABP alone and in complex with PAM2 peptides from PABP-interacting protein 2. The structures provide a detailed view of hydrophobic determinants of the MLLE binding coded by PAM2 positions 3, 5, 7, 10, and 12 and reveal novel intermolecular polar contacts. In particular, the side chain of the invariant MLLE residue K580 forms hydrogen bonds with the backbone of PAM2 residues 5 and 7. The structures also show that peptide residues outside of the conserved PAM2 motif contribute to binding. Altogether, the structures provide a significant advance in understanding the molecular basis for the binding of PABP by PAM2-containing proteins involved in translational control, mRNA deadenylation, and other cellular processes.