Towards a structural understanding of the smallest known oncoprotein: investigation of the bovine papillomavirus E5 protein using solution-state NMR

The homodimeric E5 protein from bovine papillomavirus activates the platelet-derived growth factor β receptor through transmembrane (TM) helix-helix interactions leading to uncontrolled cell growth. Detailed structural information for the E5 dimer is essential if we are to uncover its unique mechanism of action. In vivo mutagenesis has been used to identify residues in the TM domain critical for dimerization, and we previously reported that a truncated synthetic E5 peptide forms dimers via TM domain interactions. Here we extend this work with the first application of high-resolution solution-state NMR to the study of the E5 TM domain in SDS micelles. Using selectively 15N-labelled peptides, we first probe sample homogeneity revealing two predominate species, which we interpret to be monomer and dimer. The equilibrium between the two states is shown to be dependent on detergent concentration, revealed by intensity shifts between two sets of peaks in 15N-(1)H HSQC experiments, highlighting the importance of sample preparation when working with these types of proteins. This information is used to estimate a free energy of association (ΔGx°=-3.05 kcal mol(-1)) for the dimerization of E5 in SDS micelles. In addition, chemical shift changes have been observed that indicate a more pronounced change in chemical environment for those residues expected to be at the dimer interface in vivo versus those that are not. Thus we are able to demonstrate our in vitro dimer is comparable to that defined in vivo, validating the biological significance of our synthetic peptide and providing a solid foundation upon which to base further structural studies. Using detergent concentration to modulate oligomeric state and map interfacial residues by NMR could prove useful in the study of other homo-oligomeric transmembrane proteins.     

The composition rather than position of polar residues (QxxS) drives aspartate receptor transmembrane domain dimerization in vivo

Transmembrane (TM) helix association is an important process affecting the function of many integral membrane proteins. Consequently, aberrations in this process are associated with diseases. Unfortunately, our knowledge of the factors that control this oligomerization process in the membrane milieu is limited at best. Previous studies have shown a role for polar residues in the assembly of synthetic peptides in vitro and the association of de novo-designed TM helices in vivo. Here we examined, for the first time, the involvement of polar residues in the dimerization of a biological TM domain in its natural environment. We analyzed both the involvement of polar residues in the dimerization process and whether their influence is position-dependent. For this purpose, we used the TM domain of the Escherichia coli aspartate receptor (Tar) and 10 single and double mutants. Polar to nonpolar mutations in the sequence demonstrated the role of the QxxS motif in the dimerization of the Tar TM domain. Moreover, creating a GxxxG motif, instead of the polar motif, almost completely abolished dimerization. Swapping positions between two wild-type polar residues did not affect dimerization, implying a similar contribution from both positions. Interestingly, mutants that contain two identical strong polar residues, EE and QQ, demonstrated a substantially higher level of dimerization than a QE mutant, although all three TM domains contain two strong polar residues. This result suggests that, in addition to the polarity of the residues, the formation of symmetric bonds also plays a role in dimer stability. The results of this study may facilitate a rational modulation of membrane protein function for therapeutic purposes.     

The strong dimerization of the transmembrane domain of the fibroblast growth factor receptor (FGFR) is modulated by C‐terminal juxtamembrane residues

The fibroblast growth factor receptor 3 (FGFR3) is a member of the FGFR subfamily of the receptor tyrosine kinases (RTKs) involved in signaling across the plasma membrane. Generally, ligand binding leads to receptor dimerization and activation. Dimerization involves the transmembrane (TM) domain, where mutations can lead to constitutive activation in certain cancer types and also in skeletal malformations. Thus, it has been postulated that FGFR homodimerization must be inherently weak to allow regulation, a feature reminiscent of α and β integrin TM interactions. However, we show herein that in FGFR3‐TM, four C‐terminal residues, CRLR, have a profound destabilizing effect in an otherwise strongly dimerizing TM peptide. In the absence of these four residues, the dimerizing propensity of FGFR3‐TM is comparable to glycophorin, as shown using various detergents. In addition, the expected enhanced dimerization induced by the mutation associated to the Crouzon syndrome A391E, was observed only when these four C‐terminal residues were present. In the absence of these four residues, A391E was dimer‐destabilizing. Finally, using site specific infrared dichroism and convergence with evolutionary conservation data, we have determined the backbone model of the FGFR3‐TM homodimer in model lipid bilayers. This model is consistent with, and correlates with the effects of, most known pathological mutations found in FGFR‐TM.     

The single transmembrane domains of ErbB receptors self-associate in cell membranes.

Members of the epidermal growth factor receptor, or ErbB, family of receptor tyrosine kinases have a single transmembrane (TM) alpha-helix that is usually assumed to play a passive role in ligand-induced dimerization and activation of the receptor. However, recent studies with the epidermal growth factor receptor (ErbB1) and the erythropoietin receptor have indicated that interactions between TM alpha-helices do contribute to stabilization of ligand-independent and/or ligand-induced receptor dimers. In addition, not all of the expected ErbB receptor ligand-induced dimerization events can be recapitulated using isolated extracellular domains, suggesting that other regions of the receptor, such as the TM domain, may contribute to dimerization in vivo. Using an approach for analyzing TM domain interactions in Escherichia coli cell membranes, named TOXCAT, we find that the TM domains of ErbB receptors self-associate strongly in the absence of their extracellular domains, with the rank order ErbB4-TM > ErbB1-TM equivalent to ErbB2-TM > ErbB3-TM. A limited mutational analysis suggests that dimerization of these TM domains involves one or more GXXXG motifs, which occur frequently in the TM domains of receptor tyrosine kinases and are critical for stabilizing the glycophorin A TM domain dimer. We also analyzed the effect of the valine to glutamic acid mutation in ErbB2 that constitutively activates this receptor. Contrary to our expectations, this mutation reduced rather than increased ErbB2-TM dimerization. Our findings suggest a role for TM domain interactions in ErbB receptor function, possibly in stabilizing inactive ligand-independent receptor dimers that have been observed by several groups.     

Structural requirements of the extracellular to transmembrane domain junction for erythropoietin receptor function

The erythropoietin receptor (EpoR) is crucial for erythrocyte formation. The x-ray crystal structures of the EpoR extracellular domain lack the juxtamembrane (JM) region and the junction to the transmembrane (TM) domain. Yet the JM-TM regions are important for transmitting the conformational change imposed on the receptor dimer by Epo binding. Cysteine-scanning mutagenesis of the JM-TM regions identified three novel constitutively active mutants, demonstrating close disulfide-bonded juxtapositioning of these residues in the JM (L223C) and N-terminal TM domain (L226C, I227C). Chemical cross-linking defined the interface of the active helical TM dimer and revealed that the JM-TM segment encompassing Leu(226)-Leu(230) is non-helical. Molecular dynamics and NMR studies indicated that the TM-JM junction forms an N-terminal helix cap. This structure is important for EpoR function because replacement of this motif by consecutive leucines rendered the receptor constitutively active.     

A region in the seven-transmembrane domain of the human Ca2+ receptor critical for response to Ca2+

Of 12 naturally occurring, activating mutations in the seven-transmembrane (7TM) domain of the human Ca2+ receptor (CaR) identified previously in subjects with autosomal dominant hypocalcemia (ADH), five appear at the junction of TM helices 6 and 7 between residue Ile819 and Glu837. After identifying a sixth activating mutation in this region, V836L, in an ADH patient, we studied the remaining residues in this region to determine whether they are potential sites for activating mutations. Alanine-scanning mutagenesis revealed five additional residues in this region that when substituted by alanine led to CaR activation. We also found that, whereas E837A did not activate the receptor, E837D and E837K mutations did. Thus, region Ile819-Glu837 of the 7TM domain represents a "hot spot" for naturally occurring, activating mutations of the receptor, and most of the residues in this region apparently maintain the 7TM domain in its inactive configuration. Unique among the residues in this region, Pro823, which is highly conserved in family 3 of the G protein-coupled receptors, when mutated to either alanine or glycine, despite good expression severely impaired CaR activation by Ca2+. Both the P823A mutation and NPS 2143, a negative allosteric modulator that acts on the 7TM through a critical interaction with Glu837, blocked activation of the CaR by various ADH mutations. These results suggest that the 7TM domain region Ile819-Glu837 plays a key role in CaR activation by Ca2+. The implications of our finding that NPS 2143 corrects the molecular defect of ADH mutations for treatment of this disease are also discussed.     

Peptide design and structural characterization of a GPCR loop mimetic

G protein-coupled receptors (GPCRs) control fundamental aspects of human physiology and behaviors. Knowledge of their structures, especially for the loop regions, is limited and has principally been obtained from homology models, mutagenesis data, low resolution structural studies, and high resolution studies of peptide models of receptor segments. We developed an alternate methodology for structurally characterizing GPCR loops, using the human S1P(4) first extracellular loop (E1) as a model system. This methodology uses computational peptide designs based on transmembrane domain (TM) model structures in combination with CD and NMR spectroscopy. The characterized peptides contain segments that mimic the self-assembling extracellular ends of TM 2 and TM 3 separated by E1, including residues R3.28(121) and E3.29(122) that are required for sphingosine 1-phosphate (S1P) binding and receptor activation in the S1P(4) receptor. The S1P(4) loop mimetic peptide interacted specifically with an S1P headgroup analog, O-phosphoethanolamine (PEA), as evidenced by PEA-induced perturbation of disulfide cross-linked coiled-coil first extracellular loop mimetic (CCE1a) (1)H and (15)N backbone amide chemical shifts. CCE1a was capable of weakly binding PEA near biologically relevant residues R29 and E30, which correspond to R3.28 and E3.29 in the full-length S1P(4) receptor, confirming that it has adopted a biologically relevant conformation. We propose that the combination of coiled-coil TM replacement and conformational stabilization with an interhelical disulfide bond is a general design strategy that promotes native-like structure for loops derived from GPCRs.     

Role of charged amino acids conserved in the vasoactive intestinal polypeptide/secretin family of receptors on the secretin receptor functionality

The secretin receptor is a member of a large family of G-protein-coupled receptors that recognize polypeptide hormone and/or neuropeptides. Charged, conserved residues might play a key role in their function, either by interacting with the ligand or by stabilizing the receptor structure. Of the four charged amino acids that are conserved in the whole secretin receptor family, D49 and R83 (in the N-terminal domain) were probably important for the secretin receptor structure: replacement of D49 by H or R and of R83 by D severely reduced both the maximal response to secretin and its potency. No functional secretin receptor could be detected after replacement of R83 by L. Mutation of D49 to E, A, or N had no effect or reduced 5-fold the potency of secretin. The highly conserved positive charges found at the extracellular ends of TM III (K194) and IV (R255) were important for the secretin receptor function, as K194 mutation to A or Q and R255 mutation to Q or D decreased the secretin's affinity 15- to 1000-fold, respectively. Six extracellular charged residues are conserved in closely related receptors but not in the whole family. K121 (TM I) and R277 (TM V) were not important for functional secretin receptor expression. D174 (TM II) was necessary to stabilize the active receptor structure: the D174N mutant receptors were unable to stimulate normally the adenylate cyclase in response to secretin, and functional D174A receptors could not be found. Mutation of R255, E259 (second extracellular loop), and E351 (third extracellular loop) to uncharged residues reduced only 10- to 100-fold the secretin potency without changing its efficacy: these residues either stabilized the active receptor conformation or formed hydrogen rather than ionic bonds with secretin. Mutation of K121 (TM I) to Q or L and of R277 (TM V) to E or Q did not affect the receptor functional properties.     

Residues within the transmembrane domain of the glucagon-like peptide-1 receptor involved in ligand binding and receptor activation: modelling the ligand-bound receptor

The C-terminal regions of glucagon-like peptide-1 (GLP-1) bind to the N terminus of the GLP-1 receptor (GLP-1R), facilitating interaction of the ligand N terminus with the receptor transmembrane domain. In contrast, the agonist exendin-4 relies less on the transmembrane domain, and truncated antagonist analogs (e.g. exendin 9-39) may interact solely with the receptor N terminus. Here we used mutagenesis to explore the role of residues highly conserved in the predicted transmembrane helices of mammalian GLP-1Rs and conserved in family B G protein coupled receptors in ligand binding and GLP-1R activation. By iteration using information from the mutagenesis, along with the available crystal structure of the receptor N terminus and a model of the active opsin transmembrane domain, we developed a structural receptor model with GLP-1 bound and used this to better understand consequences of mutations. Mutation at Y152 [transmembrane helix (TM) 1], R190 (TM2), Y235 (TM3), H363 (TM6), and E364 (TM6) produced similar reductions in affinity for GLP-1 and exendin 9-39. In contrast, other mutations either preferentially [K197 (TM2), Q234 (TM3), and W284 (extracellular loop 2)] or solely [D198 (TM2) and R310 (TM5)] reduced GLP-1 affinity. Reduced agonist affinity was always associated with reduced potency. However, reductions in potency exceeded reductions in agonist affinity for K197A, W284A, and R310A, while H363A was uncoupled from cAMP generation, highlighting critical roles of these residues in translating binding to activation. Data show important roles in ligand binding and receptor activation of conserved residues within the transmembrane domain of the GLP-1R. The receptor structural model provides insight into the roles of these residues.     

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and forster resonance energy transfer suggest weak interactions between fibroblast growth factor receptor 3 (FGFR3) transmembrane domains in the absence of extracellular domains and ligands

Lateral dimerization of membrane proteins has evolved as a means of signal transduction across the plasma membrane for all receptor tyrosine kinases (RTKs). The transmembrane (TM) domains of RTKs are proposed to play an important role in the dimerization process. We have investigated whether the TM domains of one RTK, fibroblast growth factor receptor 3 (FGFR3), dimerize in lipid vesicles in the absence of the extracellular domains and ligands. We have performed sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with peptides produced via solid-phase peptide synthesis that correspond to the TM domain of FGFR3. We have carried out Forster resonance energy transfer (FRET) measurements using two donor-acceptor pairs, fluorescein/rhodamine and Cy3/Cy5, as a function of peptide concentration and donor-to-acceptor mole ratios. Our results suggest that FGFR3 TM domains form sequence-specific dimers in lipid bilayers. However, the dimerization propensity of FGFR3 TM domain is much weaker than the dimerization propensity of glycophorin A (GpA), the well-characterized "membrane dimer standard". We discuss our findings in the context of cell signaling across the plasma membrane and diseases or disorders that occur due to single amino acid mutations in the TM domain of FGFR3.     

The transmembrane domain of the E5 oncoprotein contains functionally discrete helical faces

The E5 protein of bovine papillomavirus is a 44-amino acid, Golgi-resident, type II transmembrane protein that efficiently transforms immortalized mouse fibroblasts. The transmembrane (TM) domain of E5 is not only critical for biological activity, it also regulates interactions with cellular targets including the platelet derived growth factor receptor (PDGF-R) and the 16-kDa subunit of the vacuolar proton ATPase (V-ATPase). In order to define the specific TM amino acids essential for E5 biological and biochemical activity, we performed scanning alanine mutagenesis on 25 of the 30 potential TM residues and genetically mapped discrete alpha-helical domains which separately regulated the ability of E5 to bind PDGF-R, activate PDGF-R, and to form oligomers. Alanine substitutions at positions 17, 21, and 24 (which lie on the same helical face) greatly inhibited E5 association with the PDGF-R, suggesting that this region comprises the receptor binding site. PDGF-R activation also mapped to a specific but broader domain in E5; mutant proteins with alanines on one helical face (positions 8, 9, 11, 16, 19, 22, and 23) continued to induce PDGF-R tyrosine phosphorylation, whereas mutant proteins with alanines on the opposite helical face (positions 7, 10, 13, 17, 18, 21, 24, and 25) did not, indicating that the latter helical face was critical for mediating receptor transphosphorylation. Interestingly, these "activation-defective" mutants segregated into two classes: 1) those that were unable to form dimers but that could still form higher order oligomers and transform cells, and 2) those that were defective for PDGF-R binding and were transformation-incompetent. These findings suggest that the ability of E5 to dimerize and to bind PDGF-R is important for receptor activation. However, since several transformation-competent E5 mutants were defective for binding and/or activating PDGF-R, it is apparent that E5 must have additional activities to mediate cell transformation. Finally, alanine substitutions also defined two separate helical faces critical for E5/E5 interactions (homodimer formation). Thus, our data identify distinct E5 helical faces that regulate homologous and heterologous intramembrane interactions and define two new classes of biologically active TM mutants.     

Open and Closed Conformations of the Isolated Transmembrane Domain of Death Receptor 5 Support a New Model of Activation

It has long been presumed that activation of the apoptosis-initiating Death Receptor 5, as well as other structurally homologous members of the TNF-receptor superfamily, relies on ligand-stabilized trimerization of noninteracting receptor monomers. We and others have proposed an alternate model in which the TNF-receptor dimer—sitting at the vertices of a large supramolecular receptor network of ligand-bound receptor trimers—undergoes a closed-to-open transition, propagated through a scissorslike conformational change in a tightly bundled transmembrane (TM) domain dimer. Here we have combined electron paramagnetic resonance spectroscopy and potential-of-mean force calculations on the isolated TM domain of the long isoform of DR5. The experiments and calculations both independently validate that the opening transition is intrinsic to the physical character of the TM domain dimer, with a significant energy barrier separating the open and closed states.     

Ligand-induced conformational changes in the Bacillus subtilis chemoreceptor McpB determined by disulfide crosslinking in vivo

Previously, we characterized the organization of the transmembrane (TM) domain of the Bacillus subtilis chemoreceptor McpB using disulfide crosslinking. Cysteine residues were engineered into serial positions along the two helices through the membrane, TM1 and TM2, as well as double mutants in TM1 and TM2, and the extent of crosslinking determined to characterize the organization of the TM domain. In this study, the organization of the TM domain was studied in the presence and absence of ligand to address what ligand-induced structural changes occur. We found that asparagine caused changes in crosslinking rate on all residues along the TM1-TM1' helical interface, whereas the crosslinking rate for almost all residues along the TM2-TM2' interface did not change. These results indicated that helix TM1 rotated counterclockwise and that TM2 did not move in respect to TM2' in the dimer on binding asparagine. Interestingly, intramolecular crosslinking of paired substitutions in 34/280 and 38/273 were unaffected by asparagine, demonstrating that attractant binding to McpB did not induce a "piston-like" vertical displacement of TM2 as seen for Trg and Tar in Escherichia coli. However, these paired substitutions produced oligomeric forms of receptor in response to ligand. This must be due to a shift of the interface between different receptor dimers, within previously suggested trimers of dimers, or even higher order complexes. Furthermore, the extent of disulfide bond formation in the presence of asparagine was unaffected by the presence of the methyl-modification enzymes, CheB and CheR, or the coupling proteins, CheW and CheV, demonstrating that these proteins must have local structural effects on the cytoplasmic domain that is not translated to the entire receptor. Finally, disulfide bond formation was also unaffected by binding proline to McpC. We conclude that ligand-binding induced a conformational change in the TM domain of McpB dimers as an excitation signal that is likely propagated within the cytoplasmic region of receptors and that subsequent adaptational events do not affect this new TM domain conformation.     

The Importance of TM3-4 Loop Subdomains for Functional Reconstitution of Glycine Receptors by Independent Domains

Truncated glycine receptors that have been found in human patients suffering from the neuromotor disorder hyperekplexia or in spontaneous mouse models resulted in non-functional ion channels. Rescue of function experiments with the lacking protein portion expressed as a separate independent domain demonstrated restoration of glycine receptor functionality in vitro. This construct harbored most of the TM3-4 loop, TM4, and the C terminus and was required for concomitant transport of the truncated α1 and the complementation domain from the endoplasmic reticulum toward the cell surface, thereby enabling complex formation of functional glycine receptors. Here, the complementation domain was stepwise truncated from its N terminus in the TM3-4 loop. Truncation of more than 49 amino acids led again to loss of functionality in the receptor complex expressed from two independent domain constructs. We identified residues 357–418 in the intracellular TM3-4 loop as being required for reconstitution of functional glycine-gated channels. All complementation constructs showed cell surface protein expression and correct orientation according to glycine receptor topology. Moreover, we demonstrated that the truncations did not result in a decreased protein-protein interaction between both glycine receptor domains. Rather, deletions of more than 49 amino acids abolished conformational changes necessary for ion channel opening. When the TM3-4 loop subdomain harboring residues 357–418 was expressed as a third independent construct together with the truncated N-terminal and C-terminal glycine receptor domains, functionality of the glycine receptor was again restored. Thus, residues 357–418 represent an important determinant in the process of conformational rearrangements following ligand binding resulting in channel opening.     

Molecular identification of the human melanocortin-2 receptor responsible for ligand binding and signaling

The melanocortin-2 receptor (MC2R), also known as the adrenocorticotropic hormone (ACTH) receptor, plays an important role in regulating and maintaining adrenocortical function, specifically steroidogenesis. Mutations of the human MC2R (hMC2R) gene have also been identified in humans with familial glucocorticoid deficiency; however, the molecular basis responsible for hMC2R ligand binding and signaling remains unclear. In this study, both truncated ACTH peptides and site-directed mutagenesis studies were used to determine molecular mechanisms of hMC2R binding ACTH and signaling. Our results indicate that ACTH1-16 is the minimal peptide required for hMC2R binding and signaling. Mutations of common melanocortin receptor family amino acid residues E80 in transmembrane domain 2 (TM2), D107 in TM3, F178 in TM4, F235 and H238 in TM6, and F258 in TM7 significantly reduced ACTH-binding affinity and signaling. Furthermore, mutations of unique amino acids D104 and F108 in TM3 and F168 and F178 in TM4 significantly decreased ACTH binding and signaling. In conclusion, our results suggest that the residues in TM2, TM3, and TM6 of hMC2R share similar binding sites with other MCRs but the residues identified in TM4 and TM7 of hMC2R are unique and required for ACTH selectivity. Our study suggests that hMC2R may have a broad binding pocket in which both conserved and unique amino acid residues are required, which may be the reason why alpha-MSH was not able to bind hMC2R.     

Exploring the binding domain of EmrE, the smallest multidrug transporter

EmrE is a small multidrug transporter in Escherichia coli that extrudes various positively charged drugs across the plasma membrane in exchange with protons, thereby rendering cells resistant to these compounds. Biochemical experiments indicate that the basic functional unit of EmrE is a dimer where the common binding site for protons and substrate is formed by the interaction of an essential charged residue (Glu14) from both EmrE monomers. Previous studies implied that other residues in the vicinity of Glu14 are part of the binding domain. Alkylation of Cys replacements in the same transmembrane domain inhibits the activity of the protein and this inhibition is fully prevented by substrates of EmrE. To monitor directly the reaction we tested also the extent of modification using fluorescein-5-maleimide. While most residues are not accessible or only partially accessible, four, Y4C, I5C, L7C, and A10C, were modified at least 80%. Furthermore, preincubation with tetraphenylphosphonium reduces the reaction of two of these residues by up to 80%. To study other essential residues we generated functional hetero-oligomers and challenged them with various methane thiosulfonates. Taken together the findings imply the existence of a binding cavity accessible to alkylating reagents where at least three residues from TM1, Tyr40 from TM2, and Trp63 in TM3 are involved in substrate binding.     

Defining the regions of Escherichia coli YidC that contribute to activity

The YidC/Oxa1/Alb3 family of proteins catalyzes membrane protein insertion in bacteria, mitochondria, and chloroplasts. In this study, we investigated which regions of the bacterial YidC protein are important for its function in membrane protein biogenesis. In Escherichia coli, YidC spans the membrane six times, with a large 319-residue periplasmic domain following the first transmembrane domain. We found that this large periplasmic domain is not required for YidC function and that the residues in the exposed hydrophilic loops or C-terminal tail are not critical for YidC activity. Rather, the five C-terminal transmembrane segments that contain the three consensus sequences in the YidC/Oxa1/Alb3 family are important for its function. However, by systematically replacing all the residues in transmembrane segment (TM) 2, TM3, and TM6 with serine and by swapping TM4 and TM5 with unrelated transmembrane segments, we show that the precise sequence of these transmembrane regions is not essential for in vivo YidC activity. Single serine mutations in TM2, TM3, and TM6 impaired the membrane insertion of the Sec-independent procoat-leader peptidase protein. We propose that the five C-terminal transmembrane segments of YidC function as a platform for the translocating substrate protein to support its insertion into the membrane.     

FGFR3 Transmembrane Domain Interactions Persist in the Presence of Its Extracellular Domain

Isolated receptor tyrosine kinase transmembrane (TM) domains have been shown to form sequence-specific dimers in membranes. Yet, it is not clear whether studies of isolated TM domains yield knowledge that is relevant to full-length receptors or whether the large glycosylated extracellular domains alter the interactions between the TM helices. Here, we address this question by quantifying the effect of the pathogenic A391E TM domain mutation on the stability of the fibroblast growth factor receptor 3 dimer in the presence of the extracellular domain and comparing these results to the case of the isolated TM fibroblast growth factor receptor 3 domains. We perform the measurements in plasma membrane-derived vesicles using a Förster-resonance-energy-transfer-based method. The effect of the mutation on dimer stability in both cases is the same (∼−1.5 kcal/mol), suggesting that the interactions observed in simple TM-peptide model systems are relevant in a biological context.     

Crystal structure of the Japanese encephalitis virus envelope protein

Japanese encephalitis virus (JEV) is the leading global cause of viral encephalitis. The JEV envelope protein (E) facilitates cellular attachment and membrane fusion and is the primary target of neutralizing antibodies. We have determined the 2.1-Å resolution crystal structure of the JEV E ectodomain refolded from bacterial inclusion bodies. The E protein possesses the three domains characteristic of flavivirus envelopes and epitope mapping of neutralizing antibodies onto the structure reveals determinants that correspond to the domain I lateral ridge, fusion loop, domain III lateral ridge, and domain I-II hinge. While monomeric in solution, JEV E assembles as an antiparallel dimer in the crystal lattice organized in a highly similar fashion as seen in cryo-electron microscopy models of mature flavivirus virions. The dimer interface, however, is remarkably small and lacks many of the domain II contacts observed in other flavivirus E homodimers. In addition, uniquely conserved histidines within the JEV serocomplex suggest that pH-mediated structural transitions may be aided by lateral interactions outside the dimer interface in the icosahedral virion. Our results suggest that variation in dimer structure and stability may significantly influence the assembly, receptor interaction, and uncoating of virions.     

Hepatitis C Virus RNA Replication and Virus Particle Assembly Require Specific Dimerization of the NS4A Protein Transmembrane Domain

Hepatitis C virus (HCV) NS4A is a single-pass transmembrane (TM) protein essential for viral replication and particle assembly. The sequence of the NS4A TM domain is highly conserved, suggesting that it may be important for protein-protein interactions. To test this hypothesis, we measured the potential dimerization of the NS4A TM domain in a well-characterized two-hybrid TM protein interaction system. The NS4A TM domain exhibited a strong homotypic interaction that was comparable in affinity to glycophorin A, a well-studied human blood group antigen that forms TM homodimers. Several mutations predicted to cluster on a common surface of the NS4A TM helix caused significant reductions in dimerization, suggesting that these residues form an interface for NS4A dimerization. Mutations in the NS4A TM domain were further examined in the JFH-1 genotype 2a replicon system; importantly, all mutations that destabilized NS4A dimers also caused defects in RNA replication and/or virus assembly. Computational modeling of NS4A TM interactions suggests a right-handed dimeric interaction of helices with an interface that is consistent with the mutational effects. Furthermore, defects in NS4A oligomerization and virus particle assembly of two mutants were rescued by NS4A A15S, a TM mutation recently identified through forward genetics as a cell culture-adaptive mutation. Together, these data provide the first example of a functionally important TM dimer interface within an HCV nonstructural protein and reveal a fundamental role of the NS4A TM domain in coordinating HCV RNA replication and virus particle assembly.