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* Department of Biochemistry and Cell Biology, Center for Structural Biology and Department of Physics and Astronomy, Stony Brook University, Stony Brook, New York 11794; and Ludwig Institute for Cancer Research, Belgium Christian de Duve Institute of Cellular Pathology, MEXP Unit, Université de Louvain, Brussels 1200, Belgium
Correspondence: Address reprint requests to Steven O. Smith, Dept. of Biochemistry and Cell Biology Z = 5215, Stony Brook University, Stony Brook, NY 11794-5215; Tel.: 631-632-1210; Fax: 631-632-8575; E-mail: steven.o.smith{at}sunysb.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESACKNOWLEDGEMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESACKNOWLEDGEMENTSREFERENCES |
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–3), which is responsible for the initial proliferative stage of erythroleukemia (1,4–8). gp55-P is able to induce cell proliferation by constitutively activating the erythropoietin (Epo) receptor (1,9–11), a member of the cytokine receptor superfamily that regulates erythrocyte maturation (12).
Both gp55-P and the Epo receptor are integral membrane proteins that span membrane bilayers with a single TM helix. Both proteins are dimeric, and they interact with one another to form a stable complex in the membranes of erythroid progenitors infected by SFFV (9,13–16). Genetic studies have shown that the TM domain and the small C-terminal juxtamembrane region of gp55-P are required for complex formation with the Epo receptor and signaling (1,12,17,18). Moreover, these regions of gp55-P are required for leukemogenicity (6,19). The observation that the interaction between gp55-P and the Epo receptor involves their TM sequences means that gp55-P interacts with the Epo receptor in a distinctly different fashion from the Epo ligand, which binds to the extracellular domain of the receptor.
Evidence for the role of the TM domain in the activation of the Epo receptor comes from comparing variant strains of the SFFV and from mutational studies on gp55-P. Both the polycythemic (P) and anemic (A) strains of the SFFV induce erythroleukemia in adult mice. However, only gp55-P causes Epo-independent proliferation of hematopoietic cell lines (8). Moreover, although both gp55-A and gp55-P are able to specifically bind to the TM domain of the Epo receptor, only gp55-P promotes proliferation and differentiation of early erythroid progenitors, the burst forming unit-erythroids (20). These differences between gp55-A and gp55-P can be traced to their TM sequences, which only differ at six positions (6,8).
How do the six TM sequence differences between gp55-P and gp55-A lead to functional differences between these two similar viral proteins? In principle, the sequence differences may cause changes in how the TM helices interact with one another within a gp55 dimer or in how the gp55 dimer interacts with the Epo receptor dimer. Both gp55-A and gp55-P are homodimers when expressed on the cell surface (21) as a result of disulfide bond formation between cysteines in their extracellular domains (2). However, only the TM domain of gp55-P forms homodimers when assayed by the ToxR chloramphenicol acetyltransferase reporter system (21). This suggests that the ability of gp55-P to activate the Epo receptor is related, at least in part, to the ability of the TM sequence of gp55-P to mediate tight helix-helix interactions.
The human Epo receptor is not activated by gp55-P. However, mutation of a single amino acid (Leu238) in the TM domain of the human receptor to serine, its counterpart in the mouse, allows it to be activated by gp55-P (22). The converse mutation (Ser238Leu) in the mouse receptor eliminates its ability to be activated, indicating that Ser238 enables gp55-P activation of either the human or mouse receptor. Based on computational searches, we proposed that Met390 of gp55-P interacts specifically with Ser238 to activate the receptor (22). Mutation of Met390 to isoleucine (the amino acid at position 390 in gp55-A) eliminates the ability of gp55-P to activate human (Leu238Ser) and mouse Epo receptors (22). In addition, the M390L mutation was reported to induce anemia, rather than polycythemia (23). These observations suggest that the sequence differences between gp55-P and gp55-A also result in changes in specific TM contacts between gp55-P/A and the Epo receptor.
Here, we use deuterium NMR spectroscopy in combination with magic angle spinning (MAS) to determine the structure of the gp55-P TM dimer. The dimer structure provides a framework for establishing the mechanism for how the TM region of gp55-P activates the Epo receptor, as well as for understanding how sequence differences in mutant viral proteins confer different phenotypes. Deuterium NMR spectroscopy takes advantage of the sensitivity of deuterium lineshapes to molecular motion (24–26). We have previously shown that the motion of amino acids located in TM helix interfaces is restricted relative to amino acids oriented toward the lipid membrane (27). As a result, by characterizing side-chain motion as a function of the TM sequence, one can map out the interface of interacting TM helices. Typically, deuterium NMR lineshape analyses are carried out on static samples and have been used extensively to study lipid motion (28). Studies of membrane proteins have been limited by low sensitivity. To overcome this problem, we take advantage of the increased sensitivity afforded by MAS to observe changes in the quadrupolar lineshape. We propose that deuterium MAS of consecutive leucine side chains in a TM sequence provides a simple approach for studying helix-helix interactions in membrane proteins. Together with results from polarized infrared (IR) spectroscopy and computational searches, the deuterium MAS spectra of specifically deuterated leucines in the TM sequence of gp55-P are used to establish its TM dimer structure.
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESACKNOWLEDGEMENTSREFERENCES |
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,5,5-D3) leucine and deuterium-free water were purchased from Cambridge Isotope Laboratories (Andover, MA). Other amino acids and octyl-ß-glucoside were obtained from Sigma Chemical (St. Louis, MO). DMPC was obtained from Avanti Polar Lipids (Alabaster, AL) as a lyophilized powder and used without further purification.
Peptide synthesis and purification
Peptides (33 residues in length) corresponding to the TM domain of gp55-P were synthesized using solid-phase methods at the W. M. Keck Peptide Synthesis Facility at Yale University. The sequence is largely hydrophobic with Arg and Trp/His defining the N-terminal and C-terminal boundaries of the TM domain, respectively.
The crude peptide (5–15 mg) was purified by reverse-phase high-performance liquid chromatography on a C4 column using gradient elution. The gradient starts with a largely aqueous solution of 70% distilled water, 12% acetonitrile, and 18% 2-propanol and is changed to a more hydrophobic composition of 40% acetonitrile and 60% 2-propanol that elutes the peptides. The elution was monitored by the optical absorbance at 260 nm. The solutions corresponding to the peaks were collected into several fractions that were then lyophilized and checked by mass spectrometry for purity.
Purified gp55-P peptides were reconstituted by detergent dialysis by first dissolving DMPC, lyophilized peptide, and detergent (octyl-ß-glucoside) in trifluoroethanol. This mixture was incubated at 37°C for over 2 h, and the trifluoroethanol was removed by evaporation using a stream of argon gas and then placing the sample under vacuum. The dry mixture was rehydrated with phosphate buffer (10 mM phosphate and 50 mM NaCl, pH 7.4), such that the final concentration of octyl-ß-glucoside was 5% (w/v). The rehydrated sample was then stirred slowly for at least 6 h, and the octyl-ß-glucoside was removed by dialysis using Spectra-Por dialysis tubing (3500 MW cut off) for 24 h against phosphate buffer at 37°C. The samples were then pelleted, lyophilized, and rehydrated with deuterium-depleted water (50 ± 5% weight) and incubated at 37°C for 24 h. The reconstituted membranes were pelleted to form multilamellar dispersions and loaded into NMR rotors.
To characterize the influence of the hydration level on the deuterium spectrum, parallel samples were run of Leu396 at 40% and 60% deuterium-depleted water (by weight). The relative intensities of the MAS side bands were not significantly different between these samples (data not shown).
Polarized IR spectroscopy
Polarized attenuated total reflection (ATR) Fourier transform infrared (FTIR) spectra were obtained on a Bruker IFS 66V/S spectrometer. Multilamellar vesicle dispersions at a concentration of 10 mg lipid/mL were layered on a germanium internal-reflection element using a slow flow of air directed at an oblique angle to the ATR plate to form an oriented multilamellar lipid-peptide film. Each sample spectrum represents the average of 1000 scans acquired at a resolution of 4 cm–1.
The dichroic ratio (RATR) is defined as the ratio between absorption of parallel (A||) and perpendicular (A
) polarized light (29). The observed dichroic ratio is used to calculate the order parameter Smeas using the equation,
 | (1) |
Order parameters (Smeas) of 1.0 and –0.5 correspond to helical orientations parallel and perpendicular to the membrane normal, respectively. The order parameter depends on the electric field amplitudes, Ex = 1.399, Ey = 1.514, and Ez = 1.621, in the thick film limit which is applicable for our experiments. 
is the angle between the helix director and the normal of the internal-reflection element, and 
is the angle between the helix director and the transition-dipole moment of the amide I vibrational mode.
To calculate the angle between the helix director and the normal of the ATR plate, one must know the value of the transition moment angle . There has been a wide range of values for reported in the literature over the years (e.g., from 20° (30) to 40° (31)) due to the lack of good model systems for polarized IR studies where the orientation and disorder of the amide bonds were known independently from the IR measurements. Recently, Marsh and co-workers have converged on a value of in two very different systems: 38° for a block co-polymer poly(
-methyl-L-glutamate)x-co-(-n-octadecyl-L-glutamate)y (32) and 39.5° for the membrane protein bacteriorhodopsin (33). These results indicate that the transition moment angle for the amide I vibration is independent of the specific system used. Importantly, Marsh and Pali (33) show that there are only limited structural deviations between the TM helices of bacteriorhodopsin and a standard -helix. Consequently, the value of 39.5° obtained for bacteriorhodopsin is applicable to standard helices.
We typically obtain polarized IR spectra of bacteriorhodopsin before measurements on reconstituted TM peptides. This approach provides an independent check of our experimental setup and validates the use of 39.5° for the transition moment angle . Bacteriorhodopsin is an integral membrane protein that forms two-dimensional crystalline arrays that orient well on germanium ATR surfaces. The disorder in orientation typically corresponds to an Smem of 0.8–0.9 based on measurements of mosaic spread (30). Since we cannot independently measure the disorder in the orientation of our reconstituted TM peptides, we use a value of Smem equal to 0.85 as the upper limit in our calculations. We assume that the disorder in the gp55-P sample is equal to the disorder in bacteriorhodopsin. If the disorder is greater in our reconstituted sample, then the measured dichroic ratio will be less than the true dichroic ratio, and the true crossing angle will be smaller for the gp55-P helices.
One way to indirectly assess the quality of our reconstitutions is to measure the dichroic ratios of the lipids. Dichroic ratios of the asymmetric and symmetric lipid CH2 stretching modes at 2924 cm–1 and 2852 cm–1 were 1.15 and 1.10, respectively, which correspond to order parameters of 0.65 and 0.67, and are consistent with well-oriented lipids (34).
NMR spectroscopy
Deuterium NMR spectra were obtained at a 2H frequency of 55.26 MHz using MAS. MAS spectra yield much higher sensitivity compared to conventional static spectra in membrane-reconstituted samples where the concentration of deuterated peptide is low. A MAS frequency of 3 kHz was used to have a sufficient number of spinning side bands to define the static lineshape. Single pulse excitation was employed using a 3.7-µs 90° pulse, followed by a 4.5-µs delay before data acquisition. The repetition delay was 250 ms. A total of 600,000–800,000 transients were averaged for each spectrum and processed using a 200-Hz exponential line broadening function. Spectra were obtained at 25°C.
MAS deuterium spectra were simulated using the program SIMPSON version 1.1.0 (35) with a spin rate of 3.0 kHz. The asymmetry parameter, 
, was set to 1.0. Only the quadrupole coupling constant was varied to quantify the breadth of the lineshape.
Computational searches
The computational search strategy using the program CHI has been described previously (36). Low-energy conformations of helix dimers were searched by rotating each helix through rotation angles 
1 and 2 from 0° to 360° with sampling step sizes of 25°–45° and with interhelical separations of 9.5 Å, 10.0 Å, and 10.5 Å. The 1 and 2 angles are equivalent to the and ß angles originally described by Adams et al. (36). Molecular dynamics simulations were run with simulated annealing at each rotational orientation of the dimer using the program X-PLOR along with the united atom topology and parameters sets, TOPH19 and PARAM19, respectively. Calculations were carried out for helices with both left-handed (LH) and right-handed (RH) geometries with initial crossing angles of 25°. Four different runs were carried out for each starting geometry using torsional angle dynamics. The rotation and crossing angles were allowed to vary during the simulations. To maintain an -helical conformation, distance restraints were applied between Oi and Ni+4 atoms along the backbone.
RESULTS AND DISCUSSION
Peptides corresponding to the TM domains of membrane proteins are able to independently fold and associate in membrane bilayers (37). To determine the structure of the TM dimer of gp55-P, we first establish that gp55-P TM peptides adopt an -helical TM conformation in bilayers using polarized IR spectroscopy. Second, deuterium NMR spectroscopy of gp55-P containing specifically 2H-labeled leucine is used to establish the rotational orientation and crossing angle of the TM helices. Third, computational searches are undertaken to generate possible low-energy dimer structures that are consistent with the IR and NMR data.
TM -helical structure of gp55-P
Fig. 1 presents polarized IR spectra of the TM domain of gp55-P reconstituted into model dimyristoylphosphocholine (DMPC) bilayers. The amide I vibration is observed at 1657 cm–1, a frequency characteristic of -helical secondary structure. Fourier deconvolution of the amide I band reveals no significant nonhelical structure. The dichroic ratio of the amide I band is sensitive to the orientation of the gp55-P helix relative to the plane of membrane. The observed dichroic ratio of 3.0 corresponds to a helix orientation of 
25° relative to the membrane normal (see Methods). These data indicate that peptides corresponding to the gp55-P TM domain are reconstituted into DMPC bilayers in a helical TM conformation.
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Previous deuterium NMR studies of side-chain motion in TM helices have focused on leucine (25), valine (27), and alanine (26). Leucine containing a single deuterated methyl group (5,5,5-2H-Leu) has several advantages over other deuterated amino acids for probing molecular motion. First, leucine is the most abundant amino acid in TM helices (38). There are often strings of consecutive leucines in membrane spanning sequences, allowing side-chain motion on different faces of a helix to be compared in a sequential manner. Second, leucine has a long flexible side chain (C-Cß-C-CD3). The high degree of flexibility of the side chain results in a large difference in the deuterium spectrum of leucine restricted in a helix interface when compared to leucine oriented toward a lipid interface. In contrast, the side chain of alanine is much less flexible. The alanine methyl group has a defined orientation relative to the helix backbone and is only averaged by rotation about the C-Cß bond. For valine, steric interactions between the side chain methyl groups and the backbone restrict motion about the C-Cß bond. Only subtle differences are observed in the lineshapes of valines located in interfacial and noninterfacial positions (27).
The approach taken here is to compare spectra of deuterated leucines at different positions in the gp55-P TM helix in parallel samples. For instance, four consecutive leucines (e.g., Leu396-Leu397-Leu398-Leu399) in the TM sequence constitute one full turn of the gp55-P -helix. At least one of the leucine side chains will be facing the helix dimer interface and exhibit a broader deuterium spectrum due to restricted motion.
The intensities of the side bands in the MAS deuterium spectra trace out the broad deuterium lineshapes. The high flexibility, which makes deuterated leucine a sensitive probe of molecular motion, limits analyses of the deuterium lineshape by detailed spectral simulation. To quantify the observed differences in the width of these lineshapes, we simulate the MAS deuterium spectra using the quadrupole coupling constant as the only free parameter. The simulations assume that the lineshapes have an asymmetry parameter () of 1 due to high motional averaging (39). Importantly, the simulations are simple to run using a program that is readily available (http://nmr.imsb.au.dk/bionmr/software/simpson.php).
Leu399 is in the gp55-P TM dimer interface
The strategy to determine the interface of the gp55-P dimer is to target a consecutive stretch of leucines in the middle of the TM domain: Leu396-Leu397-Leu398-Leu399. The string of consecutive amino acids facilitates a direct comparison. The left column of Fig. 2 shows the deuterium spectra of the gp55-P TM peptides labeled at Leu396 through Leu399. The large central resonance in the spectrum is due to residual 1H2HO. The sharp MAS side bands are spaced at the spinning frequency (3 kHz) and trace out the envelope of the deuterium lineshape. Comparison of the deuterium lineshapes (i.e., the broad envelopes traced out by the intensities of the MAS side bands) in Fig. 2 shows that the lineshape of Leu399 is considerably broader than Leu396, Leu397, or Leu398. The broader lineshape of Leu399 is indicative of restricted motion compared to the other three leucines, which in turn implies that Leu399 is located in the dimer interface. Leu396 and Leu398 are more restricted than Leu397, consistent with their packing against Leu399 (see structural model below).
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-Cß, Cß-C, and C-CD3 bonds of leucine. The right column of Fig. 2 presents simulations of the experimental 2H spectra that quantify the differences in lineshape using an asymmetry parameter of 1.0 and varying the quadrupolar coupling constant to obtain the best fit to the observed side band intensities. Based on the simulations, Leu397 has the narrowest lineshape with a quadrupolar coupling constant of 14 kHz, whereas Leu399 has the broadest lineshape with a quadrupolar coupling constant of 25 kHz.
The gp55-P TM dimer has an RH crossing angle
Comparison of the deuterium spectra in Fig. 2 indicates that Leu399 is in the dimer interface of gp55-P. With a single point of contact in the center of the TM sequence, one can establish the full dimer interface if one knows whether the helices cross with an LH or RH crossing angle. Both types of configurations are common in membrane proteins (40). The crossing angle of the two helices in the gp55-P dimer can be determined based on deuterium spectra of leucines that are positioned at the ends of the TM helices. If Leu399 is in the interface and roughly at the crossing point of the two helices in the center of the TM sequence, then the position of Leu385 and Leu407 with respect to the interface will depend on the crossing angle (Fig. 3). If Leu399 is in an interfacial "a" or "d" position of an LH coiled coil, then Leu385 would lie in an "a" or "d" position. In contrast, Leu407 would lie in a "b" or "e" position outside of the dimer interface. In a similar fashion, in an RH geometry with Leu399 in the interface, Leu385 would be oriented toward the lipid and Leu407 would lie in the dimer interface.
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) are –52° and +23°, respectively. The tilt angle of 25° relative to the bilayer normal, inferred from the amide I dichroic ratio, corresponds to a crossing angle of 50° consistent with an RH crossing geometry.
An important point to emphasize here is that the sign of the crossing angle (i.e., RH versus LH) is determined from the NMR data presented in Fig. 4. Comparison of the Leu385 and Leu407 lineshapes is consistent with an RH crossing angle but is not consistent with an LH crossing angle. An estimate of the magnitude of the crossing angle is derived from the polarized ATR-FTIR data. The calculated crossing angle of 50° is consistent with the NMR determination that the crossing angle is RH. However, as noted in the Methods section the measured dichroic ratio corresponds to the maximum tilt angle of the gp55-P helix. If the reconstitution is not optimal, then the observed dichroic ratio may be lower than the true dichroic ratio. This would yield a smaller crossing angle for the gp55-P helices.
The deuterium MAS spectra and simulations also serve to emphasize two additional points. First, the simulations assume that the lineshapes have an asymmetry parameter of 1. Both the Leu399 and Leu407 lineshapes exhibit spectra where the ±4 side bands have greater intensity than the ±3 side bands. These features are well reproduced in the simulations and support the assumption of equal to 1. Second, the Leu399 and Leu407 lineshapes are remarkably similar. In both cases, the broadening of the lineshape is attributed to restriction of leucine side-chain motion due to packing in the gp55-P dimer interface. This suggests that leucine motion and the associated deuterium MAS analyses are not dependent on the position of the deuterated leucine relatively to the membrane surface.
Structural model of the gp55-P TM dimer
The deuterium NMR data presented in Figs. 2 and 4 provide structural constraints for evaluating models of the gp55-P TM dimer that emerge from computational searches for low-energy helix dimers (36). The NMR constraints were not used to guide or otherwise influence the searches.
In our computational studies, low-energy conformations of gp55-P helix dimers having both RH and LH crossing angles were searched by rotating each helix in the dimer relative to the other (see Methods). Fig. 5 A presents the results of a computational search for RH crossing angles. The figure shows only those structures that fall into a "cluster", where a cluster is defined as a minimum of five structures with a root mean square deviation of 1 Å or less. These are the final minimized conformations that have migrated from their initial geometries into low-energy wells. Of the initial 512 structures in the computational search, only one symmetric cluster was found with Leu399 in the dimer interface (circled in Fig. 5). The low-energy structure is robust. Similar structures were found in simulations with helix separations of 9.5 Å, 10.0 Å, and 10.5 Å. The following results are based on a helix-helix separation of 10.0 Å.
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35°. The structure provides a framework for interpreting mutational and biochemical data on gp55-P. The working model is that a stable TM dimer structure is required for activation and that amino acids oriented away from the dimer interface interact directly with the TM domain of the Epo receptor.
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).
There are two sites in the gp55-P dimer interface, Gly391 and Leu399, where mutation blocks the ability of gp55-P to activate the Epo receptor. Gly391 is predicted to be in the dimer interface of the RH gp55-P structure (Fig. 3). Mutation of Gly391 to leucine blocks the ability of gp55-P to activate the Epo receptor (22). Glycine often serves as a molecular notch for mediating TM helix interactions (43). For instance, in the TM helix dimer of glycophorin A, two glycines are found in the helical interface (29,44), and mutation of either is sufficient to disrupt dimerization (45).
Mutation of Leu399 to isoleucine, the corresponding amino acid in gp55-A, also abolishes the ability of gp55-P to activate the Epo receptor (22). The deuterium data in Fig. 2 clearly show that Leu399 is well packed in the dimer interface. Whereas the mutation of Leu399 to isoleucine is conservative, in glycophorin A, it was found that mutation of Leu75, which is in the dimer interface, to isoleucine was sufficient to disrupt dimerization (45). In the structural model of the gp55-P dimer, replacement of isoleucine for leucine at position 399 results in steric clash between the isoleucine ß-methyl groups. The effects of mutations at similar amino acids in the dimer interfaces of gp55-P and glycophorin A suggest that a stable gp55-P dimer structure is required for activation of the Epo receptor.
The TM dimer structure of gp55-P in Fig. 6 shows that several amino acids thought to mediate TM interactions with the Epo receptor are oriented toward the lipid interface. We have previously proposed that Met390 is oriented away from the dimer interface and interacts with Ser238 in the Epo receptor (22). Mutation of Met390 to isoleucine, the corresponding amino acid in gp55-A, abolishes the ability to activate the Epo receptor (22). The gp55-P dimer structure confirms our previous proposal.
The isoleucine in position 404 is not directly in the dimer interface and can tolerate substitution with Leu or Phe (22). However, deletion of Ile404 results in loss of gp55-P-induced activation of the Epo receptor (21). The gp55-P dimer structure in Fig. 6 orients Trp405, a large aromatic amino acid, away from the dimer interface. Deletion of Ile404 would likely change the orientation of the C-terminal end of the gp55-P helix such that Trp405 on opposing helices would rotate toward the dimer interface and clash, possibly disrupting dimerization or the local helical secondary structure. These data suggest that the short C-terminal end of gp55-P also interacts with the Epo receptor. This would not be surprising because the juxtamembrane sequence of the Epo receptor is highly conserved and has been proposed as a switch region for regulating receptor activity (46). The deuterium NMR data, showing that Leu407 is well packed in the helical interface of gp55-P, provides compelling evidence that the end of the gp55-P helix is not unraveled and that both Trp405 and His408 are in a position to interact with the juxtamembrane sequence of the receptor.
The low-energy structure of the gp55-P dimer along with the analysis of the mutations above suggests that the gp55-P dimer structure does not change upon interaction with the Epo receptor. Rather, gp55-P catalyzes the conversion of an inactive Epo receptor TM dimer to an active dimer. Deuterium MAS NMR studies are in progress to directly test this model.
Comparison of gp55-P and gp55-A suggests that novel sequences may in principle be identified that could bind and activate the Epo receptor to different extents. We plan to exploit the productive interaction between the Epo receptor and gp55-P TM domains as a selection system to design and test sequences for their ability to bind the TM domain of the Epo receptor and activate receptor signaling. Such genetic systems have previously been described only for bacteria (47–49) and could lead to new insights into specific TM interactions in mammalian cell membranes.
Finally, the TM sequence of gp55-P is almost identical to that of the fusiogenic envelope proteins of the mink cell focus-inducing viruses and murine leukemia viruses. The major exception is at position 392, where there is a conserved proline in place of Ser in gp55-P and gp55-A (50). Position 392 is relatively tolerant to mutation in gp55-P (S. N. Constantinescu, unpublished results). However, mutation of Pro392 to alanine in the fusiogenic envelope proteins blocks viral fusion with the cell membrane but not incorporation of the envelope protein into virions (50). Langosch and co-workers (51) have proposed that structural flexibility introduced by Pro392 is required for fusogenicity. The structure of the gp55-P TM dimer provides a basis for understanding how small sequence differences in the TM domain between gp55-P and the viral envelope proteins affect function. The gp55 proteins are not able to mediate membrane fusion but have evolved the capability to activate membrane receptors.
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ACKNOWLEDGEMENTS |
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This work was supported by the National Institutes of Health-National Science Foundation instrumentation grants (S10 RR13889 and DBI-9977553), a grant from the National Institutes of Health (GM-46732) to S.O.S., and by grants from the Fonds National de la Recherche Scientifique (FNRS) and the Fédération Belge contre le Cancer and the de Hovre Foundation (SNC) to S.C. S.N.C. is a Research Associate of the FNRS Belgium.
Submitted on December 13, 2004; accepted for publication April 25, 2005.
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