Impact of histidine residues on the transmembrane helices of viroporins

Abstract

The role of histidine in channel-forming transmembrane (TM) helices was investigated by comparing the TM helices from Virus protein ‘u' (Vpu) and the M2 proton channel. Both proteins are members of the viroporin family of small membrane proteins that exhibit ion channel activity, and have a single TM helix that is capable of forming oligomers. The TM helices from both proteins have a conserved tryptophan towards the C-terminus. Previously, alanine 18 of Vpu was mutated to histidine in order to artificially introduce the same HXXXW motif that is central to the proton channel activity of M2. Interestingly, the mutated Vpu TM resulted in an increase in helix tilt angle of 11° in lipid bilayers compared to the wild-type Vpu TM. Here, we find the reverse, when histidine 37 of the HXXXW motif in M2 was mutated to alanine, it decreased the helix tilt by 10° from that of wild-type M2. The tilt change is independent of both the helix length and the presence of tryptophan. In addition, compared to wild-type M2, the H37A mutant displayed lowered sensitivity to proton concentration. We also found that the solvent accessibility of histidine-containing M2 is greater than without histidine. This suggests that the TM helix may increase the solvent exposure by changing its tilt angle in order to accommodate a polar/charged residue within the hydrophobic membrane region. The comparative results of M2, Vpu and their mutants demonstrated the significance of histidine in a transmembrane helix and the remarkable plasticity of the function and structure of ion channels stemming from changes at a single amino acid site.     

Determinants of tetherin antagonism in the transmembrane domain of the human immunodeficiency virus type 1 Vpu protein

Tetherin (BST2/CD317) potently restricts the particle release of human immunodeficiency virus type 1 (HIV-1) mutants defective in the accessory gene vpu. Vpu antagonizes tetherin activity and induces its cell surface downregulation and degradation in a manner dependent on the transmembrane (TM) domains of both proteins. We have carried out extensive mutagenesis of the HIV-1 NL4.3 Vpu TM domain to identify three amino acid positions, A14, W22, and, to a lesser extent, A18, that are required for tetherin antagonism. Despite the mutants localizing indistinguishably from the wild-type (wt) protein and maintaining the ability to multimerize, mutation of these positions rendered Vpu incapable of coimmunoprecipitating tetherin or mediating its cell surface downregulation. Interestingly, these amino acid positions are predicted to form one face of the Vpu transmembrane alpha helix and therefore potentially contribute to an interacting surface with the transmembrane domain of tetherin either directly or by modulating the conformation of Vpu oligomers. While the equivalent of W22 is invariant in HIV-1/SIVcpz Vpu proteins, the positions of A14 and A18 are highly conserved among Vpu alleles from HIV-1 groups M and N, but not those from group O or SIVcpz that lack human tetherin (huTetherin)-antagonizing activity, suggesting that they may have contributed to the adaption of HIV-1 to human tetherin.     

Three-dimensional structure of the transmembrane domain of Vpu from HIV-1 in aligned phospholipid bicelles

The three-dimensional backbone structure of the transmembrane domain of Vpu from HIV-1 was determined by solid-state NMR spectroscopy in two magnetically-aligned phospholipid bilayer environments (bicelles) that differed in their hydrophobic thickness. Isotopically labeled samples of Vpu(2-30+), a 36-residue polypeptide containing residues 2-30 from the N-terminus of Vpu, were incorporated into large (q = 3.2 or 3.0) phospholipid bicelles composed of long-chain ether-linked lipids (14-O-PC or 16-O-PC) and short-chain lipids (6-O-PC). The protein-containing bicelles are aligned in the static magnetic field of the NMR spectrometer. Wheel-like patterns of resonances characteristic of tilted transmembrane helices were observed in two-dimensional (1)H/(15)N PISEMA spectra of uniformly (15)N-labeled Vpu(2-30+) obtained on bicelle samples with their bilayer normals aligned perpendicular or parallel to the direction of the magnetic field. The NMR experiments were performed at a (1)H resonance frequency of 900 MHz, and this resulted in improved data compared to lower-resonance frequencies. Analysis of the polarity-index slant-angle wheels and dipolar waves demonstrates the presence of a transmembrane alpha-helix spanning residues 8-25 in both 14-O-PC and 16-O-PC bicelles, which is consistent with results obtained previously in micelles by solution NMR and mechanically aligned lipid bilayers by solid-state NMR. The three-dimensional backbone structures were obtained by structural fitting to the orientation-dependent (15)N chemical shift and (1)H-(15)N dipolar coupling frequencies. Tilt angles of 30 degrees and 21 degrees are observed in 14-O-PC and 16-O-PC bicelles, respectively, which are consistent with the values previously determined for the same polypeptide in mechanically-aligned DMPC and DOPC bilayers. The difference in tilt angle in C14 and C16 bilayer environments is also consistent with previous results indicating that the transmembrane helix of Vpu responds to hydrophobic mismatch by changing its tilt angle. The kink found in the middle of the helix in the longer-chain C18 bilayers aligned on glass plates was not found in either of these shorter-chain (C14 or C16) bilayers.     

Tilt angle of a trans-membrane helix is determined by hydrophobic mismatch

In order to investigate the compensation mechanism of a trans-membrane helix in response to hydrophobic mismatch, the tilt and rotation angles of the trans-membrane helix of Vpu aligned in lipid bilayers of various thickness were determined using orientation-dependent frequencies obtained from solid-state NMR experiments of aligned samples. A tilt angle of 18 degrees was observed in 18:1-O-PC/DOPG (9:1) lipid bilayers, which have a hydrophobic thickness that approximately matches the hydrophobic length of the trans-membrane helix of Vpu. Upon decreasing the hydrophobic thickness of lipid bilayers, no significant change in rotation angle was observed. However, the tilt angle increased systematically with increasing positive mismatch to 27 degrees in 14:0-O-PC/DMPG (9:1), 35 degrees in 12:0-O-PC/DLPG (9:1), and 51 degrees in 10:0 PC/10:0 PG (9:1) lipid bilayers, indicating that the change in tilt angle of the trans-membrane helix is a principal compensation mechanism for hydrophobic mismatch. In addition, the distinctive kink in the middle of the helix observed in 18:1 bilayers disappears in thinner bilayers. Although the opposite of what might be expected, this finding suggests that a helix kink may also be a part of the hydrophobic matching mechanism for trans-membrane helices.     

Three-dimensional structure of the channel-forming trans-membrane domain of virus protein "u" (Vpu) from HIV-1

The three-dimensional structure of the channel-forming trans-membrane domain of virus protein "u" (Vpu) of HIV-1 was determined by NMR spectroscopy in micelle and bilayer samples. Vpu(2-30+) is a 36-residue polypeptide that consists of residues 2-30 from the N terminus of Vpu and a six-residue "solubility tag" at its C terminus that facilitates the isolation, purification, and sample preparation of this highly hydrophobic minimal channel-forming domain. Nearly all of the resonances in the two-dimensional 1H/15N HSQC spectrum of uniformly 15N labeled Vpu(2-30+) in micelles are superimposable on those from the corresponding residues in the spectrum of full-length Vpu, which indicates that the structure of the trans-membrane domain is not strongly affected by the presence of the cytoplasmic domain at its C terminus. The two-dimensional 1H/15N PISEMA spectrum of Vpu(2-30+) in lipid bilayers aligned between glass plates has been fully resolved and assigned. The "wheel-like" pattern of resonances in the spectrum is characteristic of a slightly tilted membrane-spanning helix. Experiments were also performed on weakly aligned micelle samples to measure residual dipolar couplings and chemical shift anisotropies. The analysis of the PISA wheels and Dipolar Waves obtained from both weakly and completely aligned samples show that Vpu(2-30+) has a trans-membrane alpha-helix spanning residues 8-25 with an average tilt of 13 degrees. The helix is kinked slightly at Ile17, which results in tilts of 12 degrees for residues 8-16 and 15 degrees for residues 17-25. A structural fit to the experimental solid-state NMR data results in a three-dimensional structure with precision equivalent to an RMSD of 0.4 A. Vpu(2-30+) exists mainly as an oligomer on PFO-PAGE and forms ion-channels, a most frequent conductance of 96(+/- 6) pS in lipid bilayers. The structural features of the trans-membrane domain are determinants of the ion-channel activity that may be associated with the protein's role in facilitating the budding of new virus particles from infected cells.     

HIV-1 Vpu protein antagonizes innate restriction factor BST-2 via lipid-embedded helix-helix interactions

The Vpu protein of HIV-1 antagonizes BST-2 (tetherin), a broad spectrum effector of the innate immune response to viral infection, by an intermolecular interaction that maps genetically to the α-helical transmembrane domains (TMDs) of each protein. Here we utilize NMR spectroscopy to describe key features of the helix-helix pairing that underlies this interaction. The antagonism of BST-2 involves a sequence of three alanines and a tryptophan spaced at four residue intervals within the Vpu TMD helix. Responsiveness to Vpu involves bulky hydrophobic residues in the C-terminal region of the BST-2 TMD helix that likely fit between the alanines on the interactive face of Vpu. These aspects of Vpu and BST-2 form an anti-parallel, lipid-embedded helix-helix interface. Changes in human BST-2 that mimic sequences found in nonhuman primate orthologs unresponsive to Vpu change the tilt angle of the TMD in the lipid bilayer without abrogating its intrinsic ability to interact with Vpu. These data explain the mechanism by which HIV-1 evades a key aspect of innate immunity and the species specificity of Vpu using an anti-parallel helix-helix packing model.     

Solution structure and orientation of the transmembrane anchor domain of the HIV-1-encoded virus protein U by high-resolution and solid-state NMR spectroscopy

The structure of the membrane anchor domain (VpuMA) of the HIV-1-specific accessory protein Vpu has been investigated in solution and in lipid bilayers by homonuclear two-dimensional and solid-state nuclear magnetic resonance spectroscopy, respectively. Simulated annealing calculations, using the nuclear Overhauser enhancement data for the soluble synthetic peptide Vpu1-39 (positions Met-1-Asp-39) in an aqueous 2,2,2-trifluoroethanol (TFE) solution, afford a compact well-defined U-shaped structure comprised of an initial turn (residues 1-6) followed by a linker (7-9) and a short helix on the N-terminal side (10-16) and a further longer helix on the C-terminal side (22-36). The side chains of the two aromatic residues (Trp-22 and Tyr-29) in the longer helix are directed toward the center of the molecule around which the hydrophobic core of the folded VpuMA is positioned. As the observed solution structure is inconsistent with the formation of ion-conductive membrane pores defined previously for VpuMA in planar lipid bilayers, the isolated VpuMA domain as peptide Vpu1-27 was investigated in oriented phospholipid bilayers by proton-decoupled 15N cross polarization solid-state NMR spectroscopy. The line widths and chemical shift data of three selectively 15N-labeled peptides are consistent with a transmembrane alignment of a helical polypeptide. Chemical shift tensor calculations imply that the data sets are compatible with a model in which the nascent helices of the folded solution structure reassemble to form a more regular linear alpha-helix that lies parallel to the bilayer normal with a tilt angle of 相似文献   

Bundles consisting of extended transmembrane segments of Vpu from HIV-1: computer simulations and conductance measurements

Part of the genome of the human immunodeficiency virus type 1 (HIV-1) encodes for a short membrane protein Vpu, which has a length of 81 amino acids. It has two functional roles: (i) to downregulate CD4 and (ii) to support particle release. These roles are attributed to two distinct domains of the peptide, the cytoplasmic and transmembrane (TM) domains, respectively. It has been suggested that the enhanced particle release function is linked to the ion channel activity of Vpu, with a slight preference for cations over anions. To allow ion flux across the membrane Vpu would be required to assemble in homooligomers to form functional water-filled pores. In this study molecular dynamics simulations are used to address the role of particular amino acids in 4, 5, and 6 TM helix bundle structures. The helices (Vpu(6-33)) are extended to include hydrophilic residues such as Glu, Tyr, and Arg (EYR motif). Our simulations indicate that this motif destabilizes the bundles at their C-terminal ends. The arginines point into the pore to form a positive charged ring that could act as a putative selectivity filter. The helices of the bundles adopt slightly higher average tilt angles with decreasing number of helices. We also suggest that the helices are kinked. Conductance measurements on a peptide (Vpu(1-32)) reconstituted into lipid membranes show that the peptide forms ion channels with several conductance levels.     

Expression, purification, and activities of full-length and truncated versions of the integral membrane protein Vpu from HIV-1

Vpu is an 81-residue accessory protein of HIV-1. Because it is a membrane protein, it presents substantial technical challenges for the characterization of its structure and function, which are of considerable interest because the protein enhances the release of new virus particles from cells infected with HIV-1 and induces the intracellular degradation of the CD4 receptor protein. The Vpu-mediated enhancement of the virus release rate from HIV-1-infected cells is correlated with the expression of an ion channel activity associated with the transmembrane hydrophobic helical domain. Vpu-induced CD4 degradation and, to a lesser extent, enhancement of particle release are both dependent on the phosphorylation of two highly conserved serine residues in the cytoplasmic domain of Vpu. To define the minimal folding units of Vpu and to identify their activities, we prepared three truncated forms of Vpu and compared their structural and functional properties to those of full-length Vpu (residues 2-81). Vpu(2-37) encompasses the N-terminal transmembrane alpha-helix; Vpu(2-51) spans the N-terminal transmembrane helix and the first cytoplasmic alpha-helix; Vpu(28-81) includes the entire cytoplasmic domain containing the two C-terminal amphipathic alpha-helices without the transmembrane helix. Uniformly isotopically labeled samples of the polypeptides derived from Vpu were prepared by expression of fusion proteins in E. coli and were studied in the model membrane environments of lipid micelles by solution NMR spectroscopy and oriented lipid bilayers by solid-state NMR spectroscopy. The assignment of backbone resonances enabled the secondary structure of the constructs corresponding to the transmembrane and the cytoplasmic domains of Vpu to be defined in micelle samples by solution NMR spectroscopy. Solid-state NMR spectra of the polypeptides in oriented lipid bilayers demonstrated that the topology of the domains is retained in the truncated polypeptides. The biological activities of the constructs of Vpu were evaluated. The ion channel activity is confined to the transmembrane alpha-helix. The C-terminal alpha-helices modulate or promote the oligomerization of Vpu in the membrane and stabilize the conductive state of the channel, in addition to their involvement in CD4 degradation.     

Structure and dynamics of the second and third transmembrane domains of human glycine receptor

A 61-residue polypeptide resembling the second and third transmembrane domains (TM23) of the alpha-1 subunit of human glycine receptor and its truncated form, both with the wild-type loop linking the two TM domains (the "23" loop), were studied using high-resolution NMR. Well-defined domain structures can be identified for the TM2, 23 loop, and TM3 regions. Contrary to the popular model of a long and straight alpha-helical structure for the pore-lining TM2 domain for the Cys-loop receptor family, the last three residues of the TM2 domain and the first eight residues of the 23 loop (S16-S26) seem to be intrinsically nonhelical and highly flexible even in trifluoroethanol, a solvent known to promote and stabilize alpha-helical structures. The six remaining residues of the 23 loop and most of the TM3 domain exhibit helical structures with a kinked pi-helix (or a pi-turn) from W34 to C38 and a kink angle of 159 +/- 3 degrees . The tertiary fold of TM3 relative to TM2 is defined by several unambiguously identified long-range NOE cross-peaks within the loop region and between TM2 and TM3 domains. The 20 lowest-energy structures show a left-handed tilt of TM3 relative to TM2 with a tilting angle of 44 +/- 2 degrees between TM2 (V1-Q14) and TM3 (L39-E48) helix axes. This left-handed TM2-TM3 arrangement ensures a neatly packed right-handed quaternary structure of five subunits to form an ion-conducting pore. This is the first time that two TM domains of the glycine receptor linked by the important 23 loop have ever been analyzed at atomistic resolution. Many structural characteristics of the receptor can be inferred from the structural and dynamical features identified in this study.     

Molecular dynamics simulation of human immunodeficiency virus protein U (Vpu) in lipid/water Langmuir monolayer

Virus protein U (Vpu) is an accessory membrane protein encoded by human immunodeficiency virus type 1 (HIV-1). Various NMR and CD studies have shown that the transmembrane domain of Vpu has a helical conformation and that the cytoplasmic domain adopts the helix-loop-helix-turn motif. This 3.5-ns molecular dynamics (MD) simulation of Vpu in a lipid/membrane environment has fully reproduced these structural characteristics. Membrane propensities of two amphipathic helices in the cytoplasmic domain are further compared here to understand better their complicated orientational behavior known from experiment. This study first reveals that the highly conserved loop region in the cytoplasmic domain can be closely associated with the membrane surface. It is known from the simulation that Vpu is associated with 34 lipids in this Langmuir monolayer. The lipids that are located between the Vpu transmembrane helix and the first helix in the cytoplasmic domain are pushed up by Vpu. These elevated lipids have increased P-N tilt angles for the head groups but unchanged acyl-chain tilt angles compared with lipids that do not interact with Vpu. This study verifies the significance of applying MD simulation in refining protein structure and revealing detailed protein-lipid interaction in membrane/water environment. Figure XZ view of a snapshot of Vpu/DLGPC/water system after 3.5 ns NP(N)gamma T MD simulation. Coloring scheme: Vpu, red; C, green; H, pink; N, blue; O, orange; P, magenta; water, light blue     

Mitochondrial phosphate transport protein. Reversions of inhibitory conservative mutations identify four helices and a nonhelix protein segment with transmembrane interactions and Asp39, Glu137, and Ser158 as nonessential for transport

The mitochondrial phosphate transport protein (PTP) has six (A--F) transmembrane (TM) helices per subunit of functional homodimer with all mutations referring to the subunit of the homodimer. In earlier studies, conservative replacements of several residues located either at the matrix end (Asp39/helix A, Glu137/helix C, Asp236/helix E) or at the membrane center (His32/helix A, Glu136/helix C) of TM helices yielded inactive single mutation PTPs. Some of these residues were suggested to act as phosphate ligands or as part of the proton cotransport path. We now show that the mutation Ser158Thr, not part of a TM helix but located near the center of the matrix loop (Ile141--Ser171) between TM helices C and D, inactivates PTP and is thus also functionally relevant. On the other side of the membrane, the single mutation Glu192Asp at the intermembrane space end of TM helix D yields a PTP with 33% wild-type activity. We constructed double mutants by adding this mutation to the six transport-inactivating mutations. Transport was detected only in those with Asp39Asn, Glu137Gln, or Ser158Thr. We conclude that TM helix D can interact with TM helices A and C and matrix loop Ile141--Ser171 and that Asp39, Glu137, and Ser158 are not essential for phosphate transport. Since our results are consistent with residues present in all 12 functionally identified members of the mitochondrial transport protein (MTP) family, they lead to a general rule that specifies MTP residue types at 7 separate locations. The conformations of all the double mutation PTPs (except that with the matrix loop Ser158Thr) are significantly different from those of the single mutation PTPs, as indicated by their very low liposome incorporation efficiency and their requirement for less detergent (Triton X-100) to stay in solution. These dramatic conformational differences also suggest an interaction between TM helices D and E. The results are discussed in terms of TM helix movements and changes in the PTP monomer/dimer ratio.     

Experimentally based orientational refinement of membrane protein models: A structure for the Influenza A M2 H+ channel

The 97-residue M2 protein from Influenza A virus forms H+-selective ion channels which can be attributed solely to the homo-tetrameric alpha-helical transmembrane domain. Site-directed infrared dichroism spectra were obtained for the transmembrane domain of M2, reconstituted in lipid vesicles. Data analysis yielded the helix tilt angle beta=31.6(+/-6.2) degrees and the rotational pitch angle about the helix axis for residue Ala29 omegaAla29=-59.8(+/-9.9) degrees, whereby omega is defined as zero for a residue located in the direction of the helix tilt. A structure was obtained from an exhaustive molecular dynamics global search protocol in which the orientational data are utilised directly as an unbiased refinement energy term. Orientational refinement not only allowed selection of a unique structure but could also be shown to increase the convergence towards that structure during the molecular dynamics procedure. Encouragingly, the structure obtained is highly consistent with all available mutagenesis and conductivity data and offers a direct chemical insight that relates the altered functionality of the channel to its structure.     

Structure-function correlates of Vpu,a membrane protein of HIV-1

Vpu, a membrane protein from human immunodeficiency virus-1, folds into two distinct structural domains with different biological activities: a transmembrane (TM) helical domain involved in the budding of new virions from infected cells, and a cytoplasmic domain encompassing two amphipathic helices, which is implicated in CD4 degradation. The molecular mechanism by which Vpu facilitates virion budding is not clear. This activity of Vpu requires an intact TM helical domain. And it is known that oligomerization of the VPU TM domain results in the formation of sequence-specific, cation-selective channels. It has been shown that the channel activity of Vpu is confined to the TM domain, and that the cytoplasmic helices regulate the lifetime of the Vpu channel in the conductive state. Structure-function correlates based on the convergence of information about the channel activity of Vpu reconstituted in lipid bilayers and on its 3-D structure in membranes by a combination of solution and solid-state nuclear magnetic resonance spectroscopy may provide valuable insights to understand the role of Vpu in the pathogenesis of AIDS and for drug design aimed to block channel activity.     

The two biological activities of human immunodeficiency virus type 1 Vpu protein involve two separable structural domains.

The human immunodeficiency virus type 1 (HIV-1) Vpu protein is an integral membrane phosphoprotein that induces CD4 degradation in the endoplasmic reticulum and enhances virus release from the cell surface. CD4 degradation is specific, requires phosphorylation of Vpu, and involves the interaction between Vpu and the CD4 cytoplasmic domain. In contrast, regulation of virus release is less specific and not restricted to HIV-1 and may be mechanistically-distinct from CD4 degradation. We show here that a mutant of Vpu, Vpu35, lacking most of its cytoplasmic domain has residual biological activity for virus release but is unable to induce CD4 degradation. This finding suggests that the N terminus of Vpu encoding the transmembrane (TM) anchor represents an active domain important for the regulation of virus release but not CD4 degradation. To better define the functions of Vpu's TM anchor and cytoplasmic domain, we designed a mutant, VpuRD, containing a scrambled TM sequence with a conserved amino acid composition and alpha-helical structure. The resulting protein was integrated normally into membranes, was able to form homo-oligomers, and exhibited expression levels, protein stability, and subcellular localization similar to those of wild-type Vpu. Moreover, VpuRD was capable of binding to CD4 and to induce CD4 degradation with wild-type efficiency, confirming proper membrane topology and indicating that the alteration of the Vpu TM domain did not interfere with this function of Vpu. However, VpuRD was unable to enhance the release of virus particles from infected or transfected cells, and virus encoding VpuRD had replication characteristics in T cells indistinguishable from those of a Vpu-deficient HIV-1 isolate. Mutation of the phosphorylation sites in VpuRD resulted in a protein which was unable to perform either function of Vpu. The results of our experiments suggest that the two biological activities of Vpu operate via two distinct molecular mechanisms and involve two different structural domains of the Vpu protein.     

The role of a conserved proline residue in mediating conformational changes associated with voltage gating of Cx32 gap junctions.

We have explored the role of a proline residue located at position 87 in the second transmembrane segment (TM2) of gap junctions in the mechanism of voltage-dependent gating of connexin32 (Cx32). Substitution of this proline (denoted Cx32P87) with residues G, A, or V affects channel function in a progressive manner consistent with the expectation that a proline kink (PK) motif exists in the second transmembrane segment (TM2) of this connexin. Mutations of the preceding threonine residue T86 to S, A, C, V, N, or L shift the conductance-voltage relation of wild-type Cx32, such that the mutated channels close at smaller transjunctional voltages. The observed shift in voltage dependence is consistent with a reduction in the open probability of the mutant hemichannels at a transjunctional voltage (Vj) of 0 mV. In both cases in which kinetics were examined, the time constants for reaching steady state were faster for T86N and T86A than for wild type at comparable voltages, suggesting that the T86 mutations cause the energetic destabilization of the open state relative to the other states of the channel protein. The structural underpinnings of the observed effects were explored with Monte Carlo simulations. The conformational space of TM2 helices was found to differ for the T86A, V, N, and L mutants, which produce a less bent helix ( approximately 20 degrees bend angle) compared to the wild type, which has a approximately 37 degrees bend angle. The greater bend angle of the wild-type helix reflects the propensity of the T86 residue to hydrogen bond with the backbone carbonyl of amino acid residue I82. The relative differences in propensity for hydrogen bonding of the mutants relative to the wild-type threonine residue in the constructs we studied (T86A, V, N, L, S, and C) correlate with the shift in the conductance-voltage relation observed for T86 mutations. The data are consistent with a structural model in which the open conformation of the Cx32 channel corresponds to a more bent TM2 helix, and the closed conformation corresponds to a less bent helix. We propose that the modulation of the hydrogen-bonding potential of the T86 residue alters the bend angle of the PK motif and mediates conformational changes between open and closed channel states.     

Interaction of amiloride and one of its derivatives with Vpu from HIV-1: a molecular dynamics simulation

Vpu is an 81-residue membrane protein, with a single transmembrane segment that is encoded by HIV-1 and is involved in the enhancement of virion release via formation of an ion channel. Cyclohexamethylene amiloride (Hma) has been shown to inhibit ion channel activity. In the present 12-ns simulation study a putative binding site of Hma blockers in a pentameric model bundle built of parallel aligned helices of the first 32 residues of Vpu was found near Ser-23. Hma orientates along the channel axis with its alkyl ring pointing inside the pore, which leads to a blockage of the pore.     

vpu transmembrane peptide structure obtained by site-specific fourier transform infrared dichroism and global molecular dynamics searching.

The recently developed method of site-directed Fourier transform infrared dichroism for obtaining orientational constraints of oriented polymers is applied here to the transmembrane domain of the vpu protein from the human immunodeficiency virus type 1 (HIV-1). The infrared spectra of the 31-residue-long vpu peptide reconstituted in lipid vesicles reveal a predominantly alpha-helical structure. The infrared dichroism data of the (13)C-labeled peptide yielded a helix tilt beta = (6.5 +/- 1.7) degrees from the membrane normal. The rotational pitch angle omega, defined as zero for a residue located in the direction of the helix tilt, is omega = (283 +/- 11) degrees for the (13)C labels Val(13)/Val(20) and omega = (23 +/- 11) degrees for the (13)C labels Ala(14)/Val(21). A global molecular dynamics search protocol restraining the helix tilt to the experimental value was performed for oligomers of four, five, and six subunits. From 288 structures for each oligomer, a left-handed pentameric coiled coil was obtained, which best fits the experimental data. The structure reveals a pore occluded by Trp residues at the intracellular end of the transmembrane domain.     

Differential activities of the human immunodeficiency virus type 1-encoded Vpu protein are regulated by phosphorylation and occur in different cellular compartments.

The human immunodeficiency virus type 1 (HIV-1)-specific Vpu is an 81-amino-acid amphipathic integral membrane protein with at least two different biological functions: (i) enhancement of virus particle release from the plasma membrane of HIV-1-infected cells and (ii) degradation of the virus receptor CD4 in the endoplasmic reticulum (ER). We have previously found that Vpu is phosphorylated in infected cells at two seryl residues in positions 52 and 56 by the ubiquitous casein kinase 2. To study the role of Vpu phosphorylation on its biological activity, a mutant of the vpu gene lacking both phosphoacceptor sites was introduced into the infectious molecular clone of HIV-1, pNL4-3, as well as subgenomic Vpu expression vectors. This mutation did not affect the expression level or the stability of Vpu but had a significant effect on its biological activity in infected T cells as well as transfected HeLa cells. Despite the presence of comparable amounts of wild-type and nonphosphorylated Vpu, decay of CD4 was observed only in the presence of phosphorylated wild-type Vpu. Nonphosphorylated Vpu was unable to induce degradation of CD4 even if the proteins were artificially retained in the ER. In contrast, Vpu-mediated enhancement of virus secretion was only partially dependent on Vpu phosphorylation. Enhancement of particle release by wild-type Vpu was efficiently blocked when Vpu was artificially retained in the ER, suggesting that the two biological functions of Vpu are independent, occur at different sites within a cell, and exhibit different sensitivity to phosphorylation.     

Identification of amino acids in the human tetherin transmembrane domain responsible for HIV-1 Vpu interaction and susceptibility

Tetherin, also known as BST-2/CD317/HM1.24, is an antiviral cellular protein that inhibits the release of HIV-1 particles from infected cells. HIV-1 viral protein U (Vpu) is a specific antagonist of human tetherin that might contribute to the high virulence of HIV-1. In this study, we show that three amino acid residues (I34, L37, and L41) in the transmembrane (TM) domain of human tetherin are critical for the interaction with Vpu by using a live cell-based assay. We also found that the conservation of an additional amino acid at position 45 and two residues downstream of position 22, which are absent from monkey tetherins, are required for the antagonism by Vpu. Moreover, computer-assisted structural modeling and mutagenesis studies suggest that an alignment of these four amino acid residues (I34, L37, L41, and T45) on the same helical face in the TM domain is crucial for the Vpu-mediated antagonism of human tetherin. These results contribute to the molecular understanding of human tetherin-specific antagonism by HIV-1 Vpu.