In the absence of membranes, HIV-1 MyrMA and MyrMACA proteins appear to exist as monomers or trimers in solution (35
), consistent with the 3D crystallization of HIV-1 MA in trimer units (23
). However, CA proteins from several different retroviruses have been shown to assemble higher-order structures as hexamers, with no evidence for trimer subunits (3
). Moreover, N-terminally histidine-tagged (His-tagged) proteins composed of the HIV-1 MA, CA, and nucleocapsid (NC) domains assemble on nickel-chelating lipids in a hexamer lattice (25
), again with no clear evidence for trimers. Since it is possible that the His tag-nickel-chelating lipid interaction could influence the manner in which proteins oligomerize on membranes, we decided to examine the binding of myristoylated HIV-1 MA and MACA proteins to membranes.
For expression and purification of MyrMA and MyrMACA proteins, we followed protocols shown to produce only trace amounts of unmyristoylated species (56
). As shown in Fig. , expression of MyrMA and MyrMACA proteins and purification by virtue of their C-terminal His6
) yielded single bands on Coomassie blue-stained SDS-polyacrylamide gels (Fig. , lanes B and C). Although the mobility of MyrMA protein was slightly lower than expected for its predicted molecular mass of 15,744.8 Da, mass spectrometry analysis measured the molecular mass at 15,745.6 Da, confirming our isolation of the full-length myristoylated protein. Additional immunoblot analysis of SDS-PAGE-fractionated proteins using MA and CA specific primary antibodies verified the identities of MyrMA and MyrMACA.
FIG. 1. Purification of MyrMA and MyrMACA proteins. MyrMA (lane B) and MyrMACA (lane C) proteins were coexpressed with yeast NMT in bacteria and purified under conditions shown to yield only trace amounts of unmyristoylated products. The purified proteins were (more ...)
HIV-1 MA has been reported to bind preferentially to membranes containing PS versus PC, presumably via basic residues in its β-sheet and second helix (23
). However, HIV assembly preferentially occurs at cholesterol-rich membrane regions (24
). Thus, as a precursor to EM studies, we used liposome flotation binding assays to verify that MyrMA and MyrMACA proteins bind to membranes composed of 20% (wt/wt) cholesterol and 80% PS (data not shown). As an alternative assay, we modified the monolayer membrane binding assay described by Zuber et al. (63
), which mimics the methodology utilized in lipid monolayer 2D crystallizations (3
). For these assays, proteins are incubated in buffer beneath a lipid monolayer (Fig. ). After incubations, membrane monolayers and bound proteins are lifted onto hydrophobic Parafilm discs, after which bound and unbound protein levels are measured by immunoblotting of samples fractionated by SDS-PAGE. Using the monolayer membrane binding assay, we found less than 25% of the MyrMA protein in incubations bound to PC-cholesterol membranes, while greater than 50% of the protein bound to PS-cholesterol membranes (Fig. ). Similarly, significantly more MyrMACA bound to PS-cholesterol membranes than to PC-cholesterol membranes (Fig. ). We also observed that MyrMACA bound well to membranes containing the acidic phospholipid phosphatidic acid but bound poorly to phosphatidylethanolamine and phosphatidylinositol (data not shown).
FIG. 2. Monolayer membrane binding assays. (A) Binding of MyrMA and MyrMACA proteins to membrane monolayers was performed by incubation of proteins in buffer beneath membrane monolayers formed at the air-buffer interface. After incubations, monolayers and bound (more ...)
To analyze how MyrMA and MyrMACA proteins organize on membranes, we examined the arrangements of the proteins assembled on membranes by transmission EM, using lipid monolayer 2D crystallization methods (3
). In a procedure similar to monolayer membrane incubations (Fig. ), MyrMA and MyrMACA proteins were incubated beneath membranes composed of 80% PS and 20% cholesterol, but rather than being lifted onto Parafilm discs, the membranes and attached proteins were lifted onto lacey EM grids and processed for EM imaging. Because we previously have shown that N-terminally His-tagged HIV-1 CA and MACANC proteins assemble into hexagonal lattices on nickel-chelating lipids (4
), we anticipated that the more natural binding of myristoylated MyrMACA proteins to PS-cholesterol membranes also might yield hexagonal protein arrays. However, because little information was available concerning the membrane arrangement of MA, it was unclear whether or how MyrMA proteins might organize themselves.
Interestingly, EM analysis of negatively stained images indicated that both MyrMACA and MyrMA formed ordered arrays on PS-cholesterol membranes (Fig. ). MyrMACA arrays were difficult to detect directly from images and occasionally showed small aggregates adhering to the monolayers (Fig. ). In contrast, MyrMA arrays rarely had adherent aggregates and tended to show discernible patterns (Fig. ). Surprisingly, calculated diffraction patterns (Fourier transforms) yielded remarkably similar power spectra for the two proteins, with a hexagonal arrangement of reflections readily apparent by eye (Fig. ). Unit cell dimensions, averaged from eight images for each protein, were consistent with those of a hexagonal lattice (Table ). However, the unit cell lengths of 93.5Å for MyrMACA and 96.2 Å for MyrMA were larger than what has been observed for tightly packed HIV-1 CA or immature virus-like particles and were close to that observed with loosely packed CA tubes and sheets or the cores of mature virions (6
). To assess the space group symmetry for membrane-bound arrays of MyrMA and MyrMACA proteins, the phases of predicted symmetry-related reflections were compared and evaluated by calculating phase residuals, where a 0° residual indicates a perfect match, while a 90° residual is a random match (10
). Our analysis indicated that both MyrMACA and MyrMA 2D crystals gave excellent matches with both p3 and p6 space groups (Table ).
FIG. 3. Comparison of MyrMACA and MyrMA membrane-bound projection structures. MyrMACA (A to C) and MyrMA (D to F) proteins were assembled onto lipid monolayers composed of 4:1 PS-cholesterol, lifted onto lacey carbon grids, and negatively stained. (A and D) Crystalline (more ...)
Analysis of MyrMACA and MyrMA 2D crystalsa
Given that phase data for both MyrMACA and MyrMA membrane-bound crystals were compatible with p3 and p6 symmetry but that p6 calculations used two to three times as many symmetry comparisons, we assumed p6 symmetry in the reconstruction of averaged 2D projection maps. Merging data from seven image files for each protein produced averages that were complete to 18 Å, with low phase residuals, indicative of good data matching (10
): we did not attempt higher-resolution reconstructions due to the rapid fall-off in calculated reflection intensities. Not surprisingly, our MyrMACA projection map is reminiscent of maps generated for HIV-1 CA, His-tagged CA, and His-tagged MACANC (Fig. ). Hexamers of protein units, depicted in white, are in turn organized in a hexagonal lattice; at this resolution with our negatively stained samples, presumed capsid C-terminal domain interconnections between hexamer units (27
) were not clear. Unexpectedly, the MyrMA projection map appeared nearly identical to the MyrMACA map, with hexamer units in a hexagonal lattice (Fig. ). Although HIV-1 MA crystallizes in 3D as a trimer (23
) and MyrMA trimerizes in solution (35
), no MA trimer units were visible.
Because the lack of trimer units in membrane-bound MyrMA projection maps ran counter to expectation, several additional measures were taken. As protein controls, crystallizations were performed with several different MyrMA preparations, and unbound proteins from 2D crystallization setups were collected, subjected to SDS-PAGE, and immunoblotted to verify protein size and homogeneity (data not shown). Additionally, each of the data sets used in the MyrMA reconstruction was back transformed, assuming no symmetry constraints (p1), and evaluated to ensure that the projection map (Fig. ) accurately represented the data with regard to the hexamer rings, the hexagonal lattice, and the absence of trimer units (data not shown).
We also were successful in obtaining several unstained EM images of MyrMA arrays taken under cryo conditions. Although these crystals were small, high-quality reflections (IQ ≤ 5) (10
) occurred out to 20-Å resolution. Importantly, calculated diffraction patterns were hexagonal and gave unit cell sizes similar to those of the stained MyrMA crystals (a
= 94.2 ± 1.2 Å; γ = 120.1 ± 1.2°). Back transformation with no symmetry constraints (p1) from indexed Fourier transforms of MyrMA cryo-EM crystals gave projection maps (Fig. ) similar to the stained MyrMA 2D projection map (Fig. ), after contrast flipping. As depicted, MyrMA proteins (in white) organize in hexamer rings in a hexagonal lattice, and this observation demonstrates that staining did not introduce any gross reconstruction artifacts. A contour map of the unstained sample back transformation (Fig. ) emphasizes the hexamer nature of membrane-bound MyrMA protein associations, showing a cage of proteins surrounding trigonally symmetric and hexagonally symmetric cage holes. Comparison of the contour map with a scaled MA trimer viewed from above its putative membrane binding side (Fig. ) revealed the difficulty of fitting a trimer unit into the projection structure. In contrast, rotation of six MA monomers around an axis yielded a hexamer unit that aligned neatly onto our projection map (Fig. ). The implications of our observations are discussed below.
FIG. 4. Projection structure of unstained, membrane-bound MyrMA. MyrMA proteins were assembled onto lipid monolayers composed of 4:1 PS-cholesterol, lifted onto lacey carbon grids, plunge-frozen in liquid ethane, and imaged at −176°C to −180°C (more ...)