Sedimentation velocity studies of the high–molecular weight aggregates of the HIV gp41 ectodomain

Abstract

Accumulation of the HIV envelope protein gp41 is observed in the brain tissues of patients suffering from HIV-associated dementia. Previously, we have shown by electron microscopy that the extracellular domain of SIV gp41, which is analogous to that of HIV, forms high–molecular weight aggregates in vitro, and we have postulated that such high–molecular weight aggregates are responsible for neurological damage in a manner similar to protein deposition diseases such as Alzheimer’s and the prion diseases. In this manuscript, we have characterized the self-association of the HIV gp41 ectodomain by sedimentation velocity. We show that discreet species of the gp41 high–molecular weight aggregates are present. The maximum population occurs at 20 S, which corresponds to ~5 trimers of gp41, suggesting that five trimers are required for nucleation of the high–molecular weight aggregates. The concentration dependence of the gp41 self-association indicates that it occurs by mass action. The temperature dependence of gp41 self-association suggests that it is driven by entropy, indicating that intermolecular hydrophobic interactions between trimers of gp41 are driving the association.

HIV-associated dementia (HAD) affects 15%–20% of patients in the late stages of acquired immunodeficiency syndrome (Navia et al. 1986; Price et al. 1988; McArthur et al. 1999). Surprisingly, the incidence of HAD appears to be increasing in patients treated with highly antiretroviral therapy (Dore et al. 1999; Sacktor et al. 2001), indicating that treatment of HAD will become increasingly important in the future. The molecular determinant of HAD is presently unclear; however, the level of neurological damage is correlated with accumulation of the HIV envelope protein gp41 in brain tissues (Kure et al. 1991; Adamson et al. 1996, 1999; Rostasy et al. 1999).

The extracellular or ectodomain domain of HIV gp41, as well as its homolog in SIV, is very well characterized by biochemical and structural methods (Blacklow et al. 1995; Lu et al. 1995; Caffrey et al. 1999). Based on X-ray crystallography and nuclear magnetic resonance studies, the gp41 ectodomain is a trimer in which each monomer consists of a helix–loop–helix motif (Chan et al. 1997; Tan et al. 1997; Weissenhorn et al. 1997; Caffrey et al. 1998; Malashkevich et al. 1998; Yang et al. 1999). However, in vitro, the trimeric state of the gp41 ectodomain is only observed at low pH or on removal of the central loop region that connects the two helices (Wingfield et al. 1997; Caffrey et al. 2000). At neutral pH values, the SIV gp41 ectodomain forms high–molecular weight aggregates, as demonstrated by electron microscopy (Caffrey et al. 2000). This observation has led to the proposal that gp41 accumulates in brain tissues as high–molecular weight aggregates, and thus resembles protein deposition diseases such as Alzheimer’s and the prion diseases (Caffrey et al. 2000). In this manuscript, the association properties of the HIV gp41 ectodomain have been further characterized by sedimentation velocity.

Results and Discussion

Sedimentation velocity provides hydrodynamic information about biomolecule size and shape (for review, see Laue 2001). The sedimentation velocity profile of the HIV gp41 ectodomain is shown in Figure 1A ▶. The color of the lines in Figure 1A ▶—green, red, and black—represent three different protein concentrations as measured by absorbance at 280 nm: 26, 13, and 3μM, respectively. Strikingly, there is a distribution of species between 20 and 120 S that corresponds to 200–1200 kDa (i.e., ~5 to 30 trimers, with a trimer mass of ~45 kDa). The major population occurs at 20 S, which corresponds to about five trimers. The absence of peaks at lower S values indicates that there is a minimum nucleation size of about five trimers. Note that the expected peak for the HIV gp41 ectodomain trimer would be 4 S, and indeed at pH 3.0, which has been the condition of previous characterizations of the gp41 ectodomain containing the central loop (Wingfield et al. 1997; Caffrey et al. 1998, 2000), there is a dominant peak at 4 S as expected (data not shown). Interestingly, the profile at the highest concentration suggests the presence of discreet species. For example, the most prominent populations occur at 20, 28, 36, 52, 64, 80, and 120 S. The presence of increased populations of higher mass at higher protein concentration is indicative of a mass action association, which is what is expected if the species are specific associations. The inset shows the ratio of the major species as a function of total protein at three different protein concentrations and then at both 4° and 20°C. It is clear that there is greater association over all concentrations at the higher temperature, and this suggests that the formation of the higher-order species is driven by entropy. This idea is consistent with the hydrophobic nature of the gp41 loop region (Caffrey et al. 1998), which has been shown to be responsible for the self-association (Caffrey et al. 2000).

Figure 1.

(A) Sedimentation velocity study of the HIV gp41 high–molecular weight species. The buffer was 100 mM sodium bicarbonate at pH 8.0. (B) Model of the formation of free extracellular gp41 and its subsequent nucleation in the formation of higher–molecular weight species at physiological pH. The structure of the gp41 ectodomain is taken from Caffrey et al. 1998.

Based on the present observations, a model for the involvement of HIV gp41 in HAD is diagrammed in Figure 1B ▶. In the native state, gp41 is a trimer that is anchored into the cell or viral membrane by its transmembrane domain. The activity of extracellular proteolytic enzymes in the brain releases the gp41 ectodomain from its membrane-bound state (step 1 of Figure 1B ▶). The free gp41 ectodomain accumulates and allows, by the law of mass action, nucleation of the high–molecular weight aggregates (step 2 of Figure 1B ▶). The high–molecular weight aggregates then accumulate and cause protein deposition, resulting in neurological damage. The observation that about five trimers represent the minimum high–molecular weight aggregate may imply that this is the minimum number of trimers that is necessary to occlude the loop region from solvent. An attractive aspect of the occlusion of the loop region is that it would also occlude it from proteolysis, thereby stabilizing the high–molecular weight aggregate. As discussed above, high–molecular weight aggregates of gp41 have been implicated in HAD, which presents itself with symptoms very similar to the protein deposition diseases. For example, prion, Parkinson’s, and Alzheimer’s diseases have all been correlated to a mass action event that occurs at a certain critical concentration of the proteins that undergo misfolding and nucleation to form aggregates that impair brain cell function (Singleton et al. 2004).

Materials and methods

The HIV gp41 ectodomain (residues 540–665 of HIV-1 strain HXB2) was prepared by PCR subcloning into the T7 TOPO/CT vector (Invitrogen). To eliminate problems associated with non-specific disulfide formation (Caffrey et al. 1997), cysteines 598 and 604 have been substituted with alanine, using the QuikChange Mutagenesis Kit (Stratagene). Recombinant gp41 was expressed and purified as previously described (Wingfield et al. 1997) and subsequently dialyzed against buffer containing 100 mM NaHCO₃/pH 8.0. The identity of the gp41 construct was confirmed by DNA sequencing (University of Chicago Cancer Research Center DNA Sequencing Facility) and by mass spectrometry (Research Resources Center, University of Illinois at Chicago). Sedimentation velocity experiments were performed on a Beckman XLI ultracentrifuge. Experiments were conducted at 4° and 20°C and at 50,000 RPM. The partial specific volume and buffer density were calculated using the software program Sednterp. Accordingly, vbar was 0.7384 at 20°C and 0.7364 at 4°C, and the molecular weight calculated from the amino acid composition was 14,778 Da.

Abbreviations

• HAD, HIV-associated dementia

• HIV, human immunodeficiency virus

• SIV, simian immunodeficiency virus.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04916704.