ALIX-CHMP4 interactions in the human ESCRT pathway

Abstract

The ESCRT pathway facilitates membrane fission events during enveloped virus budding, multivesicular body formation, and cytokinesis. To promote HIV budding and cytokinesis, the ALIX protein must bind and recruit CHMP4 subunits of the ESCRT-III complex, which in turn participate in essential membrane remodeling functions. Here, we report that the Bro1 domain of ALIX binds specifically to C-terminal residues of the human CHMP4 proteins (CHMP4A-C). Crystal structures of the complexes reveal that the CHMP4 C-terminal peptides form amphipathic helices that bind across the conserved concave surface of ALIX(Bro1). ALIX-dependent HIV-1 budding is blocked by mutations in exposed ALIX(Bro1) residues that help contribute to the binding sites for three essential hydrophobic residues that are displayed on one side of the CHMP4 recognition helix (M/L/IxxLxxW). The homologous CHMP1-3 classes of ESCRT-III proteins also have C-terminal amphipathic helices, but, in those cases, the three hydrophobic residues are arrayed with L/I/MxxxLxxL spacing. Thus, the distinct patterns of hydrophobic residues provide a "code" that allows the different ESCRT-III subunits to bind different ESCRT pathway partners, with CHMP1-3 proteins binding MIT domain-containing proteins, such as VPS4 and Vta1/LIP5, and CHMP4 proteins binding Bro1 domain-containing proteins, such as ALIX.

Fig. 1.

Mapping the ALIX binding sites of CHMP4 proteins. (A) Binding isotherms and sensorgrams (Inset) showing ALIX_Bro1-V binding to GST-CHMP4A (K_D = 30 ± 11 μM) (Inset), GST-CHMP4A_205-222 (K_D = 44 ± 6 μM), GST-CHMP4A_L217A (binding not detectable), and GST-CHMP4A_1-204 (binding not detectable). The K_D estimates are averages from best fits to single-site binding models (mean ± SD, n ≥ 3). The shorter ALIX_Bro1 construct also bound to CHMP4A_205-222 (K_D = 40 ± 0.6 μM) and to the longer CHMP4A_195-222 (K_D = 40.5 ± 0.4 μM) and CHMP4A_174-222 (K_D = 36.5 ± 0.4 μM) C-terminal constructs with comparable affinities (dissociation constant and error were estimated from a statistical fit of a single binding isotherm derived from duplicate measurements at 10 different ALIX_Bro1-V concentrations; data not shown). Error bars are indicated on all biosensor figures, but are often too small to be readily visible. (B) ALIX_Bro1 binds the C termini of CHMP4A, CHMPB, and CHMP4C. Binding isotherms showing ALIX_Bro1-V binding to immobilized C-terminal peptides from CHMP4A-C. Estimated dissociation constants were: GST-CHMP4A_205-222, 44 ± 6 μM (mean ± SD, n = 6); GST-CHMP4B_205-224, 48 ± 6 μM (mean ± range, n = 2); GST-CHMP4C_216-233, 41 ± 10 μM (mean ± range, n = 2). Binding to a control GST surface was negligible (data not shown).

Fig. 2.

Ribbon diagram showing the ALIX_Bro1 domain (blue) in complex with the C-terminal helix from CHMP4A (purple).

Fig. 3.

ALIX_Bro1-CHMP4 interfaces. (A) Stereoview of the ALIX_Bro1-CHMP4A interface. The CHMP4A helix is oriented N to C from top to bottom, ALIX residues within the binding interface are shown explicitly, dashed lines indicate hydrogen bonds or salt bridges, and key hydrophobic CHMP4 residues are underlined. (B) Stereo-view showing an overlay of the bound CHMP4A-C helices. The orientation is the same as in A, and the three key hydrophobic CHMP4 binding residues are shown in sticks. Note that the CHMP4A (purple) and CHMP4C (orange) helices overlay well, whereas the CHMP4B helix (green) is rotated by ≈20°.

Fig. 4.

Molecular recognition in ALIX-CHMP4 complexes. (A) Isotherms showing ALIX_Bro1-V binding to immobilized WT CHMP4A_205-222 and to CHMP4A_205-222 constructs with the following mutations: W220A, L217A, L214A, and E209A. For the E209A mutant, K_D = 95 ± 2 μM (dissociation constant and error were estimated from a statistical fit of a single binding isotherm derived from duplicate measurements at 10 different ALIX_Bro1-V concentrations.). (B) Sequences of the C termini of human CHMP4 proteins are shown (Upper), together with a bar graph showing sites of >50% identity across metazoan CHMP4 proteins, the resulting CHMP4 consensus sequence (see also Table S2) and the distinct consensus sequence of the C-terminal amphipathic helices of CHMP1–3 proteins (Lower) (47). (C) Model of the ALIX_Bro1-CHMP4A interaction, with mutation sites that block ALIX binding and ALIX-dependent HIV-1 budding highlighted in yellow on the ALIX surface. (D) Overlay of the C-terminal recognition helices from CHMP4A (magenta) and CHMP1A (green, PDB 2jq9), extracted from the bound ALIX_Bro1-CHMP4A_205-222 and VPS4A MIT-CHMP1A_180-196 complexes. The three key hydrophobic residues from each recognition helix are shown explicitly, and the figure emphasizes that the first hydrophobic residue is positioned differently in the two helices.