Structure of the Month: August 2008 [see all]
ALIX-CHMP4 interactions in the human ESCRT pathway
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. We have shown that the Bro1 domain of ALIX binds specifically to C-terminal residues of the human CHMP4 proteins and, using both synchrotron and in-lab X-ray diffraction sources, we determined the crystal structures of the ALIX(Bro1) domain in complex with CHMP4 C-terminal peptides (CHMP4A-C).
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ALIXBro1 in complex with the C-terminal CHMP4B helix. Ribbon diagram showing the complex with the CHMP4B peptide colored cyan. |
Crystal structures of the complexes reveal that the CHMP4 C-terminal peptides form amphipathic helices that bind across a 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.
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ALIXBro1 in complex with the C-terminal CHMP4B helix. CHMP4B is shown in sticks and ALIXBro1 is shown as a surface. An Fo-Fc peptide omit map is shown in pink, contoured at 2 x rmsd and displayed over the peptide. The omit map was generated by deleting the peptide from the model, applying random shifts (0.3 Å in x, y, and z) and then re-refining the model in the absence of the peptide in REFMAC. |
This work was performed in the laboratories of Dr. Wesley Sundquist and Dr. Christopher Hill in the Department of Biochemistry, University of Utah School of Medicine, as part of a comprehensive program to understand HIV and host factors involved in HIV infection (CHEETAH - Center for the Structural Biology of Cellular Host Elements in Egress, Trafficking, and Assembly of HIV). This work was supported by National Institutes of Health Grants GM082534 (to W.I.S. and C.P.H.) and AI051174 (to W.I.S.).
X-ray diffraction data were collected to 2.0 Å resolution in-house using a Rigaku MicroMax 007 HF copper rotating anode generator with Varimax HR confocal optic and R-AXIS IV detector. X-ray diffraction data were also collected to 2.0 Å resolution at the Stanford Synchrotron Radiation Laboratory (SSRL).
Data collection details
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Sample |
ALIXBro1-CHMP4B complex |
ALIXBro1-CHMP4C complex |
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PDB ID |
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Space group |
C2 |
C2 |
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Unit cell |
a=120.5 Å b=62.6 Å c=76.1 Å |
a=120.9 Å b=62.4 Å c=76.4 Å |
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Crystal size |
0.2 x 0.2 x 0.1 mm |
0.2 x 0.2 x 0.1 mm |
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Radiation |
Cu Kα |
Cu Kα |
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Generator |
MicroMax-007 HF |
MicroMax-007 HF |
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Optic |
VariMax HR |
VariMax HR |
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Detector |
R-AXIS IV |
R-AXIS IV |
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Low temperature system |
Oxford Cryostream |
Oxford Cryostream |
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Crystal-to-detector distance |
150 mm |
150 mm |
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Exposure time per frame |
10 min |
10 min |
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Oscillation width |
0.5° |
0.5° |
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Number of frames |
751 |
697 |
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Total oscillation angle |
375.5° |
348.5° |
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Data processing |
DENZO/SCALEPACK |
DENZO/SCALEPACK |
Resolution |
2.1 - 50 Å |
2.0 - 50 Å |
Reference
McCullough, J., Fisher, R.D., Whitby, F.G., Sundquist, W.I., Hill, C.P. (2008) ALIX-CHMP4 interactions in the human ESCRT pathway. Proc.Natl.Acad.Sci.USA 105: 7687-7691.