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.

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ALIX-CHMP4 interactions in the human
ESCRT pathway
John McCullough, Robert D. Fisher, Frank G. Whitby, Wesley I. Sundquist*, and Christopher P. Hill*
Department of Biochemistry, University of Utah, Salt Lake City, UT 84112-5650.
Edited by Axel T. Brunger, Stanford University, Stanford, CA, and approved March 25, 2008 (received for review February 16, 2008)
The ESCRT pathway facilitates membrane fission events during
enveloped virus budding, multivesicular body formation, and cy-
tokinesis. 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 remod-
elingfunctions.Here,wereportthattheBro1domainofALIXbinds
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 ALIXBro1. ALIX-dependent
HIV-1 budding is blocked by mutations in exposed ALIXBro1 resi-
dues 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.
cytokinesis ? ESCRT-III ? HIV ? mutivesicular body
T
kinesis (2), and in intralumenal vesicle formation at the late
endosome or multivesicular body (MVB) (3, 4). Involvement in
these seemingly diverse biological processes can be rationalized
if the ESCRT machinery encodes membrane remodeling and
fission activities that are required to resolve the thin membrane
‘‘necks’’ created during the final stages of virus budding, MVB
vesicle formation, and cell division. Although mechanistic de-
tails are still lacking, there is increasing evidence that the
ESCRT-III proteins may mediate such vesicle extrusion and/or
membrane fission activities, possibly in conjunction with the
AAA ATPase VPS4 (for example, see ref. 5). Humans express
11 related, but distinct ESCRT-III proteins (termed the CHMP
proteins) that can be subdivided into seven different families
(CHMP1–7) based on their similarities to one another and to the
six ESCRT-III-like proteins in yeast. The different ESCRT-III
proteins apparently adopt similar folds (6) and can copolymerize
together on membranes, yet have evolved to interact differently
with other ESCRT pathway components. For example, only the
three human CHMP4 proteins (CHMP4A-C) can bind ALIX
(yeast Bro1p), another protein in the ESCRT pathway (7–16).
The ESCRT machinery functions at different membranes, and
ALIX plays important roles in targeting the pathway to function
in retrovirus budding by binding directly to viral Gag proteins
(16, 17) and to function in abscission by binding the midbody
protein CEP55 (2). In both cases, ALIX must also bind the
CHMP4 proteins, because ALIX point mutations that block
CHMP4 binding inhibit HIV-1 budding (10, 11) and abscission
(18). Thus, ALIX can serve as an adaptor that recruits CHMP4/
ESCRT-III complexes to function at distinct biological mem-
branes. Conversely, CHMP4 proteins can apparently recruit
he ESCRT pathway functions in the budding of HIV-1 and
other lentiviruses (1), in the final abscission stage of cyto-
ALIX to membranes because the membrane-bound Snf7p (yeast
CHMP4) brings Bro1p/ALIX to the endosome to function in
MVB vesicle formation (19).
In addition to its involvement in HIV budding and cytokinesis,
ALIX has been implicated in a variety of biological processes
that may reflect other ESCRT pathway functions or possibly
ESCRT-independent ALIX functions. These functions include
lysobisphosphatidic acid (LBPA) binding (reviewed in ref. 20),
endophilin binding (21), receptor trafficking (22–24), endosome
distribution (25), cell motility/adhesion (26, 27), apoptosis (re-
viewed in ref. 28), actin and microtubule binding (26, 29) and
regulation of JNK signaling (30). Thus, ALIX appears to play
widespread roles in membrane biology and cell signaling. Sim-
ilarly, ALIX has been implicated in the release of several other
classes of enveloped viruses, including hepatitis B virus (31),
human parainfluenza virus (32), and possibly Sendai virus (33)
(however, see ref. 34). Thus, ALIX may play widespread roles in
the release of highly divergent enveloped viruses.
ALIX has three distinct regions: an N-terminal Bro1 domain
(residues 1–358), a central ‘‘V’’ domain (362–702), and a C-
terminal proline-rich region (703–868). Crystal structures of
different ALIX constructs (9, 10, 35) have revealed that the
banana-shaped Bro1 domain is organized about a core of
tetratricopeptide helical hairpins and that the V domain is
composed of two extended helical arms that fold in the shape of
the letter V. YP(Xn)L sequence motifs within retroviral Gag
proteins bind on the inner face of the second arm of the V
domain (10, 17, 35–37). The proline-rich region contains binding
epitopes for a number of other cellular factors, including
TSG101 (13, 15, 16), endophilins (21), and ALG-2 (38, 39). The
Bro1 domain contains binding sites for both HIV-1 NC (40) and
the CHMP4 proteins (7–11). Mutations that inhibit CHMP4
binding cluster within an exposed hydrophobic patch on the
concave surface of the Bro1 domain, which is thought to be the
CHMP4 binding site (9–11). This important interaction has yet
to be characterized in molecular detail, however, and, we have
therefore mapped the ALIX binding sites on the three human
CHMP4 proteins and determined crystal structures of the
relevant ALIXBro1-CHMP4 complexes.
Results
CHMP4-ALIX Interactions. Deletion analyses were used in conjunc-
tion with biosensor binding experiments to map the ALIX
Author contributions: J.M. and R.D.F. contributed equally to this work; J.M., R.D.F., W.I.S.,
and C.P.H. designed research; J.M., R.D.F., and F.G.W. performed research; and J.M., W.I.S.,
and C.P.H. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The atomic coordinates and structure factors have been deposited in the
Protein Data Bank, www.pdb.org [ID codes 3C3O (CHMP4A), 3C3Q (CHMP4B), and 3C3R
(CHMP4C)].
*To whom correspondence may be addressed. E-mail: wes@biochem.utah.edu or
chris@biochem.utah.edu.
This article contains supporting information online at www.pnas.org/cgi/content/full/
0801567105/DCSupplemental.
© 2008 by The National Academy of Sciences of the USA
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binding site on CHMP4A. As summarized in Fig. 1A, an ALIX
constructspanningtheBro1andVdomains(ALIXBro1-V)bound
both full-length CHMP4A and a minimal C-terminal
CHMP4A205-222construct with similar affinities (30 ? 11 ?M
and 44 ? 6 ?M), but did not bind detectably to a CHMP4A
construct that lacked the final 18 residues (CHMP1-204) or to a
full-length CHMP4A protein that harbored a single point mu-
tation within this region (CHMP4AL217A) (15). Thus, the ALIX-
Bro1-Vbinding site maps to the final 18 residues of CHMP4A.
The three human CHMP4 family members have similar but
distinct C-terminal sequences, so we tested whether peptides
corresponding to the termini of CHMP4B and CHMP4C also
bound ALIXBro1-V. As shown in Fig. 1B, terminal fragments
from all three CHMP4 proteins bound ALIXBro1-Vwith similar
affinities (44, 48, and 41 ?M, respectively), demonstrating that
all three CHMP4 proteins encode C-terminal ALIX binding
sites. Similar binding data were also obtained for the shorter
ALIXBro1construct (see Fig. 1 legend), and the Bro1 domain of
ALIX,therefore,bindstheCterminiofallthreehumanCHMP4
proteins.
Crystal Structures of ALIXBro1-CHMP4 Complexes. Crystal structures
of ALIXBro1 in complex with peptides corresponding to the
C-terminal binding sites from each of the three human CHMP4
proteins were determined in order to visualize the molecular
basis for ALIX-CHMP4 interactions [Figs. 2 and 3 and support-
ing information (SI) Figs. S1–S3]. All three complexes crystal-
lized isomorphously in space group C2 with a single ALIXBro1-
CHMP4 complex in the asymmetric unit. The CHMP4A-C
complexes were refined to resolutions of 2.15, 2.10, and 2.02 Å,
respectively, with good geometries and Rfreevalues ?30% (Table
S1).
As shown in Fig. 2, CHMP4A205-222 forms an amphipathic
helix that binds across the concave surface of ALIXBro1, con-
Concentration (M)
Percent Saturation
80
70
60
50
40
30
20
10
0
10-5
10-4
10-6
10-7
CHMP4A205-222
CHMP4B205-224
CHMP4C216-233
A
Percent Saturation
Concentration (M)
Time (s)
Response (RU)
80
70
60
50
40
30
20
10
0
10-5
10-4
10-6
10-7
CHMP4A205-222
CHMP4AL217A
CHMP4A1-204
CHMP4A
50
100
150
200
0
-50 5 10 15 20 25 30
B
Fig. 1.
isothermsandsensorgrams(Inset)showingALIXBro1-VbindingtoGST-CHMP4A
(KD ? 30 ? 11 ?M) (Inset), GST-CHMP4A205-222 (KD ? 44 ? 6 ?M), GST-
CHMP4AL217A(binding not detectable), and GST-CHMP4A1-204(binding not
detectable).TheKDestimatesareaveragesfrombestfitstosingle-sitebinding
models (mean ? SD, n ? 3). The shorter ALIXBro1construct also bound to
CHMP4A205-222(KD? 40 ? 0.6 ?M) and to the longer CHMP4A195-222(KD?
40.5 ? 0.4 ?M) and CHMP4A174-222(KD? 36.5 ? 0.4 ?M) 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 ALIXBro1-Vconcentrations; data not shown).
Error bars are indicated on all biosensor figures, but are often too small to be
readily visible. (B) ALIXBro1 binds the C termini of CHMP4A, CHMPB, and
CHMP4C. Binding isotherms showing ALIXBro1-Vbinding to immobilized C-
terminal peptides from CHMP4A-C. Estimated dissociation constants were:
GST-CHMP4A205-222, 44 ? 6 ?M (mean ? SD, n ? 6); GST-CHMP4B205-224, 48 ?
6 ?M (mean ? range, n ? 2); GST-CHMP4C216-233, 41 ? 10 ?M (mean ? range,
n ? 2). Binding to a control GST surface was negligible (data not shown).
Mapping the ALIX binding sites of CHMP4 proteins. (A) Binding
C
α1
α2
α3
α4
α5
α6
α7
α8
α9
α10
β1
β2
C
N
Fig. 2.
the C-terminal helix from CHMP4A (purple).
Ribbon diagram showing the ALIXBro1domain (blue) in complex with
A
CHMP4B
CHMP4C
CHMP4A/B/C
CHMP4B
CHMP4C
CHMP4A/B/C
B
D143
K147
K202
D206
M208
F199
L216
I212
α5
α6
W220
L217
L214
L214/I225/M214
L217/228/217
W220/231/220
L214/I225/M214
L217/228/217
W220/231/220
M224
M224
D143
K147
K202
D206
M208
F199
L216
I212
α5
α6
W220
L217
L214
Fig. 3.
interface. The CHMP4A helix is oriented N to C from top to bottom, ALIX
residues within the binding interface are shown explicitly, dashed lines indi-
catehydrogenbondsorsaltbridges,andkeyhydrophobicCHMP4residuesare
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)
andCHMP4C(orange)helicesoverlaywell,whereastheCHMP4Bhelix(green)
is rotated by ?20°.
ALIXBro1-CHMP4 interfaces. (A) Stereoview of the ALIXBro1-CHMP4A
7688 ?
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tacting helices 5–7 and the extended C-terminal strand that
traverses the domain. ALIXBro1does not change conformation
notablyuponCHMP4binding,althoughseveralsidechainsinthe
binding site shift slightly, with the largest adjustment (?1.5Å)
being made by the ALIX Phe-199 ring. The CHMP4A205-222and
CHMP4C216-233peptides have similar lengths and sequences and
bind ALIXBro1 in very similar fashions, whereas the longer
CHMP4B205-224peptide binds at the same site, but in a slightly
different fashion (see below).
In all three complexes, important interactions are made by
hydrophobic residues located on three successive turns of the
CHMP4 recognition helix (CHMP4A residues Leu-214, Leu-
217, and Trp-220). The most distinctive contact is made by the
indole ring of Trp-220, which binds in a hydrophobic pocket
located between helices 5 and 6 (Fig. 3A). The indole nitrogen
also forms a hydrogen bond with the conserved ALIX Asp-143
carboxylate, which in turn is buttressed by a salt bridge with the
ALIX Lys-202 side chain. Leu-217 binds in an adjacent hydro-
phobic pocket located between ALIX helices 6 and 7, whereas
Leu-214 binds on a more open hydrophobic surface of ALIX
helix 6. No other CHMP4A side chains make substantial con-
tacts, and the structures therefore indicate that the three hy-
drophobic residues of the CHMP4A recognition helices are the
primary determinants of ALIX binding and specificity. The
terminal carboxylates of CHMP4A and CHMP4C probably also
contribute to ALIX recognition, because they make water-
mediated interactions with the ALIX Lys-151 side chain.
The ALIXBro1-CHMP4B205-224 Complex. The longer CHMP4B205-224
peptide binds ALIXBro1at the same site and forms an amphi-
pathic helix that places the same three hydrophobic side chains
in analogous binding sites. In this case, however, the helix is
rotated by ?20° relative to the CHMP4A/C helices, which
displaces the N and C termini of the CHMP4B helix by ?5Å and
3Å, respectively. (Fig. 3B and Figs. S2B and S3). The C-terminal
displacement allows the final two CHMP4B residues (not
present in the shorter CHMP4A/C helices) to make unique
interactions that appear to dictate the helix orientation. Specif-
ically, the CHMP4B Ser-223 hydroxyl caps the helix and hydro-
gen bonds with the ALIX Lys-147 side chain (whereas ALIX
Lys-147 hydrogen bonds with the Trp-220 main-chain carbonyl
in the CHMP4A and 4C complexes). The terminal CHMP4B
Met-224 residue, in turn, contacts a hydrophobic patch between
ALIX helices 5 and 6 (Fig. 3B and Fig. S3B). Thus, the
C-terminal helices of all three human CHMP4 proteins bind the
same site on ALIX, although the detailed interactions can differ
depending on the length of the recognition helix.
Mutational Analyses of the ALIXBro1-CHMP4A Interaction. Biosensor
binding assays were also performed to test the importance of the
three conserved hydrophobic residues of the CHMP4 recogni-
tion helix. As shown in Fig. 4A, single alanine substitutions of
CHMP4A residues Leu-214, Leu-217, and Trp-220 abolished
ALIXBro1-Vbinding, confirming the energetic importance of all
three residues. A cluster of acidic residues is also conserved at
the N-terminal end of the CHMP4 recognition helix (Fig. 4B).
These residues do not contact ALIX directly in the crystal
structures, but do approach basic surface residues Lys-209 and
Lys-215, and could therefore also contribute to binding. Muta-
tion of the glutamate residue present in all three human CHMP4
proteins (CHMP4A Glu-209) reduced ALIXBro1-Vbinding af-
finity by twofold, indicating that hydrophilic flanking residues
can also contribute to ALIXBro1binding, albeit modestly.
Discussion
Our biochemical and structural analyses show that ALIX binds
amphipathic C-terminal helices on all three human CHMP4 pro-
teins. Three hydrophobic residues on the CHMP4 recognition
R
K
E
E
E
D
D
D
M
K
LW
A
D
E
Percent
Occurrence
0%
25%
50%
100%
75%
4A
4B
4C
205PKVDEDEEALKQLAEWVS222
205KKKEEEDDDMKELENWAGSM224
216QRAEEEDDDIKQLAAWAT233
---Lxx+LAALxx
xKxEEEDDDMKxLxxWAx
1-3
C
F199
I212
L216
N
C
D
A
Concentration (M)
Percent Saturation
205-222
205-222E209A
205-222W220A
205-222L214A
205-222L217A
10-5
10-4
10-6
80
70
60
50
40
30
20
10
0
B
Fig. 4.
ing ALIXBro1-Vbinding to immobilized WT CHMP4A205-222and to CHMP4A205-222
constructs with the following mutations: W220A, L217A, L214A, and E209A. For
the E209A mutant, KD ? 95 ? 2 ?M (dissociation constant and error were
estimatedfromastatisticalfitofasinglebindingisothermderivedfromduplicate
measurementsat10differentALIXBro1-Vconcentrations.).(B)SequencesoftheC
terminiofhumanCHMP4proteinsareshown(Upper),togetherwithabargraph
showing sites of ?50% identity across metazoan CHMP4 proteins, the resulting
CHMP4 consensus sequence (see also Table S2) and the distinct consensus se-
quenceoftheC-terminalamphipathichelicesofCHMP1–3proteins(Lower)(47).
(C) Model of the ALIXBro1-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 ALIXBro1-
CHMP4A205-222and VPS4A MIT-CHMP1A180-196complexes. The three key hydro-
phobic residues from each recognition helix are shown explicitly, and the figure
emphasizesthatthefirsthydrophobicresidueispositioneddifferentlyinthetwo
helices.
Molecular recognition in ALIX-CHMP4 complexes. (A) Isotherms show-
McCullough et al.
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helices contact ALIX extensively, and their energetic importance
was confirmed by mutagenesis. The C-terminal Leu and Trp
residues are invariant in metazoan CHMP4 proteins, whereas the
first hydrophobic position of the helix can vary between Met, Leu,
Ile, and Phe (Fig. 4B and Table S2). This pattern of conservation
isnicelyexplainedbytheALIXBro1-CHMP4structures,whichshow
that the terminal Leu and Trp residues of the CHMP4 recognition
helix bind in well defined pockets, whereas the first hydrophobic
residueinthehelixbindsagainstaflathydrophobicsurfacethatcan
presumably tolerate greater side chain variability. Several sets of
flanking hydrophilic residues are also conserved in the recognition
helix, including an upstream acidic cluster (residues 208–212 in
CHMP4A) and two basic residues (CHMP4A Lys-206 and 215).
These side chains are solvent exposed and do not contact ALIX
extensively, but a mutation within the CHMP4A acidic cluster did
reduce ALIXBro1binding slightly, indicating that the cluster may
interactfavorablywiththebasicALIXBro1surface.Itisalternatively
possible, however, that flanking hydrophilic residues are conserved
primarily because they make important contacts when CHMP4
proteins adopt alternative conformations or bind other partners.
CHMP4 binding appears to be a conserved function of Bro1
domains, because three other Bro1 domain-containing proteins,
Rim20p, HD-PTP, and Brox, also bind CHMP4 proteins (41,
42). Alignment of the Bro1 domains from metazoan ALIX
proteins, human Brox and HD-PTP, and yeast Rim20p and
Bro1p reveal strong, although not absolute conservation of
CHMP4 contact residues, suggesting that most Bro1 domains
will bind CHMP4 proteins in a similar fashion (Fig. S4). For
example, the ALIXBro1 Asp-143 and Lys-202 residues, which
form a salt bridge that buttresses the ALIXBro1 Asp-143-
CHMP4A Trp-220 hydrogen bond, are absolutely conserved as
an acidic/basic residue pair in all of the aligned Bro1 domains.
ALIX can be recruited to facilitate enveloped virus budding
and release, and this function has been studied most extensively
for HIV-1 (10, 11). These studies have identified three different
ALIX point mutations (F199D, I212D, and L216D) that block
both CHMP4 binding and ALIX-mediated release of HIV-1
constructs that cannot recruit TSG101/ESCRT-I. All three of
these residues map to the CHMP4 binding interface, and the
crystal structures are consistent with the observed loss of
CHMP4 binding for Asp mutations at these positions (Fig. 4C).
The ALIX I212D mutation has also been shown to block the
ALIX-dependent abscission step of cytokinesis (18). Thus, the
crystallographic ALIX-CHMP4 interface visualized here is es-
sential for ALIX-dependent steps in HIV-1 budding and cyto-
kinesis. This interaction recruits CHMP4 proteins, which in turn
presumably recruit additional ESCRT-III subunits and VPS4
complexes to function in membrane remodeling and fission.
The recruiting order appears to be reversed in the case of
MVB vesicle formation, where copolymerization of the different
ESCRT-III subunits on endosomal membranes creates a surface
that recruits other ESCRT pathway proteins, including ALIX,
VPS4,Doa4p,IST1,andVta1p/LIP5.LiketheCHMP4subunits,
the CHMP1–3 subunits of ESCRT-III also have C-terminal
amphipathic helices [MIT interacting motifs (MIM)], but in
these cases the helices bind the MIT domains of VPS4, Vta1p/
LIP5, and AMSH and thereby recruit ATPase and deubiquity-
lating activities to the membrane (43–48). Hence, the terminal
helices of different ESCRT-III subunits must display distinct
binding surfaces to ensure specificity in protein recruitment.
This is borne out by the observation that the C-terminal
CHMP4A helix binds ALIXBro1but not the VPS4A MIT do-
main, whereas the C-terminal CHMP1B helix binds the VPS4A
MIT domain but not ALIXBro1(Fig. S5). As shown in Fig. 4D,
the MIM helices of the CHMP1–3 proteins display three key
leucine/hydrophobic residues that make important MIT domain
contacts (47, 48). In these cases, the three hydrophobic residues
are spaced by three and two intervening residues, whereas the
three hydrophobic residues of the CHMP4 recognition helix are
each separated by two intervening residues. As a result, the
initial hydrophobic residues of the CHMP1–3 and CHMP4
recognition helices occupy different relative positions. Further-
more, the terminal hydrophobic CHMP4 residue is a Trp,
whereas the terminal CHMP1–3 hydrophobic residue is a Leu.
Thus, the different identities and positions of the hydrophobic
residues in their terminal recognition helices help ensure that
each ESCRT-III subunit recruits its proper binding partner(s).
Methods
Expression Constructs and Plasmids. Expression constructs for GST-CHMP4A
(WISP06–197) and GST-CHMP4A L217A (WISP06–61) are described in ref. 15.
Quikchange mutagenesis (Stratagene) was used to create expression con-
structs for GST-CHMP4A1-204(WISP06–198), GST-CHMP4A205-222(WISP06–60),
GST-CHMP4B205-224 (WISP06–201), GST-CHMP4C216-233 (WISP06–202), GST-
CHMP4A205-222W220A (WISP06–203), GST-CHMP4A205-222L217A (WISP06–204),
GST-CHMP4A205-222L214A(WISP06–205),andGST-CHMP4A205-222E209A(WISP06–
206), using expression constructs for GST-CHMP4A, GST-CHMP4B (WISP06–
199), and GST-CHMP4C (WISP06–200) as templates.
ALIX and CHMP4 Protein Expression and Purification. CHMP4 peptides used for
crystallization studies were synthesized with an N-terminal acetyl capping
group: 205PKVDEDEEALKQLAEWVS222 (CHMP4A), 205KKKEEEDDDMKELEN-
WAGSM224(CHMP4B),and216QRAEEEDDDIKQLAAWAT233(CHMP4C).ALIXBro1
(residues1–359)andALIXBro1-V(residues1–702)proteinsusedinbiosensorand
crystallization studies were expressed and purified as described in ref. 10.
GST-CHMP4 proteins used in biosensor experiments were expressed in
BL21(DE3) Codon?(RIPL) Escherichia coli cells grown in autoinduction media,
ZYP-50502 (49). Cells were grown at 37°C for 4 h, then transferred to 23°C or
17°Cforgrowthtosaturation.Subsequentpurificationstepswereperformed
at 4°. Cells from 100 ml of cultures were harvested, resuspended in 4 ml of 20
mMsodiumphosphate(pH7.2),150mMNaCl,5mM?-mercaptoethanol,and
protease inhibitors (Roche Diagnostics) and lysed by the addition of 1 mg/ml
lysozyme followed by sonication. The lysate was clarified by centrifugation,
and GST-CHMP4 proteins were affinity purified by incubation with 500 ?l of
glutathionebeadsinbiosensorrunningbuffer[20mMsodiumphosphate(pH
7.2), 150 mM NaCl, 0.01% P20, 0.2 mg/ml BSA, 5 mM ?-mercaptoethanol] for
30 min, washed extensively, and eluted with 20 mM reduced glutathione in
running buffer.
Biosensor Binding Studies. Binding experiments used Biacore 2000 and T100
opticalbiosensorinstruments.Research-gradeCM5sensorchipswerederivatized
withanti-GSTantibody,usingaminecoupling,andwereusedtocaptureaffinity
purified GST-CHMP4 proteins or GST alone (reference) at surface densities of
300-3030 response units (RU). Similar binding data were obtained for both
ALIXBro1and ALIXBro1-Vconstructs, but the ALIXBro1-Vconstruct was more soluble
and therefore allowed more complete sampling of the binding isotherms. Pure
ALIXBro1-V (diluted in running buffer to the designated concentrations) was
injected in duplicate (50 ?l/min, 20°C) and binding data were collected at 2 Hz
during the 30-s association and dissociation phases. All interactions reached
equilibriumrapidlyanddissociatedwithinsecondsduringthedissociationphase,
and all were studied at more than one surface density to rule out crowding and
mass transport effects. Binding responses at 10–20 sec were fit to simple 1:1
binding isotherms to obtain equilibrium constants.
Crystallization. ALIXBro1-CHMP4 crystals were grown by sitting drop vapor diffu-
sionat4°Catprotein:peptidemolarratiosof1:1.2atfinalALIXBro1concentrations
of 25 mg/ml (CHMP4A) or 23 mg/ml (CHMP4B/C). Crystals grew from drops
containing0.5?lofproteinsolutionmixedwith0.5?lofreservoirsolution:15%
PEG 8000, 100 mM Na MES (pH 6.5), and 200 mM Na Acetate (CHMP4A) from
dropscontaining1.2?lofproteinsolutionmixedwith0.7?lofreservoirsolution
[15%PEG8000,100mMNaMES(pH6.5),200mMNaAcetate(CHMP4B)]orfrom
dropscontaining1.2?lofproteinsolutionmixedwith0.7?lofreservoirsolution
[10% PEG 2000, 100 mM Na MES (pH 6.5) (CHMP4C)].
Data Collection and Structure Refinement. Crystals were cryoprotected in
reservoirsolutionsmadeupwith20%glycerol,suspendedinnylonloops,and
flash frozen in liquid nitrogen. Data were collected on a copper rotating
anodegeneratorwithconfocaloptics(Rigaku;MicroMax007HF)andaRigaku
R-AxisIVimageplate(CHMP4BandCHMP4C)andatbeamlines11-1(MAR325,
0.97607 Å wavelength) and 7-1 (Quantum 315, 0.98397 Å wavelength) of the
Stanford Synchrotron Radiation Laboratory (CHMP4A). Crystals were main-
tained at 100 K during data collection. Data were integrated and scaled with
7690 ?
www.pnas.org?cgi?doi?10.1073?pnas.0801567105McCullough et al.

Page 5

Denzo and Scalepack, using the HKL2000 suite (50). ALIXBro1-CHMP4 crystals
wereisomorphouswithcrystalsofunligandedALIXBro1(PDBentry2OEW)(10).
The unliganded ALIXBro1structure was used as a starting model for all three
refinements, which were performed with REFMAC, using the maximum like-
lihoodtargetfunctionandincorporatingTLSanalysis(51,52)asimplemented
intheCCP4package(53).ModelswerebuiltinO(54)andCOOT(55),geometry
wasanalyzedwithPROCHECK(56),andfiguresweregeneratedinPyMOL(57).
The following residues were modeled in the different CHMP4 complexes:
CHMP4A, 210–222; CHMP4B, 207–224; and CHMP4C, 221–233. Crystallo-
graphic statistics are presented in Table S1.
ACKNOWLEDGMENTS.BiosensoranalyseswereperformedattheUniversity
of Utah core facility, and x-ray data were collected at the Stanford Syn-
chrotron Radiation Laboratory (SSRL). The SSRL is a national user facility
operated by Stanford University on behalf of the U.S. Department of
Energy, Office of Basic Energy Sciences. The SSRL Structural Molecular
Biology Program is supported by the Department of Energy Office of
Biological and Environmental Research, the National Institutes of
Health National Center for Research Resources, Biomedical Technology
Program,andNationalInstituteofGeneralMedicalSciences.Thisworkwas
supported by National Institutes of Health Grants GM082534 (to W.I.S. and
C.P.H.) and AI051174 (to W.I.S.).
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