Structure
Article
Solution Structure of the ESCRT-I and -II
Supercomplex: Implications
for Membrane Budding and Scission
2,4 Hoi Sung Chung,2 Dawn Z. Herrick,3 Bertram Canagarajah,1 David S. Cafiso,3
_
Evzen Boura,1,4 Bartosz Rózycki,
William A. Eaton,2 Gerhard Hummer,2,* and James H. Hurley1,*
1Laboratory
of Molecular Biology
of Chemical Physics
National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA
3Department of Chemistry, Biophysics Program and Center for Membrane Biology, University of Virginia, Charlottesville, VA 22904-4319, USA
4These authors contributed equally to this work
*Correspondence: [email protected] (G.H.), [email protected] (J.H.H.)
DOI 10.1016/j.str.2012.03.008
2Laboratory
SUMMARY
The ESCRT-I and ESCRT-II supercomplex induces
membrane buds that invaginate into the lumen of
endosomes, a process central to the lysosomal
degradation of ubiquitinated membrane proteins.
The solution conformation of the membranebudding ESCRT-I-II supercomplex from yeast was
refined against small-angle X-ray scattering (SAXS),
single-molecule Förster resonance energy transfer
(smFRET), and double electron-electron resonance
(DEER) spectra. These refinements yielded an
ensemble of 18 ESCRT-I-II supercomplex structures
that range from compact to highly extended. The
crescent shapes of the ESCRT-I-II supercomplex
structures provide the basis for a detailed mechanistic model, in which ESCRT-I-II stabilizes membrane buds and coordinates cargo sorting by lining
the pore of the nascent bud necks. The hybrid refinement used here is general and should be applicable
to other dynamic multiprotein assmeblies.
INTRODUCTION
Much of contemporary cell biology focuses on the visualization
of complex processes at ever-increasing spatial and temporal
resolution. At the same time, contemporary structural biology
is concerned with understanding ever larger, more complex,
and more dynamic systems. The biogenesis of multivesicular
bodies (MVBs) by the ESCRT complexes is an example of
a cell process that has been the target of intensive efforts using
approaches from both fields (Hurley and Hanson, 2010; Williams
and Urbé, 2007). MVBs are a key intermediate in the trafficking of
ubiquitinated membrane proteins to their degradation in the
lysosome. MVBs form by the budding of intralumenal vesicles
(ILVs) into the lumen of the endosome. The budding occurs
with the opposite topology relative to the cytosol as compared
to coated vesicle budding events (Hurley and Hanson, 2010).
In human cells, the ESCRTs are hijacked by HIV-1 and other enveloped viruses that bud away from the cytosol (Morita and
Sundquist, 2004), and the ESCRTs are also required for cytokinesis (Carlton and Martin-Serrano, 2007).
The ESCRT complexes divide the tasks of MVB biogenesis as
follows. ESCRT-0 is primarily responsible for clustering ubiquitinated cargo into microdomains and delivering it to the downstream ESCRTs (Raiborg and Stenmark, 2009; Wollert and
Hurley, 2010). ESCRT-I and -II (Babst et al., 2002b; Katzmann
et al., 2001) drive bud formation by forming an assembly at the
bud neck and thereby stabilizing it (Wollert and Hurley, 2010).
ESCRT-III is responsible for membrane scission (Hanson et al.,
2008; Wollert et al., 2009). ESCRT-III forms spiral or domeshaped filamentous structures that are thought to mediate
membrane scission (Fabrikant et al., 2009; Hanson et al., 2008;
Lata et al., 2008). The 1:1 complex of the ESCRT-I and -II
complexes (hereafter referred to as the ‘‘supercomplex’’; Gill
et al., 2007) connects the ESCRT-0-cargo domains to ESCRTIII and is therefore the linchpin that coordinates the entire
pathway.
The structure and function of the individual domains and the
core assemblies of ESCRT-I and -II are now well understood.
Yeast ESCRT-I consists of one copy each of four subunits,
Vps23, Vps28, Vps37, and Mvb12 (Gill et al., 2007; Kostelansky
et al., 2007). Vps23 contains an ubiquitin E2 variant (UEV) domain
(Katzmann et al., 2001) at its N-terminus. The Vps23 UEV domain
binds to (S/T)DP motifs of ESCRT-0 and ubiquitin at nonoverlapping sites (Ren and Hurley, 2011; Teo et al., 2004b). The UEV
domain is linked to the core portion of ESCRT-I by a 50 residue
proline-rich region (PRR). The ESCRT-I core is 180 Å long and
consists of a stalk and a headpiece region. Vps23 and Vps37
participate in both regions, Vps28 participates in the headpiece,
and Mvb12 participates in the stalk. The core has no known
binding partners and is thought to function at least in part as
a rigid mechanical element that controls the spatial organization
of the other domains. Vps28 contains at its C-terminus a fourhelix bundle (CTD) that is the primary binding site for ESCRT-II
(Gill et al., 2007; Kostelansky et al., 2006; Teo et al., 2006). A
30-residue flexible linker connects the Vps28 core and CTD
regions. The N-terminus of Vps37 contains a putative basic helix
that is involved in contacts with acidic membrane lipids (Kostelansky et al., 2007) and is flexibly attached to the stalk portion
of Vps37. All of these flexibly attached regions are dynamic in
solution with respect to the ESCRT-I core (Boura et al., 2011).
874 Structure 20, 874–886, May 9, 2012 ª2012 Elsevier Ltd All rights reserved
Structure
Structure of the ESCRT-I-II Supercomplex
Yeast ESCRT-II consists of two copies of Vps25 and one each
of Vps22 and Vps36, arranged in the shape of the capital letter
‘‘Y’’ (Hierro et al., 2004; Teo et al., 2004a). The Vps25 subunits
make up the stem and one branch of the Y, whereas the core
portions of Vps22 and Vps36 make up the second branch. The
Vps25 subunits each bind one copy of the early-acting
ESCRT-III subunit Vps20 at the tip most distal to the rest of the
Y-shaped core (Im et al., 2009). The N-termini of Vps22 and
Vps36 project away from one tip of the Y. At this tip, a basic helix
of Vps22 is exposed and contributes to membrane binding
(Im and Hurley, 2008). The N-terminus of yeast Vps36 is
complex. Most of this region is a variant PH domain referred to
as a ‘‘GLUE’’ domain, which is responsible for binding 3-phosphoinositides (Slagsvold et al., 2005; Teo et al., 2006). Within
an internal loop in the yeast GLUE domain, two Npl4-type zinc
fingers (NZF1 and NZF2) are inserted. NZF1 binds to the
C-terminal domain of Vps28 of ESCRT-I (Gill et al., 2007),
whereas NZF2 binds to ubiquitin (Alam et al., 2004). Hydrodynamic analysis and modeling suggest that the GLUE domain is
in a relatively compact conformation with respect to the core
(Im and Hurley, 2008), yet the GLUE-core linker is accessible
to proteolysis in solution (Hierro et al., 2004) and no crystals
have been obtained for any ESCRT-II construct containing
both the core and the GLUE domain. One of the objectives of
this study was to obtain an improved structural model for the
full-length ESCRT-II complex in solution, taking into account
its flexible elements.
ESCRT-II assembles into a 1:1 complex with ESCRT-I with
a Kd value of 30–50 nM (Gill et al., 2007) via the interface
between its Vps36 NZF1 domain and the CTD of ESCRT-I
Vps28. Deformation of the membrane into buds is only carried
out efficiently by ESCRT-I and -II in combination (Hurley and
Hanson, 2010; Wollert and Hurley, 2010). Visualization of the
structure of the ESCRT-I-II supercomplex is thus a prerequisite
to understanding the deformation mechanism. Its molecular
weight of 244 kDa puts the supercomplex at the lower margin
of feasibility for current cryoEM analysis yet renders it too large
for conventional solution NMR of uniformly labeled samples.
However, the most significant obstacle to structural analysis of
the intact ESCRT-I-II supercomplex is not its size but rather
the way in which two ordered cores and several autonomously
folded domains are tethered to one another by intrinsically disordered regions. The dynamics of the ‘‘spaghetti’’-like regions
seem to be important for their function but render them intractable to X-ray crystallography. The combination of ordered and
disordered structural elements seems to be inherent in many
dynamic cellular processes.
To address this class of problems, we developed a quantitative
scheme for the multimodal refinement of simultaneously ordered
and disordered structures against small-angle X-ray scattering
(SAXS), single-molecule Förster resonance energy transfer
(smFRET), and double electron-electron resonance (DEER)
data, starting from known crystal structures of the ordered
portions. SAXS provides constraints on the size and shape of
the supercomplex, whereas DEER and smFRET yield distibutions
of distances between pairs of electron spin and between fluorophore labels, respectively, at defined positions in the polypeptide
sequence. Building on an earlier multimodal structural analysis
of intact yeast ESCRT-I in solution using this approach (Boura
et al., 2011), here we deduce the solution conformations of yeast
ESCRT-II and the ESCRT-I-II supercomplex. This structure of the
ESCRT-I-II supercomplex fills in a final missing link in the structural connectivity of the pathway, allowing us to propose a
start-to-finish structural mechanism for MVB biogenesis.
RESULTS
Small-Angle X-Ray Scattering
To determine the overall conformation of ESCRT-II and the
ESCRT-I-II supercomplex in solution, SAXS data were collected
(Figures 1A and 1B). Data were collected at multiple concentrations, and no concentration-dependence was noted up to the
highest concentrations tested (Figures S1A and S1B available
online). The Kratky plot (Figure 1C) for ESCRT-II shows a modest
but discernable rise beginning at q = 0.23 Å1, consistent with
the presence of a limited amount of nonglobular structure (Figure 1E). The ESCRT-I-II supercomplex manifested substantial
nonglobular structure on the basis of the Kratky plot (Figure 1D)
and was highly extended based on the experimental Dmax value
of 375 Å (Figure 1F).
Single-Molecule FRET
Domain-specific conformational distributions were probed by
engineering Cys pairs such that one label was in the core of
ESCRT-II and the other in the Vps36 GLUE domain (Figures 2A
and 2B). The GLUE domain is the primary membrane-targeting
domain in ESCRT-II (Teo et al., 2006). Cys residues were
introduced in the most N-terminal ordered helix of the core
portion of Vps22 (Cys34), the GLUE domain (Vps36 Cys96
and Cys282), and the first winged helix (WH) domain of the
core of Vps36 (Cys417). The labeled pairs were analyzed to
provide three independent measurements of the position and
dynamics of the GLUE domain relative to the core of ESCRT-II
(Figure 3A). The FRET efficiency distribution was broad for all
three FRET pairs, demonstrating that the GLUE domain is in
multiple conformations with respect to the core of the ESCRTII complex.
In order to probe the conformations of the ESCRT-I-II supercomplex, the FRET efficiency distributions of the labeled
ESCRT-II samples were measured in the presence of a 104fold excess of unlabeled ESCRT-I (Figure 3B). The distributions
resembled those seen in the absence of ESCRT-I; thus,
ESCRT-I does not substantially perturb the orientation of the
GLUE domain. The conformation of ESCRT-I in the presence
of ESCRT-II was examined by smFRET analysis of the Vps28
Cys65-Cys151 pair, which probes the distance between the
core and Vps28 CTD (Figure 3C). The ESCRT-I Vps28 CTD
contains the binding site for ESCRT-II. A small decrease in the
peak height of the efficiency distribution was observed in the
presence of ESCRT-II at the margin of significance. Thus,
ESCRT-II does not change structure perceptibly when it binds
ESCRT-I. In ESCRT-I, there is a small shift to slightly longer
distances between the core and Vps28-CTD. Indeed, in one
of the closed conformations obtained previously for ESCRT-I
alone, the ESCRT-II binding site on the Vps28 CTD is sequestered against the ESCRT-I headpiece domain (Boura et al.,
2011). This type of closed conformation would be precluded in
the ESCRT-I-II supercomplex.
Structure 20, 874–886, May 9, 2012 ª2012 Elsevier Ltd All rights reserved 875
Structure
Structure of the ESCRT-I-II Supercomplex
A
B
ESCRT−II SAXS
experiment
simulation
experiment
simulation
1000
I(q)
I(q)
1000
ESCRT−I−II SAXS
100
100
10
10
1
1
0.1
0.2
0.3
0.4
0.5
0.1
0.2
−1
C
D
experiment
simulation
experiment
simulation
2
q2I(q)
q2I(q)
ESCRT−I−II SAXS
2.5
1.5
1
0.5
0
0.1
0.2
0.3
0.4
0.5
0.1
−1
0.4
0.008
0.007
0.012
0.006
0.010
ESCRT-II
0.008
0.005
P(r)
P(r)
0.3
q [Å ]
F
0.014
0.2
−1
q [Å ]
E
0.4
q [Å ]
ESCRT−II SAXS
4
3.5
3
2.5
2
1.5
1
0.5
0
0.3
−1
q [Å ]
0.006
0.003
0.004
0.002
0.002
0.001
0.000
0.000
0
50
ESCRT-I/II
0.004
100 150 200 250 300 350 400
r (Å)
0
50
100 150 200 250 300 350 400
r (Å)
Figure 1. SAXS and smFRET of ESCRT-II and ESCRT-I-II
(A and B) Scattering intensity of (A) ESCRT-II alone and (B) the ESCRT-I-II supercomplex.
(C and D) Kratky plots of (C) ESCRT-II alone and (D) the ESCRT-I-II supercomplex. The rise at high q is indicative of nonglobular structure, which is more
pronounced for the supercomplex than for ESCRT-II. The error bars represent the standard error of the mean.
(E and F) Pair distance distributions P(r) for (E) ESCRT-II alone and (F) the ESCRT-I-II supercomplex.
See also Figure S1.
DEER Spectroscopy
To explore the conformation of ESCRT-I in the presence
of ESCRT-II, DEER spectra were collected for three dually
spin-labeled samples of yeast ESCRT-I (Figures 4A–4C). These
consist of the Vps28 Cys65-Cys151 sample described previously (Figure 4A), Vps23 Cys108-Cys256, which probes the
ESCRT-I UEV domain-core distance (Figure 4B), and Vps37
Cys12- Vps23 Cys 223, which probes the Vps37 N-terminal
helix-core distance (Figure 4C). The UEV domain binds to ubiquitin and ESCRT-0, whereas the Vps37 N-terminal helix is
involved in membrane interactions. As compared to free
ESCRT-I, the Vps37 Cys12- Vps23 Cys223 pair, remote from
the ESCRT-II binding site, does not change (Figure 4F). The
Vps28 Cys65-Cys151 and Vps23 Cys108-Cys256 pairs show
a population loss in the short distance states when bound to
ESCRT-II (Figures 4D and 4E). Interspin distances of around
2 nm contribute to the fast initial decay of the measured DEER
signal. The DEER decay slows in the ESCRT-I-II supercomplex
for Vps28 Cys65-Cys151 and Vps23 Cys108-Cys256 spin-label
pairs. These data show that the closed population of ESCRT-I is
somewhat reduced when bound to ESCRT-II in the mixture of
open and closed states.
Solution Structure of ESCRT-II
We performed unbiased replica exchange Monte Carlo (REMC)
simulations of the ESCRT-II complex in solution and then refined
876 Structure 20, 874–886, May 9, 2012 ª2012 Elsevier Ltd All rights reserved
Structure
Structure of the ESCRT-I-II Supercomplex
A
UEV domain
H0 helix
ESCRT-I core
Vps28 CTD
Vps28 T65C
Vps23 N108C
Vps28 K151C
Vps28 T65C
Vps37
Q12C
Vps36 E417C
Vps23 E223C Vps23E256C
Vps22
D34C
Vps36
D96C
ESCRT-II core
Vps36 E282C
Figure 2. Introduced Cys Residues Used for
Labeling
(A) Structures shown are the basic predicted helix
from the N-terminus of Vps37 (model), the UEV
domain of Vps23 (Protein Data Bank [PDB] ID code
1UZX; Teo et al., 2004b), the heterotetrameric core
of ESCRT-I (PDB ID code 2P22; Kostelansky et al.,
2007), the C-terminal domain of Vps28 (PDB ID
code 2J9U; Gill et al., 2007), the Y-shaped core of
ESCRT-II (PDB ID code 1U5T; Hierro et al., 2004),
and the GLUE domain of Vps36 (PDB ID code
2CAY; Teo et al., 2006).
(B) Schematic of the domain and core organization
of ESCRT-I and -II.
Vps36 GLUE domain
B
core
ESCRT-I
stalk
UEV
Vps28
headpiece
Mvb12
Vps23
Vps37
PRR
H0
CTD
5
s2
Vp
V
Vps25
core
s22
GLUE
F1
6
ps3
NZ
F2
NZ
Ub
Vp
ESCRT-II
the resulting simulation ensemble using the experimental SAXS
and FRET data. In Figure S2, we compare the simulation data
obtained before any refinement to match the experimental
data for ESCRT-II. We find that for ESCRT-II, the simulation
ensemble without refinement almost fully accounts for the
SAXS data over the entire q range. The raw simulation ensemble
also accounts well for two out of the three smFRET efficiency
histograms, but overall the comparison to the smFRET data
reveals detail that is not fully accounted for by the unrefined
simulations. Whereas the smFRET histograms from the simulations do cover the full range of efficiencies observed in the experiments, the weights of structures with low smFRET efficiencies
are overestimated somewhat for labels (Vps36 Cys96– Vps22
Cys34) and significantly for (Vps36 Cys96 – Vps36 Cys417). In
effect, the refinement of the ESCRT-II structure eliminates
conformations from the initial simulation ensemble, in which
these two label pairs are distant, while further improving the
agreement with the SAXS data.
In the refinement, a representative solution ensemble was
determined based on a minimum-ensemble approach (Boura
et al., 2011; Figure S2). An ensemble of 15 structures best
represents the conformations of ESCRT-II (Figure 5). The Dmax
values for various structures in the ESCRT-II ensemble range
from 138 to 183Å, consistent with the maximum dimension
in the P(r) distribution (Figure 1E). Only in the most open conformations of ESCRT-II does the GLUE domain fail to contact the
core. Roughly 90% of ESCRT-II complexes have the GLUE
domain contacting the core, although the contact modes vary.
The yeast ESCRT-II GLUE-core interaction thus seems to be weak and dynamic,
similar to the case for the human complex
(Im and Hurley, 2008). These results are in
good agreement with the broad FRET
efficiency histograms and past observations that the GLUE-core linker is highly
susceptible to proteolysis (Hierro et al.,
2004; Teo et al., 2004a).
Solution Structure of ESCRT-I
and -II
The REMC simulations described previously were extended to the ESCRT-I-II
supercomplex with the addition of a
model based on the crystal structures of
yeast Vps23 UEV (Teo et al., 2004a), the yeast ESCRT-I core
(Kostelansky et al., 2007), yeast Vps28 CTD (Gill et al., 2007),
and the Vps37 N-terminal helix and linkers. For the ESCRT-I-II
supercomplex, we again find good agreement of the unrefined
simulation data with the SAXS measurements (Figure S3).
However, somewhat larger deviations at small q values indicate
that the simulation ensemble is overweighted in fully extended
structures with a large radius of gyration. The agreement with
the DEER measurements is excellent for one of the label pairs
and good for the other two, capturing the plateau values but
being off in the initial decay by about a factor of two. For the
smFRET data, the efficiency distributions calculated from the
unrefined simulation ensembles agree quite well with experiment
for three of the four label pairs and in all cases cover the FRET
efficiency range seen in experiment (with the possible exception
of the highest efficiencies for Vps36 Cys96 – Vps36 Cys417). The
refinement of the ESCRT-I-II supercomplex structural ensemble
is thus again achieved largely by elimination of structures
inconsistent with at least some of the pair distance measurements, while improving the agreement with the SAXS data
through reweighting. The ESCRT-I-II SAXS, smFRET, and
DEER data were jointly best represented by an ensemble of 18
structures (Figures 6 and S4). The smFRET efficiency distributions calculated for this ensemble fully account both for the
measured data (Figure 3D). For cross-validation, we omitted
the Vps28 Cys65-Cys151 pair from the refinement and again
found excellent agreement between calculated and measured
smFRET data (Figure 3E).
Structure 20, 874–886, May 9, 2012 ª2012 Elsevier Ltd All rights reserved 877
Structure
Structure of the ESCRT-I-II Supercomplex
A
experiment
simulation
experiment
simulation
120
80
60
40
40
20
20
0
0
0
0.2
0.4
0.6
0.8
10
0
0.2
0.4
0.8
0
1
0.2
96 Vps36 - 34 Vps22
282 Vps36 - 34 Vps22
100
with ESCRT-I
w/o ESCRT-I
counts
40
30
20
0.8
1
96 Vps36 - 417 Vps36
with ESCRT-I
w/o ESCRT-I
80
0.4
0.6
efficiency
70
with ESCRT-I
w/o ESCRT-I
60
50
counts
50
0.6
efficiency
efficiency
B
30
20
0
1
40
counts
counts
60
experiment
simulation
50
100
80
counts
60
140
100
counts
96 Vps36 − 417 Vps36
96 Vps36 − 34 Vps22
282 Vps36 − 34 Vps22
120
60
40
40
30
20
10
20
0
0
0
0.2
0.4
0.6
efficiency
0.8
10
0
1
0.2
0.4
0.6
efficiency
0.8
0
1
0
0.2
0.4
0.6
efficiency
0.8
1
C
65 Vps28 - 151 Vps28
140
with ESCRT-II
w/o ESCRT-II
counts
120
100
80
60
40
20
0
0
D
0.2
0.4
0.6
efficiency
0.8
1
282 Vps36 − 34 Vps22 with ESCRT−I
counts
counts
30
20
10
0
0
0.2
0.4
0.6
0.8
1
efficiency
E
90
80
70
60
50
40
30
20
10
0
experiment
simulation
counts
experimet
simulation
40
96 Vps36 − 417 Vps36 with ESCRT−I
96 Vps36 − 34 Vps22 with ESCRT−I
50
0
0.2
0.4
0.6
efficiency
0.8
1
80
70
60
50
40
30
20
10
0
experiment
simulation
0
0.2
0.4
0.6
efficiency
0.8
1
Cross-validation
65 Vps28 − 151 Vps28 with ESCRT−II
120
experiment
simulation
counts
100
80
60
40
20
0
0
0.2
0.4
0.6
efficiency
0.8
1
Figure 3. Single-Molecule FRET Histograms
(A) Experimental smFRET measurements for ESCRT-II alone are compared to the simulation for the indicated Cys pairs.
(B) Experimental smFRET for ESCRT-II pairs in the presence (thick blue bars) and absence (thin red bars) of ESCRT-I, showing that binding of ESCRT-I does not
change the conformation of these regions of ESCRT-II.
878 Structure 20, 874–886, May 9, 2012 ª2012 Elsevier Ltd All rights reserved
Structure
Structure of the ESCRT-I-II Supercomplex
65 Vps28 − 151 Vps28 with ESCRT−II
A
D
1
experiment
simulation
0.98
65 Vps28 – 151 Vps28
+ ESCRT-II
- ESCRT-II
0.95
V(t)/V(0)
0.96
V(t)/V(0)
1.00
0.94
0.92
0.9
0.96
0.94
0.85
0.92
0.90
0.88
0.88
0
0.5
1
1.5
0.75
0.0
2
0.5
t [µs]
B
108 Vps23 − 256 Vps23 with ESCRT−II
E
1
V(t)/V(0)
V(t)/V(0)
0.99
1.00
0.98
0.97
0.96
0.86
+ ESCRT-II
- ESCRT-II
0.98
0.97
0.96
0.94
0.0
0.94
0
0.5
1
1.5
2
12 Vps37 − 223 Vps23 with ESCRT−II
F
1.00
1
experiment
simulation
0.99
0.5
1.0
1.5
2.0
t [µs]
t [µs]
0.98
0.99
V(t)/V(0)
V(t)/V(0)
2.0
0.95
0.95
C
1.0
1.5
t [µs]
108 Vps23 – 256 Vps23
0.99
experiment
simulation
0.98
0.90
0.80
0.86
1.00
0.97
0.96
0.95
12 Vps37 – 223 Vps23
+ ESCRT-II
- ESCRT-II
0.98
0.97
0.96
0.95
0.94
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8
t [µs]
0.94
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8
t [µs]
Figure 4. DEER of ESCRT-I Bound to ESCRT-II
(A–C) Experimental DEER measurements (red) for ESCRT-I in the presence of ESCRT-II are compared to the simulation (solid line) for the indicated Cys pairs.
(D–F) Experimental DEER measurements (red) for ESCRT-I in the presence of ESCRT-II are compared to equivalent experimental measurements in the absence
of ESCRT-II (black). In the top row, the modulation depths for the two experiments differ as shown by the dual y axis labels.
See also Figure S3.
The conformations of the ESCRT-I and -II complexes individually are qualitatively similar to their conformations in isolation.
ESCRT-I consists of a mixture of open and closed states,
whereas ESCRT-II is primarily in a relatively compact state. In
the supercomplex, 8% of the population is in a closed conformation, in which the two complexes are folded back on one
another, with a Dmax value of 208 Å, similar to that of ESCRT-I
alone (Figure 6, upper left). The remainder of the population is
in highly extended conformations, with Dmax values ranging up
to 381 Å (Figure 6), consistent with the ab initio P(r) distribution
(Figure 1F). Most of the extended conformations are partially
folded back on themselves, though without any inter-complex
interactions to tether them shut. Most of these conformations
have the shape of a crescent, which is often diagnostic of
proteins that target to membranes and induce curvature (Peter
et al., 2004; Shibata et al., 2009).
(C) smFRET for ESCRT-I in the presence (thick blue bars) and absence (thin red bars) of ESCRT-II, showing a small shift in the distribution toward longer distances,
consistent with a higher population of more open conformations in the presence of ESCRT-II.
(D) Experimental smFRET measurements for ESCRT-II in the context of the supercomplex are compared to the simulation for the indicated Cys pairs.
(E) Cross-validation of the structural refinement. The 65 Vps28 - 151 Vps28 FRET data obtained in the presence of ESCRT-II were excluded from refinement.
See also Figure S2.
Structure 20, 874–886, May 9, 2012 ª2012 Elsevier Ltd All rights reserved 879
Structure
Structure of the ESCRT-I-II Supercomplex
Figure 5. Gallery of ESCRT-II Conformations
The bulk of ESCRT-II consists of the crystallized
Vps22:Vps25:Vps36 core, which was treated as a rigid
body in the simulations. The conformational distributions
comprise different positions of the GLUE (arrows), NZF1,
and NZF2 domains of Vps36 relative to the core.
See also Figure S4.
A Model of ESCRT-I-II in the Bud Neck
The main function of the ESCRT-I-II supercomplex in MVB
biogenesis is to stabilize the neck of nascent membrane
buds, to which it localizes (Wollert and Hurley, 2010). On the
basis of membrane elasticity theory, the calculated low energy
geometry of the bud neck is a catenoid (Michalet et al., 1994).
In this minimal surface, the negative curvature along the pore
axis exactly cancels the positive curvature of the bud rim.
The radii of curvature of the supercomplex conformations are
roughly consistent with the bud neck widths of 25 nm
observed in yeast MVBs (Wemmer et al., 2011). We considered
the implications of positioning the crescent-shaped conformations of the supercomplex in terms of two major classes of
models. In the head-to-tail ring model the convex face docks
onto the negatively curved inner surface of the pore, whereas
in the spoke model their concave face docks onto the positively
curved profile of the rim. The head-to-tail ring model has
a topology analogous to the postulated mode of inverse BAR
(I-BAR) domain function in promoting negative membrane
curvature (Mattila et al., 2007). The ‘‘spoke’’ model is so-named
for its analogy to spokes of the nuclear pore
(Strambio-De-Castillia et al., 2010). The spoke
model positions the ESCRT-III-binding tips of
the ESCRT-II complex into the center of the
pore (Figures 7A and 8), whereas the locus of
ESCRT-0 binding on ESCRT-I projects outside
the pore. Unlike the head-to-tail ring model, the
spoke mode is consistent with the sequential
function of ESCRT complexes as deduced
from yeast genetics (Babst et al., 2002a,
2002b; Katzmann et al., 2001) and with the
division of labor deduced from in vitro reconstitution (Wollert
and Hurley, 2010).
ESCRT-II is a Y-shaped complex that binds the Vps20 subunit
of ESCRT-III at the tips of its two Vps25 subunits (Hierro et al.,
2004; Im et al., 2009; Teo et al., 2004a). Biological function in
MVB biogenesis requires that both tips be functional (Hierro
et al., 2004; Teis et al., 2010), but it has not been clear why
two sites are needed instead of one. The spoke model
suggests that roughly 6–10 copies of ESCRT-II could fit in the
bud neck, which would template the initiation of twice that
number of short ESCRT-III filaments that drive membrane scission (Figure 7B). The number and density of the filaments
would be critical to provide enough binding affinity and induce
the correct geometry for membrane scission. The number and
density depend directly on the number of functional copies of
ESCRT-II Vps25 lining the pore. The loss of function in singleVps25 variants of ESCRT-II could thus be explained in terms of
a halving of the number of ESCRT-III filaments. This model can
be considered a variant of the dome model for ESCRT-III-mediated membrane scission (Fabrikant et al., 2009). Presumably,
Figure 6. Conformational Ensemble of the ESCRTI-II Supercomplex
All eighteen conformations of the supercomplex are
shown, with ESCRT-I in green and ESCRT-II in magenta.
Estimated populations obtained from the fitting weights
and the maximum intra-atomic distance Dmax are indicated.
880 Structure 20, 874–886, May 9, 2012 ª2012 Elsevier Ltd All rights reserved
Structure
Structure of the ESCRT-I-II Supercomplex
Figure 7. The ESCRT-I-II Supercomplex in the
Bud Neck
(A) Six copies of an experimentally observed solution
conformation of the ESCRT-I-II supercomplex were
manually docked to a 225 Å diameter catenoidal
membrane neck, subject to the restraint of symmetry
about the axis of the pore.
(B) This spoke model for ESCRT-I-II docking in the bud
neck predicts that ESCRT-III filaments will project into
the pore in a whorl arrangement.
these short filaments would arrange themselves into whorls
(Figure 7B) because other arrangements would leave large
gaps between the filaments. Thus, one specific prediction of
this model is that under appropriate conditions ESCRT-III whorls
form in cells.
DISCUSSION
The ESCRT-I-II supercomplex, and the broader ESCRT pathway, is typical of most of the cell’s regulatory machinery in
the way that it combines rigid and flexible elements. Although
abundant and critically important in cell physiology, these
systems have been intractable by conventional approaches.
They represent a blind spot in structural-level understanding
of cell function. Here, we have applied a series of techniques
that, taken separately, provide low-resolution global information (SAXS) or high-resolution information limited to specific
sites (DEER, smFRET). All of these methods are widely used
independent of one another and are sometimes used together
in a comparative and qualitative way. Here, we have integrated these methods in a joint structural refinement, in the
context of a system where all of the component crystal structures are known. We find that a self-consistent solution is
obtainable, despite the varied nature of the experimental
conditions. Strikingly, a large number of conformations are
required to fit the experimental data. We attribute this to the
high information density in the smFRET histograms in particular, which provide a direct readout of the full conformational
distribution for each domain pair tested. In a previous analysis
of ESCRT-I alone, an ensemble of two structures was sufficient to fit SAXS data alone, while an ensemble of six was
able to fit SAXS and DEER data together. In this study, refinement was carried out against SAXS, DEER, and smFRET data
simultaneously. As compared to refinement against SAXS and
DEER data sets, larger ensembles are needed principally to
represent the detail-rich smFRET histograms. Because the
robustness of this approach was unknown, and because the
experimental data are relatively sparse, cross-validation was
a key aspect of this study. We were encouraged by the excellent agreement between the observed and computed smFRET
histogram for an ESCRT-I dye pair excluded from the
refinement.
The alignment of the ESCRT-I-II supercomplex in the bud neck, in the context of
the spoke model, allows us to rationalize
the elongated and curved shapes of the
supercomplex and present a structural
model for a number of otherwise unexplained observations (Figure 8; Movie S1). The mechanism of cargo transfer from the
ESCRT-0 coat into the nascent bud has been mysterious (Raiborg and Stenmark, 2009; Wollert and Hurley, 2010). The
discovery that the ESCRT-I-II complex is in equilibrium between
open and closed states suggests a mechanism for cargo transfer. If ESCRT-I were to transition from the open to closed states
following release of ESCRT-0, this would drag cargo along the
membrane into the neck of the nascent bud. This model also
helps explain why the budding machinery evolved as such
a complicated system, with two separate complexes, both
having substantial intrinsic flexibility. A simpler and more rigid
machinery would not permit such a large conformational transition, and would so be unable to move cargo as far as 300 Å
along the membrane.
Since the ESCRT-I-II supercomplex is the main organizer of
the neck of the nascent ILV, the structural insights allowed us
to propose a model that integrates a decades’ worth of genetic,
biochemical, and structural data. The model plausibly accounts
for ESCRT-dependent cargo sorting, vesicle budding, and
vesicle scission (Figure 8; Movie S1). The model makes testable
predictions, including the existence of ESCRT-III whorls.
The refined ensemble of these flexible and partially disordered
protein complexes has led to mechanistic insights into one of
the most complex cellular processes to be addressed in this
level of detail. The multiexperiment analysis applied in this
study bypasses obstacles to conventional structural biology
approaches, which are unsuited to describing larger dynamic
complexes containing substantial intrinsic disorder. This
approach thus helps point a way forward for mechanistic and
structural dissection for the class of complex, dynamic, and flexible systems that carry out most cell processes.
EXPERIMENTAL PROCEDURES
Protein Expression and Purification
All four subunits of the Cys-free yeast ESCRT-I (Vps23C133A,C344A, Vps28C101A,
Vps37C123A, and Mvb12C48A/C54A/C61A) and double Cys mutants were co-expressed from the pST39 polycistronic vector (Boura et al., 2011). Vps23 was
expressed as a fusion protein with an N- terminal His6 tag followed by a TEV
cleavage site. The ESCRT-I protein complex was expressed in Escherichia
coli BL21 cells by induction with 0.3 mM isopropyl thiogalactoside (IPTG) at
an optical density of 0.8 and at 28 C overnight. The protein was affinity purified
Structure 20, 874–886, May 9, 2012 ª2012 Elsevier Ltd All rights reserved 881
Structure
Structure of the ESCRT-I-II Supercomplex
Figure 8. A Speculative Model for Cargo Sorting, Membrane Budding, and Membrane Scission by ESCRTs in MVB Biogenesis
The model considers what the broader implications would be if the crescent-shaped ESCRT-I-II supercomplexes were to be aligned in the bud neck as shown in
Figure 7A. The entire process of ESCRT-mediated MVB biogenesis, from start to finish, is considered with respect to this geometry.
(A) ESCRT-0 complexes begin to cluster ubiquitinated (yellow dots) cargo (orange shapes below the translucent membrane, per their ability to cluster membranetethered ubiquitin in vitro (Wollert and Hurley, 2010) and to form flat clathrin coats in cells (Raiborg et al., 2001; Sachse et al., 2002).
(B) ESCRT-0 recruits ESCRT-I (Bache et al., 2003; Bilodeau et al., 2003; Katzmann et al., 2003; Lu et al., 2003; Pornillos et al., 2003), with a stoichiometry that is
probably 1:1 in humans (Im et al., 2010) and 1:1 or 1:2 in yeast (Ren and Hurley, 2011).
(C) ESCRT-I recruits ESCRT-II with 1:1 stoichiometry (Gill et al., 2007).
(D and E) The ESCRT-I-II supercomplex is the principal entity that stabilizes the initial membrane bud (Wollert and Hurley, 2010).
882 Structure 20, 874–886, May 9, 2012 ª2012 Elsevier Ltd All rights reserved
Structure
Structure of the ESCRT-I-II Supercomplex
using Ni-NTA resin (Qiagen, Venlo, The Netherlands) in accordance with the
manufacturer’s instructions. Immediately upon elution, the buffer was
supplemented with 1 mM ethylenediaminetetraacetic acid (EDTA) and
cleaved by TEV protease overnight. The complex was further purified by
size-exclusion chromatography on a Superdex 200 column (GE Healthcare,
Waukesha, WI, USA).
To prepare the pseudo-Cys-free yeast ESCRT-II complex, all Cys
that are not Zn ligands in the NZF1 or NZF2 domains, were replaced by Ala,
leading to the following constructs: Vps22C68A, C96A, C103A, C146A, C176A,
Vps25C38A,C103A,C176A,C181A, and Vps36C161A,C464A,C486A,C539A. Artificial
codon-optimized genes bearing these mutations were purchased from
GenScript (Piscataway, NJ). The Vps25 and Vps36 subunits were cloned
into the pST39 vector (Tan, 2001), and Vps22 subunits were cloned into the
pRSFD vector. At the 50 end of the VPS36 gene, a sequence encoding an
N- terminal His6 tag was inserted, followed by a TEV cleavage site. Double
Cys mutants were prepared using the QuikChange kit (Stratgene, La Jolla,
CA, USA) or by overlapping PCR. Yeast ESCRT-II complex wild-type and
mutants were expressed and purified as described (Wollert and Hurley,
2010). The ESCRT-II complex was expressed in E. coli BL21 cells in medium
supplemented with 10 mM ZnCl2 upon induction with 0.2 mM IPTG at optical
density 0.8 at 18 C overnight. Affinity purification on Ni-NTA resin was followed
by TEV cleavage overnight. The complex was further purified by size-exclusion
chromatography on a Superdex 200 column (GE Healthcare). Proteins were
concentrated to 5 mg/ml, flash frozen in liquid nitrogen, and stored at
80 C until use.
to measurement, samples were dialyzed overnight at 4 C against 50 mM
Tris (pH 7.4), 150 mM NaCl, 3 mM b-mercaptoethanol, 1% glycerol, and
0.18% ascorbic acid. Data were collected for a concentration series from
5 to 0.5 mg/ml at SSRL beamline BL4-2. Data reduction and analysis were
performed using the beamline software SAStool.
Labeling by MTSL and Fluorescent Dyes
For spin labeling, the double Cys mutants were incubated overnight at 4 C
with 5 mM MTSL. For fluorescent dye labeling, the ESCRT-I complex double
Cys mutant was incubated overnight at 4 C with 5-fold molar excess of
Alexa488 and Alexa594 maleimide. To label yeast ESCRT-II, the double
Cys mutants were incubated overnight at 4 C with a 2-fold molar excess
of Alexa488 and Alexa594 maleimide. Unreacted dyes were removed by
size-exclusion chromatography on a Superdex 25 column (GE Healthcare).
The labeling efficiency for MTSL-labeled proteins was 100% because we
could not detect any unlabeled material using mass spectroscopy. An
average of 1 Alexa dye per Cys was estimated spectroscopically with
a donor to acceptor (Alexa488 to Alexa594) ratio of 1:2. Labeled proteins
were concentrated to 30 mM for MTSL-labeled proteins and to 1 mM for
fluorescently labeled proteins, flash frozen in liquid nitrogen, and stored at
80 C until use.
Single-Molecule FRET Experiments
SmFRET experiments were performed using a confocal microscope system
(MicroTime200, PicoQuant, Berlin, Germany). The donor fluorophore (Alexa
Fluor 488) was excited by a linearly polarized dual mode (CW/pulsed)
485 nm diode laser (LDH-D-C-485, PicoQuant) in the CW mode at 45 mW
through an oil-immersion objective (Plan Apo, NA 1.4, 1003, Olympus,
Shinjuku, Tokyo, Japan). Donor and acceptor (Alexa Fluor 594) fluorescence,
emitted from molecules freely diffusing through the illuminated volume,
were collected by the same objective, divided into two channels, and
focused through optical filters onto single-photon avalanche diodes (SPAD;
PerkinElmer Optoelectronics SPCM-AQR-15, Waltham, MA, USa). The
labeled proteins were diluted to 40 pM for ESCRT-I complex and to 160 pM
for ESCRT-II complex into a 20 mM Tris buffer (pH 7.4) with 100 mM NaCl
and 3 mM b-mercaptoethanol with bovine serum albumin (1 mg/ml).
The measurement for ESCRT-II was performed at a higher concentration
(160 pM) to compensate for molecules lost because of adsorption on
the surface of the glass coverslip. However, from the comparison of the
frequency of fluorescence bursts between ESCRT-I and ESCRT-II, the effective concentration of ESCRT-II was estimated to be below 40 pM. An analysis
described in Gopich (2008) shows that it is very improbable to have two
molecules simultaneously in the illuminated volume in both ESCRT-I and
ESCRT-II experiments. In case of experiments done on the ESCRT-I/II
Small-Angle X-Ray Scattering
To avoid possible cysteine-mediated aggregation, the no-cysteine-residue
mutant of ESCRT-I complex (Mvb12C48A/C54A/C61A, Vps37C123A, Vps28C101A,
and Vps23C133A,C344A) and the no-nonessential-cysteine residue ESCRT-II
complex (Vps22C68A, C96A, C103A, C146A, C176A, Vps25C38A,C103A,C176A,C181A,
and Vps36C161A,C464A,C486A,C539A) were used for SAXS data collection. Prior
DEER Experiments
Pulse-EPR measurements were performed on 25–30 ml of sample loaded
into quartz capillaries with 2.00 mm i.d. by 2.40 mm o.d. (Fiber Optic Center,
Inc., New Bedford, MA, USA). The protein concentration of ESCRT-I mutants
with two nitroxide spin labels attached was 20–50 mM in a 20 mM Tris,
100 mM NaCl, (pH 7.4), and 10% w/v glycerol buffer, and the ESCRT-I:
ESCRT-II protein molar ratio was 1:1. Prior to insertion into the instrument,
the sample-containing capillaries were flash frozen in a dry ice/ isopropanol
bath. The DEER data were recorded at 50 K or 80 K on a Bruker ElexsysE580 spectrometer fitted with an ER4118X-MS3 split-ring resonator
(Bruker Biospin, Billerica, MA, USA) at X-band frequency. Data were acquired
using a four-pulse DEER sequence (Pannier et al., 2000) with a 16 ns p/2
and two 32 ns p observe pulses separated by a 28 ns p pump pulse. The
dipolar evolution times were typically 2.0–2.5 ms. The pump frequency was
set to the center maximum of the nitroxide spectrum, and the observer
frequency was set to the low field maximum, typically 65–70 MHz higher.
The phase-corrected dipolar evolution data were processed assuming a
three-dimensional background using the DeerAnalysis2009 package (Jeschke
et al., 2006).
(F) The same UEV domain of ESCRT-I binds to both ESCRT-0 and ubiquitin, suggesting the ubiquitin tags need only travel a short distance in the plane of the
membrane to be handed off to ESCRT-I.
(G) The observation that the ESCRT-I-II supercomplex is in an equilibrium between open and closed conformations suggests a mechanism for ubiquitinated
cargo transfer from the limiting membrane into the bud neck via an open-to-closed conformational change in the supercomplex.
(H) ESCRT-II initiates the assembly of ESCRT-III by binding to and activating Vps20 monomers. Two Vps20 monomers bind to each ESCRT-II complex (Im et al.,
2009; Teo et al., 2004a), and both binding events are essential for function (Hierro et al., 2004; Teis et al., 2010).
(I) ESCRT-III recruits deubiquitinating enzymes (not shown; McCullough et al., 2004; Richter et al., 2007), which replenish the pool of cytosolic ubiquitin monomers
and may also help disengage cargo from ESCRT-I and -II.
(J) ESCRT-III filaments associate laterally with one another (Hanson et al., 2008; Lata et al., 2008).
(K and L) Vps4 is not essential for membrane scission in a minimal in vitro system but has been shown by live-cell imaging of cytokinesis (Elia et al., 2011) and
HIV-1 budding (Baumgärtel et al., 2011; Jouvenet et al., 2011) to engage with ESCRT-III prior to membrane scission. Multiple Vps4 dodecamers appears to be
recruited, and (L) their assembly onto the ESCRT-III lattice has been modeled in terms of a two-dimensional hexagonal lattice (Yang and Hurley, 2010).
(M–O) ESCRT-III filament self-association, perhaps promoted by the polyvalent binding of Vps4 and its ESCRT-III binding cofactor, Vta1, deform the membrane
into a dome-shaped scission intermediate (Fabrikant et al., 2009).
(P and Q) ESCRT-I and -II dissociate at least partially in advance of ATP hydrolysis by Vps4, whereas the dissociation and recycling of ESCRT-III depends strictly
on ATP hydrolysis.
See also Movie S1.
Structure 20, 874–886, May 9, 2012 ª2012 Elsevier Ltd All rights reserved 883
Structure
Structure of the ESCRT-I-II Supercomplex
supercomplex, the labeled protein was mixed with the unlabeled binding
partner proteins at 1 mM concentration. Other experimental details can be
found in Chung et al. (2009).
from SAXS and DEER data were quantified by c2SAXS and c2DEER as in (Boura
et al., 2011). The free energy function,
X
X
c2DEERði;jÞ +
c2FRETði;jÞ + mN;
G = c2SAXS +
ði;jÞ
Single-Molecule FRET Histogram
The apparent FRET efficiencies, hEappi = nA / (nA + nD), calculated from the
number of donor (nD) and acceptor (nA) photons in 2 ms bins containing
more than 90 photons were, after correcting for background and donor
leakage into the acceptor channel, converted to true FRET efficiencies, hEi =
nA / (nA + g nD), where the correction factor g accounts for differences in the
detection efficiencies of the donor and acceptor channels and differences in
the quantum yields of the donor and acceptor dyes. g = 2 was determined
from measurements of donor lifetimes in the presence (tDA) and absence
(tD) of acceptor, which yield the true FRET efficiencies from hEi = 1 – tDA/tD
(Merchant et al., 2007). In the FRET efficiency histograms, the number of
counts at each value of the FRET efficiency represents the number of 2 ms
bins containing more than 90 photons.
ði;jÞ
measures the deviation from the different experiments, giving equal weight
to each measurement. The last term in G is a penalty for the size, N, of the
ensemble that is controlled by a parameter m. To obtain a minimal ensemble
of structures that capture the different experiments, G was minimized numerically with respect to the number, N, of structures and their relative weights, wk.
Starting from a small value, m was increased until all experiments could be fit
simultaneously within the respective statistical uncertainties, c2 z1.
SUPPLEMENTAL INFORMATION
Supplemental Information includes four figures and one movie and can be
found with this article online at doi:10.1016/j.str.2012.03.008.
ACKNOWLEDGMENTS
Ensemble Refinement
In the ensemble refinement, the number, N, of structures in the ensemble and
the weights, wk, assigned to individual structures were varied. To avoid introducing regularization-dependent artifacts into the refinement procedure, the
simulation structures were refined directly against the measured data. The
ensemble-averaged SAXS intensity was calculated from model coordinates
_
exactly as in the original EROS method (Rózycki
et al., 2011). The background-corrected, ensemble-averaged DEER dipolar evolution functions
were calculated exactly as in our recent study on ESCRT-I (Boura et al.,
2011) using the rotamer library of spin-labeled Cys (Polyhach et al., 2011).
Also the single-molecule FRET efficiency histograms were calculated as previously (Boura et al., 2011). Briefly, possible locations of the fluorescence dyes
were sampled according to a distance distribution that was previously
obtained from molecular simulations (Merchant et al., 2007). Dye positions
that overlapped with the proteins or with the other dye were excluded from
the analysis, with a distance cutoff of 6 Å. All orientations of the donor and
acceptor transition dipoles were assumed to be equally probable. The mean
FRET efficiency for a dye pair attached to residues i and j in structure k was
calculated as
Ek;ði;jÞ =
1+
rab
R0
6 1 ;
c2FRETði;jÞ = Nb
m=1
s2ði;jÞ ðEm Þ
Received: February 16, 2012
Revised: March 19, 2012
Accepted: March 19, 2012
Published online: May 3, 2012
REFERENCES
where R0 = 54 Å is the Förster radius, and the average is performed over all
positions a and b of the dyes attached to sites i and j, respectively. Using these
mean efficiencies, Ek,(i,j), and the weights, wk, assigned to structures in the
course of refinement, the FRET efficiency histograms were constructed for
all dye pairs (i,j) by the recoloring method (Gopich and Szabo, 2007) according
to the distributions in the number of photons in the individual bursts measured
in experiment. The deviations between the measured and calculated FRET
data for any residue pair (i,j) were quantified by
2
Nb
Nði;jÞ ðEm Þ Nobs
X
ði;jÞ ðEm Þ
1
We thank E. Tyler for generating Figure 8 and Movie S1. SAXS data were
collected at the Stanford Synchrotron Radiation Laboratory, 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, and by the National Institutes of Health
(NIH), National Center for Research Resources, Biomedical Technology
Program. B.R. was supported by a Marie Curie International Outgoing Fellowship within the 7th European Community Framework Programme. E.B. was
supported by an Intramural AIDS Research Fellowship. This work was supported by the Intramural Program of the NIH, National Institute of Diabetes
and Digestive and Kidney Diseases (G.H., J.H.H., and W.A.E.), the Intramural
AIDS-Targeted Antiviral Program of the Office of the Director, NIH (J.H.H.), and
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;
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