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. 2011 Jun 7;108(23):9437-42.
doi: 10.1073/pnas.1101763108. Epub 2011 May 19.

Solution structure of the ESCRT-I complex by small-angle X-ray scattering, EPR, and FRET spectroscopy

Evzen Boura  1 , Bartosz Rózycki, Dawn Z Herrick, Hoi Sung Chung, Jaroslav Vecer, William A Eaton, David S Cafiso, Gerhard Hummer, James H Hurley
Affiliations

Solution structure of the ESCRT-I complex by small-angle X-ray scattering, EPR, and FRET spectroscopy

Evzen Boura et al. Proc Natl Acad Sci U S A. .

Abstract

ESCRT-I is required for the sorting of integral membrane proteins to the lysosome, or vacuole in yeast, for cytokinesis in animal cells, and for the budding of HIV-1 from human macrophages and T lymphocytes. ESCRT-I is a heterotetramer of Vps23, Vps28, Vps37, and Mvb12. The crystal structures of the core complex and the ubiquitin E2 variant and Vps28 C-terminal domains have been determined, but internal flexibility has prevented crystallization of intact ESCRT-I. Here we have characterized the structure of ESCRT-I in solution by simultaneous structural refinement against small-angle X-ray scattering and double electron-electron resonance spectroscopy of spin-labeled complexes. An ensemble of at least six structures, comprising an equally populated mixture of closed and open conformations, was necessary to fit all of the data. This structural ensemble was cross-validated against single-molecule FRET spectroscopy, which suggested the presence of a continuum of open states. ESCRT-I in solution thus appears to consist of an approximately 50% population of one or a few related closed conformations, with the other 50% populating a continuum of open conformations. These conformations provide reference points for the structural pathway by which ESCRT-I induces membrane buds.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Structural domains and labeling sites. (A) Domain schematic of the four subunits of ESCRT-I, colored blue (Vps23), red (Vps28), gray (Vps37), and orange (Mvb12) showing a speculative model for their orientation relative to the neck of a membrane bud, adapted from ref. . (B) UEV domain of Vps23. (C) CTD of Vps28. (D) Core assembly. (E) NTH of Vps37. Engineered Cys residues are shown in a filled sphere model.
Fig. 2.
Fig. 2.
SAXS of ESCRT-I. (A) Fit of the simulated scattering curves [I(q)] to the observed scattering of ESCRT-I. (B) Kratky plot of the same experimental data and simulated scattering shown in A. Experimental I(q) data points in A and B represent the mean of 10 consecutive measurements of the same sample, and the error bars represent the standard error of the mean. (C) Pair distribution function [P(r)] for ESCRT-I. (D) An ab initio molecular envelope for ESCRT-I. (E) Superposition of the two EROS-refined structures of ESCRT-I. (F) Fit of experimental SAXS data of A to values computed from the structures fit to both SAXS and DEER data.
Fig. 3.
Fig. 3.
DEER of ESCRT-I. (Left) Experimentally observed (solid) modulation of V(t) together with points calculated from six structural clusters. (Right) Histograms of MTSL distances computed from the models. The green curves in the top panels show the results from Gaussian regularization.
Fig. 4.
Fig. 4.
Solution structure of ESCRT-I. Structures are shown with their relative weights obtained from fitting, indicated as percentages. Subunits are colored as in Fig. 1.
Fig. 5.
Fig. 5.
Single-molecule FRET. Comparison of experimental (light brown wide bars) and calculated (black narrow bars) FRET efficiency histograms, using the interdye distances for the model structures, for a threshold number of photons (nT) of (A) 50 and (B) 110 with a bin time (Tbin) of 2 ms. In the experimental histogram, the FRET efficiency of each bin was γ-corrected after subtraction of the average background photons (donor: 0.6 ms-1; acceptor: 1.1 ms-1) and the correction for leak of donor fluorescence into the acceptor channel (6%). The fluorescence bursts from the donor-only-labeled molecules (E < 0.18) were excluded and the data were prefiltered to remove bursts of photons in which either blinking or bleaching occurred. (C) Inferred model for an open state of ESCRT-I (Top) consisting of a continuum of conformations, in equilibrium with a closed state (Bottom) consisting of one or a few related conformations.
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