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

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.

[1]  Bethan McDonald,et al.  No strings attached: the ESCRT machinery in viral budding and cytokinesis , 2009, Journal of Cell Science.

[2]  J. Hurley,et al.  Molecular Mechanism of Multivesicular Body Biogenesis by ESCRT Complexes , 2010, Nature.

[3]  J. Bonifacino,et al.  Crystallographic and functional analysis of the ESCRT-I /HIV-1 Gag PTAP interaction. , 2010, Structure.

[4]  Peter V. Konarev,et al.  ATSAS 2.1 – towards automated and web-supported small-angle scattering data analysis , 2007 .

[5]  Gunnar Jeschke,et al.  Rotamer libraries of spin labelled cysteines for protein studies. , 2011, Physical chemistry chemical physics : PCCP.

[6]  W. Weissenhorn,et al.  The Crystal Structure of the C‐Terminal Domain of Vps28 Reveals a Conserved Surface Required for Vps20 Recruitment , 2006, Traffic.

[7]  D. Cafiso,et al.  Recent advances and applications of site-directed spin labeling. , 2006, Current opinion in structural biology.

[8]  A. Szabó,et al.  Theory of photon statistics in single-molecule Förster resonance energy transfer. , 2005, The Journal of chemical physics.

[9]  T. Obsil,et al.  Both the N-terminal Loop and Wing W2 of the Forkhead Domain of Transcription Factor Foxo4 Are Important for DNA Binding* , 2007, Journal of Biological Chemistry.

[10]  B. Schuler,et al.  Unfolded protein and peptide dynamics investigated with single-molecule FRET and correlation spectroscopy from picoseconds to seconds. , 2008, The journal of physical chemistry. B.

[11]  S. Gygi,et al.  Human ESCRT and ALIX proteins interact with proteins of the midbody and function in cytokinesis , 2007, The EMBO journal.

[12]  P. Bieniasz,et al.  HIV-1 and Ebola virus encode small peptide motifs that recruit Tsg101 to sites of particle assembly to facilitate egress , 2001, Nature Medicine.

[13]  Irina V Gopich.  Concentration effects in "single-molecule" spectroscopy. , 2008, The journal of physical chemistry. B.

[14]  R. Piper,et al.  ESCRT ubiquitin-binding domains function cooperatively during MVB cargo sorting , 2009, The Journal of cell biology.

[15]  Roger L. Williams,et al.  Structural Insights into Endosomal Sorting Complex Required for Transport (ESCRT-I) Recognition of Ubiquitinated Proteins* , 2004, Journal of Biological Chemistry.

[16]  W. Sundquist,et al.  Ubiquitin recognition by the human TSG101 protein. , 2004, Molecular cell.

[17]  L. Verplank,et al.  Tsg101, a homologue of ubiquitin-conjugating (E2) enzymes, binds the L domain in HIV type 1 Pr55Gag , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[18]  Gerhard Hummer,et al.  Coarse-grained models for simulations of multiprotein complexes: application to ubiquitin binding. , 2008, Journal of Molecular Biology.

[19]  Wesley I. Sundquist,et al.  Structure of the Tsg101 UEV domain in complex with the PTAP motif of the HIV-1 p6 protein , 2002, Nature Structural Biology.

[20]  J. Martin-Serrano,et al.  Parallels Between Cytokinesis and Retroviral Budding: A Role for the ESCRT Machinery , 2007, Science.

[21]  P. Heřman,et al.  Maximum Entropy Analysis of Analytically Simulated Complex Fluorescence Decays , 2011, Journal of Fluorescence.

[22]  Jill Trewhella,et al.  Refined solution structure of the 82-kDa enzyme malate synthase G from joint NMR and synchrotron SAXS restraints , 2008, Journal of biomolecular NMR.

[23]  W. Eaton,et al.  Characterizing the unfolded states of proteins using single-molecule FRET spectroscopy and molecular simulations , 2007, Proceedings of the National Academy of Sciences.

[24]  S. Emr,et al.  ESCRT-I Core and ESCRT-II GLUE Domain Structures Reveal Role for GLUE in Linking to ESCRT-I and Membranes , 2006, Cell.

[25]  Dmitri I. Svergun,et al.  Uniqueness of ab initio shape determination in small-angle scattering , 2003 .

[26]  B. Schuler,et al.  Protein dynamics from single-molecule fluorescence intensity correlation functions. , 2009, The Journal of chemical physics.

[27]  D I Svergun,et al.  Restoring low resolution structure of biological macromolecules from solution scattering using simulated annealing. , 1999, Biophysical journal.

[28]  William A Eaton,et al.  Experimental determination of upper bound for transition path times in protein folding from single-molecule photon-by-photon trajectories , 2009, Proceedings of the National Academy of Sciences.

[29]  J. Hurley,et al.  Membrane budding and scission by the ESCRT machinery: it's all in the neck , 2010, Nature Reviews Molecular Cell Biology.

[30]  John A. Tainer,et al.  X-ray solution scattering (SAXS) combined with crystallography and computation: defining accurate macromolecular structures, conformations and assemblies in solution , 2007, Quarterly Reviews of Biophysics.

[31]  J. Hurley,et al.  Molecular Architecture and Functional Model of the Complete Yeast ESCRT-I Heterotetramer , 2007, Cell.

[32]  H. Zimmermann,et al.  DeerAnalysis2006—a comprehensive software package for analyzing pulsed ELDOR data , 2006 .

[33]  G. Hummer,et al.  SAXS ensemble refinement of ESCRT-III CHMP3 conformational transitions. , 2011, Structure.

[34]  S. Emr,et al.  Ubiquitin-Dependent Sorting into the Multivesicular Body Pathway Requires the Function of a Conserved Endosomal Protein Sorting Complex, ESCRT-I , 2001, Cell.

[35]  Andreas Volkmer,et al.  Identification of Single Molecules in Aqueous Solution by Time-Resolved Fluorescence Anisotropy , 1999 .

[36]  Dmitri I. Svergun,et al.  Determination of the regularization parameter in indirect-transform methods using perceptual criteria , 1992 .

[37]  G. Jeschke,et al.  Dead-time free measurement of dipole-dipole interactions between electron spins. , 2000, Journal of magnetic resonance.

[38]  Harald Stenmark,et al.  The ESCRT machinery in endosomal sorting of ubiquitylated membrane proteins , 2009, Nature.

[39]  S. Emr,et al.  Structural insight into the ESCRT‐I/‐II link and its role in MVB trafficking , 2007, The EMBO journal.

[40]  A. Szabó,et al.  Decoding the pattern of photon colors in single-molecule FRET. , 2009, The journal of physical chemistry. B.

[41]  E. Freed,et al.  Overexpression of the N-terminal domain of TSG101 inhibits HIV-1 budding by blocking late domain function , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[42]  Axel T Brunger,et al.  Three-dimensional molecular modeling with single molecule FRET. , 2011, Journal of structural biology.

[43]  Wesley I. Sundquist,et al.  Tsg101 and the Vacuolar Protein Sorting Pathway Are Essential for HIV-1 Budding , 2001, Cell.

[44]  H. Masuhara,et al.  Time-Dependent Fluorescence Depolarization Analysis in Three-Dimensional Microspectroscopy , 1995 .

[45]  Matthew West,et al.  Did2 coordinates Vps4-mediated dissociation of ESCRT-III from endosomes , 2006, The Journal of cell biology.

[46]  Stanley N Cohen,et al.  Inhibition of HIV budding by a genetically selected cyclic peptide targeting the Gag-TSG101 interaction. , 2008, ACS chemical biology.

[47]  A. Szabó,et al.  FRET efficiency distributions of multistate single molecules. , 2010, The journal of physical chemistry. B.

[48]  J. Hurley,et al.  Structural and Functional Organization of the ESCRT-I Trafficking Complex , 2006, Cell.

[49]  Natalie Elia,et al.  Midbody Targeting of the ESCRT Machinery by a Noncanonical Coiled Coil in CEP55 , 2008, Science.

[50]  William A Eaton,et al.  Extracting rate coefficients from single-molecule photon trajectories and FRET efficiency histograms for a fast-folding protein. , 2011, The journal of physical chemistry. A.