to CR9114 (table S7) (7). The other Ile45Phe escape variant is naturally present in essentially all human H2N2 viruses that circulated from 1957 to 1968, and Phe45Ile substitution in a human H2 HA restores high affinity binding for both CR6261 and CR9114 (table S8). In contrast, Phe 45 occurs in only 3 of 8550 sequences from avian H2N2 isolates and the remaining 15 subtypes. 31. J. C.Krause et al., J. Virol.85,10905 (2011). 32. J. R.Whittle et al., Proc.Natl.Acad. Sci.U.S.A. 108, 14216 (2011). 33. Why influenza B viruses are not neutralized in the same way by CR9114 remains currently unknown. 34. In contrast, polyclonal sheep sera directed against B/Florida/4/2006 and B/Malaysia/2506/2004 did prevent initial infection, but not of virus from the opposite lineage. 35. L. V. Gubareva, L. Kaiser, F. G. Hayden, Lancet 355, 827 (2000). 36. Y. Bao et al., J. Virol.82,596 (2008). Acknowledgments: We thank J. Juraszek; B.Brandenburg; M.Koldijk; K. Hegmans; J. Meijer; N. Hafkemeier; A.Apetri; J. Dulos; L. Dekking; H. Vietsch; G. Perdok; H. Tien; D. Marciano; the staff of the Advanced Photon Source (APS) GM/CA-CAT 23ID-B and 23ID-D, Stanford Synchrotron Radiation Lightsource (SSRL) BL11-1 and SSRL BL7-1, and Advanced Light Source (ALS); X. Dai; R. Stanfield; W. Yu; J. P. Julien; R. N. Kirchdoerfer; and E. Brown of Ottawa University, Canada, for the mouse-adapted A/Hong Kong/1/68 strain. Crystallization experiments were carried out on the Rigaku Crystalmation system at the Joint Center for Structural Genomics, which is supported by the National Institute of General Medical Sciences (NIGMS) Protein Structure Initiative U54 GM094586. The EM and image analysis was conducted by R.K., J.H.L., and Z.M.at the National Resource for Automated Molecular Microscopy, which is supported by NIH through the National Center for Research Resources’ P41 program (RR017573). This project has been funded in part by the Area of Excellence Scheme of the University Grants Committee, Hong Kong (grant AoE/M-12/06); a predoctoral fellowship from the Achievement Rewards for College Scientists Foundation (D.C.E.); grant GM080209 from the NIH Molecular Evolution Training Program (D.C.E.); a Saper Aude Postdoc grant from the Danish Council for Independent Research, Natural Sciences (N.S.L.), and the Skaggs Institute (I.A.W.). Portions of this research were carried out at the SSRL, a national user facility operated by Stanford University on behalf of the U.S. Department of Energy (DOE), Office of Basic Energy Sciences. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research and by NIH, National Center for Research Resources, Biomedical Technology Program (P41RR001209), and the NIGMS. The GM/CA CAT 23-ID-B beamline has been funded in whole or in part with federal funds from National Cancer Institute (Y1-CO-1020) and NIGMS (Y1-GM-1104). Use of the APS was supported by the DOE, Basic Energy Sciences, Office of Science, under contract no. DE-AC02-06CH11357. The content is solely the responsibility of the authors and does not necessarily represent the official views of NIGMS or the NIH.The ALS is supported by the director, Office of Science, Office of Basic Energy Sciences, of the DOE under contract no.DE-AC02-05CH11231. Coordinates and structure factors are deposited in the Protein Data Bank (PDB code 4FQH for CR9114 Fab, 4FQI for CR9114 Fab-A/H5 HA, 4FQJ for CR8071 Fab-B/Florida HA1, 4FQK for CR8059 Fab-B/Brisbane HA, 4FQL for CR8033 Fab, 4FQM for B/Brisbane HA, 4FQY for CR9114 Fab-A/H3 HA, and 4FQV for CR9114 Fab-A/H7 HA). Image reconstructions have been deposited at the EMData under accession numbers EMD-2143 (CR8033/Florida), EMD-2144 (CR8071/Florida), EMD-2145 (CR8071/Malaysia), EMD-2146 (CR9114/Florida), EMD-2147 (CR9114/H1), EMD-2148 (CR9114/H3), EMD-2149 (CR9114/H9), and EMD-2150 (CR9114/H7). Nucleotide sequences for the CR8033, CR8059 (CR8071), and CR9114 variable regions have been deposited in GenBank [accession numbers JX213635 (CR8033, V H ), JX213636 (CR8033, V k ), JX213637 (CR8059, V H ), JX213638 (CR8059, V l ), JX213639 (CR9114, V H ), and JX213640 (CR9114, V l )]. Patent applications relating to antibodies CR8033, CR8059, CR8071, and CR9114 have been filed (CR8033, CR8059, and CR8071 are claimed in EP 12158525.1 and U.S. 61/608,414. CR9114 is claimed in EP 11173953.8 and U.S. 61/572,417). Sharing will be subje to standard material transfer agreements. This is publication 21741 from the Scripps Research Institute. Supplementary Materials www.sciencemag.org/cgi/content/full/science.1222908/DC Materials and Methods Figs.S1 to S18 Tables S1 to S8 References (37–62) 4 April 2012;accepted 9 July 2012 Published online 9 August 2012; 10.1126/science.1222908 Structural Probing of a Protein Phosphatase 2A Network by Chemical Cross-Linking and Mass Spectrometry Franz Herzog, 1 * Abdullah Kahraman, 1 * Daniel Boehringer, 2 * Raymond Mak, 1 Andreas Bracher, 4 Thomas Walzthoeni, 1 Alexander Leitner, 1 Martin Beck, 3 Franz-Ulrich Hartl, 4 Nenad Ban, 2 Lars Malmström, 1 Ruedi Aebersold 1 † The identification of proximate amino acids by chemical cross-linking and mass spectrometry (XL-MS) facilitates the structural analysis of homogeneous protein complexes. We gained distance restraints on a modular interaction network of protein complexes affinity-purified from human cells by applying an adapted XL-MS protocol. Systematic analysis of human protein phosphatase 2A (PP2A) complexes identified 176 interprotein and 570 intraprotein cross-links that link specific trimeric PP2A complexes to a multitude of adaptor proteins that control their cellular functions. Spatial restraints guided molecular modeling of the binding interface between immunoglobulin binding protein 1 (IGBP1) and PP2A and revealed the topology of TCP1 ring complex (TRiC) chaperonin interacting with the PP2A regulatory subunit 2ABG. This study establishes XL-MS as an integral part of hybrid structural biology approaches for the analysis of endogenous protein complexes. C ellular processes can rarely be attributed to the activity of a single protein. Instead, proteins act in functional modules, such as macromolecular complexes or signal transduction networks. Gaining mechanistic insights into the function of multisubunit modules is facilitated by structural elucidation (1, 2). Chemical cross-linking, in combination with mass spectrometry (XL-MS), is a low-resolution structural technique for the char- acterization of the architecture of protein complexes (3,4).XL-MS provides distance information by identifying spatially proximate lysine residues that have been covalently linked by a bifunctional cross- linking reagent. This approach has been successful- ly applied to homogenous protein complexes purified for crystallographic purposes (5–8). In contrast to high-resolution structural biology techniques, XL-MS has the potential to acquire spatial restraints from heterogeneous protein samples. We thus ap- plied this method to characterize the topolog signaling network by systematic analysis of en- dogenous protein complexes (Fig. 1). The abundant protein phosphatase 2A (PP is involved in diverse signaling pathways reg cell growth, apoptosis, differentiation, cell mo DNA damage response, and cell cycle progre (9–12). In eukaryotes, active PP2A holoenzymes are heterotrimers composed of a catalytic su (C, human isoforms PP2AA and PP2AB), a sca fold subunit (A, human isoforms 2AAA and 2A and one of a large array of regulatory subuni B', BW, and BW', each subfamily consisting o five isoforms) (Fig. 2A and table S1) that sele ly target PP2A subpopulations to specific sub (13). To reveal the topology of PP2A holoenzy in complex with the adaptor proteins that reg their phosphatase activity and subcellular loc tion,we purified these complexes from human cells and analyzed their topology by combini XL-MS and computational molecular modelin We first determined the proteins copurifyin with trimeric PP2A holoenzymes by isolating proteins, previously detected in PP2A comple (14, 15), through an N-terminal His 6 -HA-StrepII- tag from human embryonic kidney cells (fig. and table S2). The associated protein comple were identified by mass spectrometry (Fig. 1 resulting in a dense interaction network of 94 proteins (Fig. 2A and tables S1 and S3). To elucidate the topology of the PP2A net- work, we probed the complexes by XL-MS. Cr linking was achieved by immobilizing the PP2 complexes on beads (16). Incubation with the propriate concentration of disuccinimidyl sub (DSS) (fig. S2) and subsequent proteolysis re in a complex mixture of tryptic peptides that 1 Department of Biology, Institute of Molecular Systems Biology, Eidgenössische Technische Hochschule Zürich, Wolfgang-Pauli Strasse 16, 8093 Zurich, Switzerland. 2 Department of Biology, Institute of Molecular Biology and Biophysics, Eidgenössische Technische Hochschule Zürich, Schafmattstrasse 20, 8093 Zu- rich,Switzerland. 3 European Molecular Biology Laboratory, Meyerhofstraße 1, 69117 Heidelberg, Germany. 4 Department of Cellular Biochemistry, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany. *These authors contributed equally to this work. †To whom correspondence should be addressed. E-mail: aebersold@imsb.biol.ethz.ch 14 SEPTEMBER 2012 VOL 337 SCIENCE www.sciencemag.org 1348 REPORTS on September 13, 2012 www.sciencemag.org Downloaded from