Structural Analysis of Multiprotein Complexes by Cross-linking, Mass Spectrometry, and Database Searching

Maiolica, Alessio; Cittaro, Davide; Borsotti, Dario; Sennels, Lau; Ciferri, Claudio; Tarricone, C.; Musacchio, Andrea; Rappsilber, Juri

doi:10.1074/mcp.m700274-mcp200

Cited by 228 publications

(306 citation statements)

References 40 publications

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“…Thus, unlike NMR, de novo structure determination is not possible and external structural information is required for a meaningful model. Previous cross-linking studies inherently suffered from lack of high-resolution knowledge on the subunit structures (25,32), their interfacing modes (23)(24)(25), overall shape of the complex (24,25,33) or combinations of these factors. In the TRiC system all these factors are known to a much higher resolution than the maximum cross-linking length (approximately 28Ǻ).…”

Section: Discussionmentioning

confidence: 99%

“…At the same time, the complexity and size of the investigated protein complexes increased (23,24), with recent studies probing the 600 kDa PolIIF transcription system (25) and the intact proteasome (26). The combination of the technique with particle reconstruction by cryo-EM appears to be especially powerful because the cross-link data is helpful in assigning specific subunits to unassigned electron density.…”

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confidence: 99%

“…For simpler systems involving cross-linking of a pair of proteins, multiple docking models were derived and then filtered by the cross-link sets (23,27). However, studies of more complex systems with multiple proteins only reported a single model in very coarse terms (24,25). The latter approach is understandable given the great computational complexity of modeling such large systems but clearly has the disadvantage of not allowing one to gauge the confidence in the reported model compared to alternative models.…”

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confidence: 99%

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Subunit order of eukaryotic TRiC/CCT chaperonin by cross-linking, mass spectrometry, and combinatorial homology modeling

Kalisman

Adams

Levitt

2012

Proc. Natl. Acad. Sci. U.S.A.

164

178

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The TRiC/CCTchaperonin is a 1-MDa hetero-oligomer of 16 subunits that assists the folding of proteins in eukaryotes. Low-resolution structural studies confirmed the TRiC particle to be composed of two stacked octameric rings enclosing a folding cavity. The exact arrangement of the different proteins in the rings underlies the functionality of TRiC and is likely to be conserved across all eukaryotes. Yet despite its importance it has not been determined conclusively, mainly because the different subunits appear nearly identical under low resolution. This work successfully addresses the arrangement problem by the emerging technique of cross-linking, mass spectrometry, and modeling. We cross-linked TRiC under native conditions with a cross-linker that is primarily reactive toward exposed lysine side chains that are spatially close in the context of the particle. Following digestion and mass spectrometry we were able to identify over 60 lysine pairs that underwent crosslinking, thus providing distance restraints between specific residues in the complex. Independently of the cross-link set, we constructed 40,320 (¼8 factorial) computational models of the TRiC particle, which exhaustively enumerate all the possible arrangements of the different subunits. When we assessed the compatibility of each model with the cross-link set, we discovered that one specific model is significantly more compatible than any other model. Furthermore, bootstrapping analysis confirmed that this model is 10 times more likely to result from this cross-link set than the next best-fitting model. Our subunit arrangement is very different than any of the previously reported models and changes the context of existing and future findings on TRiC.group II chaperonins | protein folding | violation distances I n eukaryotes and archaea the folding of nascent and mis-folded polypeptide chains is assisted by a group II chaperonin system known as the thermosome in archea and TRiC (or CCT) in eukaryotes (1). These large protein complexes consist of two stacked octameric rings (2) The open state of the complex binds the polypeptide substrate (4, 5), whereupon closing the substrate is sequestered into a large interior cavity where folding can occur. TRiC has been implicated in the folding pathways of many cytosolic proteins (6), most notably actin and tubulin (7,8).Many archaeal species have just one thermosome gene and a simple homo-oligomeric architecture (9). Other archaeal species have at most three different types of subunits, and there is evidence that the multiple genes are redundant (10). In stark contrast, the eukaryotic TRiC consists of eight different subunit types (CCT1 to CCT8), all of which are essential. The subunit specialization occurred very early in eukaryote evolution (11) and is conserved to such an extent that the sequence identity between mammalian and yeast subunits of the same type is nearly 60%, whereas the sequence identity between the different subunit types in the same organism is only 30%. The diversity of eight distinct subun...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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confidence: 99%

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Subunit order of eukaryotic TRiC/CCT chaperonin by cross-linking, mass spectrometry, and combinatorial homology modeling

Kalisman

Adams

Levitt

2012

Proc. Natl. Acad. Sci. U.S.A.

164

178

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“…Cross-linking experiments were carried out using BS3 as cross-linker. The reaction mixtures were separated into a monomer and two oligomer bands by gel electrophoresis and trypsin digested (44). Cross-linked peptides were fractionated (45), desalted (46), and analyzed on a LTQ Orbitrap Velos (Thermo Fisher Scientific) mass spectrometer (26).…”

Section: Methodsmentioning

confidence: 99%

Multiple molecular architectures of the eye lens chaperone αB-crystallin elucidated by a triple hybrid approach

Braun

Zacharias²,

Peschek³

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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150

177

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The molecular chaperone αB-crystallin, the major player in maintaining the transparency of the eye lens, prevents stress-damaged and aging lens proteins from aggregation. In nonlenticular cells, it is involved in various neurological diseases, diabetes, and cancer. Given its structural plasticity and dynamics, structure analysis of αB-crystallin presented hitherto a formidable challenge. Here we present a pseudoatomic model of a 24-meric αB-crystallin assembly obtained by a triple hybrid approach combining data from cryoelectron microscopy, NMR spectroscopy, and structural modeling. The model, confirmed by cross-linking and mass spectrometry, shows that the subunits interact within the oligomer in different, defined conformations. We further present the molecular architectures of additional well-defined αB-crystallin assemblies with larger or smaller numbers of subunits, provide the mechanism how "heterogeneity" is achieved by a small set of defined structural variations, and analyze the factors modulating the oligomer equilibrium of αB-crystallin and thus its chaperone activity.T he most prominent member of the small heat-shock protein (sHsp) family, α-crystallin, is expressed at high concentrations in the vertebrate eye lens (1) where it plays a major role in maintaining lens transparency (2). Moreover, it protects lens epithelial cells from environmental stress by preventing aggregation of stress-damaged proteins (3). In the low protein turnover milieu of the eye lens, proteins gradually deteriorate throughout the lifespan due to posttranslational modifications and become increasingly prone to aggregation leading to opacity. Thus, the chaperone action of α-crystallin is vital for maintaining the eye lens transparency. Lenticular α-crystallin is composed of two homologous polypeptides, αA-and αB-crystallin, which comprise 173 and 175 amino acid residues, respectively (1, 4). Both proteins possess a three-domain organization consisting of the α-crystallin domain (ACD), a consensus sequence of approximately 90 amino acids common to all sHsps, flanked by a diverse N-terminal region and a moderately conserved C-terminal extension (5, 6).Of the two constituents of α-crystallin, αB-crystallin is the more widespread chaperone with versatile functions: Besides the eye lens, it is abundantly expressed in other tissues (7) and upregulated by various stresses (8). There is growing evidence for its implications in several neuropathological diseases (9) including Parkinson disease, Alzheimer's disease, and multiple sclerosis (10, 11) as well as in cancer (12). In vitro, αB-crystallin prevents the stress-induced aggregation of partially folded polypeptides (3, 13).The αB-crystallin assembles into homooligomers with a variable number of subunits, primarily 24-32 (14-16), and the subunits exchange between homooligomers (17). These properties have hampered high-resolution structural studies on the functionally assembled, full-length protein. An earlier cryoelectron microscopy (cryo-EM) study (18) presented at low resolution (3...

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“…Previous cross-linking experiments and mass spectrometry data have shed light on the organization and register of the Hec1 and Nuf2 coiled-coil domains (9). Using this combined approach, it was shown that there are four distinct positions along the parallel, intertwined coiled-coil domains of Hec1 and Nuf2 that are within close contact.…”

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confidence: 99%

Complementary Interhelical Interactions between Three Buried Glu-Lys Pairs within Three Heptad Repeats Are Essential for Hec1-Nuf2 Heterodimerization and Mitotic Progression

Ngo

Guo

et al. 2013

Journal of Biological Chemistry

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Background: Hec1 and Nuf2 are components of the NDC80 complex essential for faithful chromosome segregation. Results: Three contiguous heptad repeats with buried interhelical Glu-Lys pairs are required for Hec1-Nuf2 dimerization, NDC80 complex formation, and mitotic progression. Conclusion: Interhelical electrostatic and hydrophobic interactions define specificity and stability requirements for Hec1-Nuf2 dimerization. Significance: The results elucidate how Hec1-Nuf2 dimerize and provide insight into NDC80 complex formation.

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Structural Analysis of Multiprotein Complexes by Cross-linking, Mass Spectrometry, and Database Searching

Cited by 228 publications

References 40 publications

Subunit order of eukaryotic TRiC/CCT chaperonin by cross-linking, mass spectrometry, and combinatorial homology modeling

Subunit order of eukaryotic TRiC/CCT chaperonin by cross-linking, mass spectrometry, and combinatorial homology modeling

Multiple molecular architectures of the eye lens chaperone αB-crystallin elucidated by a triple hybrid approach

Complementary Interhelical Interactions between Three Buried Glu-Lys Pairs within Three Heptad Repeats Are Essential for Hec1-Nuf2 Heterodimerization and Mitotic Progression

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