Structural Analysis of 70S Ribosomes by Cross-Linking/Mass Spectrometry Reveals Conformational Plasticity

AbstractThe ribosome is not only a highly complex molecular machine that executes translation according to the central dogma of molecular biology, but also an exceptional specimen for testing and optimizing cross-linking/mass spectrometry (XL-MS) workflows. Due to its high abundance, ribosomal proteins are frequently identified in proteome-wide XL-MS studies of cells or cell extracts. Here, we performed in-depth cross-linking of the E. coli ribosome using the amine-reactive cross-linker diacetyl dibutyric urea (DSAU). We analyzed 143 E. coli ribosomal structures, mapping a total of 10,771 intramolecular distances for 126 cross-link-pairs and 3,405 intermolecular distances for 97 protein pairs. Remarkably, 44% of intermolecular cross-links covered regions that have not been resolved in any high-resolution E. coli ribosome structure and point to a plasticity of cross-linked regions. We systematically characterized all cross-links and discovered flexible regions, conformational changes, and stoichiometric variations in bound ribosomal proteins, and ultimately remodeled 2,057 residues (15,794 atoms) in total. Our working model explains more than 95% of all cross-links, resulting in an optimized E. coli ribosome structure based on the cross-linking data obtained. Our study might serve as benchmark for conducting biochemical experiments on newly modeled protein regions, guided by XL-MS. Data are available via ProteomeXchange with identifier PXD018935.

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Comparative cross-linking and mass spectrometry of an intact F-type ATPase suggest a role for phosphorylation

Nature Communications ◽

10.1038/ncomms2985 ◽

2013 ◽

Vol 4 (1) ◽

Cited By ~ 102

Author(s):

Carla Schmidt ◽

Min Zhou ◽

Hazel Marriott ◽

Nina Morgner ◽

Argyris Politis ◽

...

Keyword(s):

Mass Spectrometry ◽

Conformational Changes ◽

Mass Spectra ◽

Nucleotide Binding ◽

Cross Linking ◽

Spinach Chloroplasts ◽

Cross Links ◽

Chemical Cross Linking

Abstract F-type ATPases are highly conserved enzymes used primarily for the synthesis of ATP. Here we apply mass spectrometry to the F1FO-ATPase, isolated from spinach chloroplasts, and uncover multiple modifications in soluble and membrane subunits. Mass spectra of the intact ATPase define a stable lipid ‘plug’ in the FO complex and reveal the stoichiometry of nucleotide binding in the F1 head. Comparing complexes formed in solution from an untreated ATPase with one incubated with a phosphatase reveals that the dephosphorylated enzyme has reduced nucleotide occupancy and decreased stability. By contrasting chemical cross-linking of untreated and dephosphorylated forms we show that cross-links are retained between the head and base, but are significantly reduced in the head, stators and stalk. Conformational changes at the catalytic interface, evidenced by changes in cross-linking, provide a rationale for reduced nucleotide occupancy and highlight a role for phosphorylation in regulating nucleotide binding and stability of the chloroplast ATPase.

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Quantitative cross-linking/mass spectrometry reveals subtle protein conformational changes

Wellcome Open Research ◽

10.12688/wellcomeopenres.9896.1 ◽

2016 ◽

Vol 1 ◽

pp. 5 ◽

Cited By ~ 16

Author(s):

Zhuo Chen ◽

Lutz Fischer ◽

Salman Tahir ◽

Jimi-Carlo Bukowski-Wills ◽

Paul Barlow ◽

...

Keyword(s):

Mass Spectrometry ◽

Conformational Changes ◽

Complement C3 ◽

Cross Linking ◽

Complement Protein ◽

Protein Protein Interaction ◽

Code Of Practice ◽

Final Maturation ◽

Cross Links ◽

Binary Switching

Quantitative cross-linking/mass spectrometry (QCLMS) probes protein structural dynamics in solution by quantitatively comparing the yields of cross-links between different conformational statuses. We have used QCLMS to understand the final maturation step of the proteasome lid and also to elucidate the structure of complement C3(H2O). Here we benchmark our workflow using a structurally well-described reference system, the human complement protein C3 and its activated cleavage product C3b. We found that small local conformational changes affect the yields of cross-linking residues that are near in space while larger conformational changes affect the detectability of cross-links. Distinguishing between minor and major changes required robust analysis based on replica analysis and a label-swapping procedure. By providing workflow, code of practice and a framework for semi-automated data processing, we lay the foundation for QCLMS as a tool to monitor the domain choreography that drives binary switching in many protein-protein interaction networks.

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Protein-induced conformational changes in 16S rRNA during assembly of the Escherichia Coli 30S ribosomal subunit: A STEM study

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100128833 ◽

1987 ◽

Vol 45 ◽

pp. 910-911

Author(s):

M. Boublik ◽

V. Mandiyan ◽

J.F. Hainfeld ◽

J.S. Wall

Keyword(s):

16S Rrna ◽

Conformational Changes ◽

Ribosomal Proteins ◽

Structural Changes ◽

Ribosomal Subunit ◽

Compact Structure ◽

E Coli ◽

Freeze Dried ◽

16S Rna ◽

30S Subunit

The aim of this study is to understand the mechanism of 16S rRNA folding into the compact structure of the small 30S subunit of E. coli ribosome. The assembly of the 30S E. coli ribosomal subunit is a sequence of specific interactions of 16S rRNA with 21 ribosomal proteins (S1-S21). Using dedicated high resolution STEM we have monitored structural changes induced in 16S rRNA by the proteins S4, S8, S15 and S20 which are involved in the initial steps of 30S subunit assembly. S4 is the first protein to bind directly and stoichiometrically to 16S rRNA. Direct binding also occurs individually between 16S RNA and S8 and S15. However, binding of S20 requires the presence of S4 and S8. The RNA-protein complexes are prepared by the standard reconstitution procedure, dialyzed against 60 mM KCl, 2 mM Mg(OAc)2, 10 mM-Hepes-KOH pH 7.5 (Buffer A), freeze-dried and observed unstained in dark field at -160°.

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Ribosomal Proteins of E. coli: Stoichiometry and Implications for Ribosome Structure

Cold Spring Harbor Symposia on Quantitative Biology ◽

10.1101/sqb.1969.034.01.007 ◽

1969 ◽

Vol 34 (0) ◽

pp. 25-38 ◽

Cited By ~ 64

Author(s):

R. R. Traut ◽

H. Delius ◽

C. Ahmad-Zadeh ◽

T. A. Bickle ◽

P. Pearson ◽

...

Keyword(s):

Ribosomal Proteins ◽

E Coli ◽

Ribosome Structure

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Investigation of the quaternary structure of Neurospora pyruvate kinase by cross-linking with bifunctional reagents: the effect of substrates and allosteric ligands

Canadian Journal of Biochemistry ◽

10.1139/o77-008 ◽

1977 ◽

Vol 55 (1) ◽

pp. 43-49 ◽

Cited By ~ 17

Author(s):

M. Kapoor ◽

M. D. O'Brien

Keyword(s):

Pyruvate Kinase ◽

Functional Groups ◽

Conformational Changes ◽

Quaternary Structure ◽

Polyacrylamide Gel ◽

Cross Linking ◽

Complex Cross ◽

Dimethyl Suberimidate ◽

Cross Links

Pyruvate kinase (EC 2.7.1.40) of Neurospora, a tetramer composed of apparently identical subunits, has been shown to be a dimer of dimers by interprotomeric cross-linking experiments in which bifunctional reagents were used. An analysis of the polyacrylamide gel profiles of the enzyme after cross-linking with glutaraldehyde, dimethyl suberimidate, and dimethyl adipimidate shows that the extent of intersubunit cross-linking is influenced markedly by the ligand bound to the enzyme. Bifunctional cross-linking reagents with a shorter distance between the two functional groups form cross-links effectively in the unliganded enzyme. In the FDP – pyruvate kinase complex, cross-linking was observed over longer distances compared with the unliganded enzyme. It is demonstrated that covalent cross-linkers can be used as sensitive indicators of conformational changes induced in pyruvate kinase by substrates and allosteric ligands.

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The Colibactin Genotoxin Generates DNA Interstrand Cross-Links in Infected Cells

mBio ◽

10.1128/mbio.02393-17 ◽

2018 ◽

Vol 9 (2) ◽

Cited By ~ 50

Author(s):

Nadège Bossuet-Greif ◽

Julien Vignard ◽

Frédéric Taieb ◽

Gladys Mirey ◽

Damien Dubois ◽

...

Keyword(s):

Escherichia Coli ◽

Colon Cancer ◽

Cross Linking ◽

High Fat ◽

E Coli ◽

High Fat Diets ◽

Interstrand Cross Links ◽

Cross Links ◽

Infected Cells ◽

Fat Diets

ABSTRACTColibactins are hybrid polyketide-nonribosomal peptides produced byEscherichia coli,Klebsiella pneumoniae, and otherEnterobacteriaceaeharboring thepksgenomic island. These genotoxic metabolites are produced bypks-encoded peptide-polyketide synthases as inactive prodrugs called precolibactins, which are then converted to colibactins by deacylation for DNA-damaging effects. Colibactins are bona fide virulence factors and are suspected of promoting colorectal carcinogenesis when produced by intestinalE. coli. Natural active colibactins have not been isolated, and how they induce DNA damage in the eukaryotic host cell is poorly characterized. Here, we show that DNA strands are cross-linked covalently when exposed to enterobacteria producing colibactins. DNA cross-linking is abrogated in aclbPmutant unable to deacetylate precolibactins or by adding the colibactin self-resistance protein ClbS, confirming the involvement of the mature forms of colibactins. A similar DNA-damaging mechanism is observedin cellulo, where interstrand cross-links are detected in the genomic DNA of cultured human cells exposed to colibactin-producing bacteria. The intoxicated cells exhibit replication stress, activation of ataxia-telangiectasia and Rad3-related kinase (ATR), and recruitment of the DNA cross-link repair Fanconi anemia protein D2 (FANCD2) protein. In contrast, inhibition of ATR or knockdown of FANCD2 reduces the survival of cells exposed to colibactin-producing bacteria. These findings demonstrate that DNA interstrand cross-linking is the critical mechanism of colibactin-induced DNA damage in infected cells.IMPORTANCEColorectal cancer is the third-most-common cause of cancer death. In addition to known risk factors such as high-fat diets and alcohol consumption, genotoxic intestinalEscherichia colibacteria producing colibactin are proposed to play a role in colon cancer development. Here, by using transient infections with genotoxicE. coli, we showed that colibactins directly generate DNA cross-linksin cellulo. Such lesions are converted into double-strand breaks during the repair response. DNA cross-links, akin to those induced by metabolites of alcohol and high-fat diets and by widely used anticancer drugs, are both severely mutagenic and profoundly cytotoxic lesions. This finding of a direct induction of DNA cross-links by a bacterium should facilitate delineating the role ofE. coliin colon cancer and engineering new anticancer agents.

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Probing conformational changes of human serum albumin due to unsaturated fatty acid binding by chemical cross-linking and mass spectrometry

Biochemical Journal ◽

10.1042/bj20041624 ◽

2005 ◽

Vol 387 (3) ◽

pp. 695-702 ◽

Cited By ~ 48

Author(s):

Bill X. HUANG ◽

Chhabil DASS ◽

Hee-Yong KIM

Keyword(s):

Mass Spectrometry ◽

Fatty Acids ◽

Fatty Acid ◽

Human Serum Albumin ◽

Serum Albumin ◽

Human Serum ◽

Conformational Changes ◽

Cross Linking ◽

X Ray ◽

Chemical Cross Linking

Mass spectrometry with chemical cross-linking was used to probe the conformational changes of HSA (human serum albumin) in solution on interaction with monounsaturated OA (oleic acid) or polyunsaturated AA (arachidonic acid) or DHA (docosahexaenoic acid). Fatty acid-free or -bound HSA was modified with lysine-specific cross-linkers and digested with trypsin. Cross-linked peptides were analysed by nano-electrospray ionization MS to localize the sites of cross-linking. Our data indicated that a local conformational change involving movement of the side chains of Lys-402 of subdomain IIIA or Lys-541 of subdomain IIIB occurred upon binding of all three fatty acids. Our data also indicated that the side chains of Lys-205 (IIA) and Lys-466 (IIIA) moved closer towards each other upon binding AA or DHA, but not OA, suggesting that the conformations of HSA when bound to mono- and poly-unsaturated fatty acids are distinctively different. While these observations agreed with previous X-ray crystallographic studies, the distances between ε-amino groups of most cross-linked lysine pairs were shorter than the crystal structure predicted, possibly reflecting a discrepancy between the solution and crystal structures. This method can serve as a useful complement to X-ray crystallography, particularly in probing the structure of a protein in solution.

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Monitoring Conformational Changes in Protein Complexes Using Chemical Cross-Linking and Fourier Transform Ion Cyclotron Resonance Mass Spectrometry: The Effect of Calcium Binding on the Calmodulin—Melittin Complex

European Journal of Mass Spectrometry ◽

10.1255/ejms.882 ◽

2007 ◽

Vol 13 (4) ◽

pp. 281-290 ◽

Cited By ~ 8

Author(s):

Petr Novak ◽

Vladimir Havlicek ◽

Peter J. Derrick ◽

Kyle A. Beran ◽

Sajid Bashir ◽

...

Keyword(s):

Mass Spectrometry ◽

Fourier Transform ◽

Conformational Changes ◽

Calcium Binding ◽

Cyclotron Resonance ◽

Structural Changes ◽

Cross Linking ◽

Ion Cyclotron Resonance ◽

Ion Cyclotron ◽

Chemical Cross Linking

Calmodulin is an EF hand calcium binding protein. Its binding affinities to various protein/peptide targets often depend on the conformational changes induced by the binding of calcium. One such target is melittin, which binds tightly to calmodulin in the presence of calcium, and inhibits its function. Chemical cross-linking combined with Fourier transform ion cyclotron resonance mass spectrometry has been employed to investigate the coordination of calmodulin and melittin in the complex at different concentrations of calcium. This methodology can be used to monitor structural changes in proteins induced by ligand binding and to study the effects these changes have on non-covalent interactions between proteins. Cross-linking results indicate that the binding place of the first melittin in the calcium-free calmodulin form is the same as in the calcium-loaded calmodulin/melittin complex.

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