Protein

Subscriber access provided by LOCK HAVEN UNIV

Article

A Novel Bio-Orthogonal Cross-Linker for Improved Protein/Protein Interaction Analysis Catherine Nury, Virginie Redeker, Sebastien Dautrey, Anthony Romieu, Guillaume Van Der Rest, Pierre-Yves Renard, Ronald Melki, and Julia Chamot-Rooke Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac503892c • Publication Date (Web): 18 Dec 2014 Downloaded from http://pubs.acs.org on December 19, 2014

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

A Novel Bio-Orthogonal Cross-Linker for Improved Protein/Protein Interaction Analysis Catherine Nury†,‡‡, Virginie Redeker§‡, Sébastien Dautreyǁ, Anthony Romieu¶,▽, Guillaume van der Rest#, Pierre-Yves Renardǁ, Ronald Melki§, Julia Chamot-Rooke†,‡* †

Structural Mass Spectrometry and Proteomics Unit, Institut Pasteur, CNRS UMR 3528, Paris, France ‡

§

ǁ

CNRS UMR 3528, Institut Pasteur, Paris, France

Neuroscience Paris-Saclay Institute, CNRS, Gif-sur-Yvette, France

Normandie Université, COBRA UMR 6014 & FR 3038; UNIV Rouen; INSA Rouen; CNRS, IRCOF, 1, rue Tesnières, 76821 Mont-Saint-Aignan Cedex, France

¶

ICMUB, UMR CNRS 6302, Université de Bourgogne, 9, Avenue Alain Savary, 21078 Dijon, France ▽

#

Institut Universitaire de France, 103, Boulevard Saint-Michel, 75005 Paris, France

Laboratoire de Chimie Physique, Université Paris Sud, 91405 Orsay Cedex, France

‡These authors contributed equally corresponding author: [email protected]

ACS Paragon Plus Environment

1


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 28

ABSTRACT The variety of protein cross-linkers developed in recent years illustrates the current requirement for efficient reagents optimized for Mass Spectrometry (MS) analysis. To date, the most widely used strategy relies on commercial cross-linkers that bear an isotopically-labeled tag and N-hydroxysuccinimid-ester (NHS-ester) moieties. Moreover, an enrichment step using liquid chromatography is usually performed after enzymatic digestion of the cross-linked proteins. Unfortunately, this approach suffers from several limitations. First, it requires large amounts of proteins. Second, NHS-ester cross-linkers are poorly efficient because of their fast hydrolysis in water. Finally data analysis is complicated because of uneven fragmentation of complex isotopic cross-linked peptide mixtures. We therefore synthesized a new type of trifunctional cross-linker to overrule these limitations. This reagent, named NNP9, comprises a rigid core and bears two activated carbamate moieties and an azido group. NNP9 was used to establish intra- and intermolecular crosslinks within creatine kinase, then to map the interaction surfaces between αSynuclein (α-Syn), which aggregation leads to Parkinson’s disease, and the molecular chaperone Hsc70. We show that NNP9 cross-linking efficiency is significantly higher than that of NHSester commercial cross-linkers. The number of cross-linked peptides identified was increased and a high quality of MS/MS spectra leading to high sequence coverage was observed. Our data demonstrate the potential of NNP9 for an efficient and straightforward characterization of protein-protein interfaces and illustrate the power of using different cross-linkers to map thoroughly the surface interfaces within protein complexes.


2

Page 3 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


INTRODUCTION The combination of chemical cross-linking (XL) and mass spectrometry (MS) has proved to be a powerful approach to obtain structural information on protein structures or protein-protein interaction interfaces. The efficiency of the cross-linking reaction and the detection and identification of the reaction products after enzymatic digestion are critical in XL-MS experiments. Many different cross-linkers have been synthesized in the last decade1,2 with various types of reactive groups2 or chemical functions allowing an easier identification of crosslinked peptides in MS spectra1 by improved MS/MS or further enrichment.3-6 Some reagents have also proved to be useful for in vivo cross-linking.7 However, a major issue in XL-MS experiments is the complexity of the peptide mixture obtained after enzymatic digestion. Indeed, a mixture of unmodified, singly modified (dead-end or mono-link) peptides and both intra- and inter-molecular cross-linked peptides (cross-link) are generated.8 Moreover, if proper conditions are used to avoid non-native, unspecific, inter-molecular interactions, minute amounts of crosslinked peptides are generated thus impacting their analytical identification. To date, one of the most widely used XL-MS approach is based on the use of homobifunctional isotopically-labeled cross-linkers9 bearing amine-reactive N-hydroxysuccinimidyl (NHS) esters. The presence in mass spectra of isotopic pairs exclusively for the cross-linked peptides facilitates their detection and identification.10 Integrated XL-MS strategies, including cross-linked peptides enrichment using various chromatographic techniques (SEC11, SCX12,13) and dedicated softwares such as xQuest14, Stavrox15, pLink16, CrossWork17 or XLink-DB18 have been shown to provide useful structural information for the analysis of very large complexes such as the RNA polymerase II subunit19, 26S proteasome20-22, protein phosphatase 2A (PP2A) network23 or the mammalian mitochondrial


3


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 28

ribosome24. However, the overall XL-MS process remains long and difficult and there is room for improvement at different stages. First, the cross-linking reaction yield is generally very low due to the rapid hydrolysis of the NHS ester function in aqueous solution. Second, large amounts of proteins are required if an enrichment step is performed. In addition, MS/MS data analysis can be complicated by the presence of the isotopic pairs. Indeed, it often happens that only the deuterated cross-link, which has a slightly shorter retention time compared to its hydrogenated counterpart, is fragmented. In that case, the identification of the cross-linked peptides will be problematic as some software tools require the fragmentation of both the deuterated and the nondeuterated peptide counterpart. Therefore the isotopic pair helps for the labeling of the crosslinked peptides among the unmodified ones in MS spectra but can miss cross-linked peptides identification when only one of the isotopic peptide pairs is selected and fragmented in automated MS/MS analysis but not the other one. Finally, despite efforts towards the development of automated software tools, confident cross-linked peptide identification still requires manual validation due to the quality of MS/MS data. To improve XL-MS strategies aimed at identifying intra- and inter-molecular interactions within/between proteins, we designed and synthesized a new bio-orthogonal cross-linker. This novel trifunctional cross-linker, named NNP9, includes (i) a rigid benzenic core (ii) two Nsuccinimidyl carbamate moieties, less prone to hydrolysis than the N-succinimidyl ester moieties, and (iii) an azido "clickable" group in order to allow further enrichment of cross-linked peptides. In NNP9, "NN" refers to the two reactive groups targeting primary amines, "P" relates to the phenyl core, and "9" adverts the number of atoms between the two reactive groups. The spacer arm length of NNP9 is ~ 10 Å.


4

Page 5 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


NNP9 was used to map the intra- and inter-molecular interactions within creatine kinase, used as a model reference protein, and to probe the interfaces between α-Synuclein (α-Syn), which aggregation is associated to Parkinson’s disease, and the molecular chaperone Hsc70. Our results were compared to those obtained under the same experimental conditions with isotopicallylabeled homobifunctional NHS-ester reagents with spacer arms of similar length. Our novel cross-linker showed a higher cross-linking efficiency than the commercial ones a general improvement in the quality of the MS/MS spectra and thus in the confidence in cross-links identification. As unique cross-links were also obtained with the commercial reagents, our study illustrates the power of using different cross-linkers to thoroughly map protein/protein interaction surfaces.

EXPERIMENTAL SECTION Synthesis of NNP9 cross-linker The trifunctional cross-linker NNP9 was synthesized in 16% overall yield through a three-step synthetic procedure from already described di-tert-butyloxycarbonyl (Boc) derivative of 3,5bis(aminomethyl)benzoic acid. The synthesis, purification and characterization of NNP9 are detailed in SI. Expression and Purification of α-Syn and Hsc70 Recombinant wild-type α-Syn (α-Synuclein) was expressed and purified as described previously.25 Pure α-Syn (1.0 mM) in 50 mM Tris-HCl, pH 7.5, 50 mM KCl was stored at -80 °C. Recombinant hexahistidine tagged wild-type Hsc70 was purified as described previously.26 Pure Hsc70 (60 µM) in 50 mM Tris-HCl, pH 7.5, 150 mM KCl, 5 mM β-mercaptoethanol, 5 mM


5


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 28

MgCl2, 1 mM EGTA, and 10% glycerol was stored at -80 °C. Hsc70’s ATPase activity, alone or in the presence of α-Syn, was monitored as previously described.27 Cross-Linking, SDS-PAGE Separation and Enzymatic Digestion Cross-linking of rabbit creatine kinase (10 µM) was carried out in PBS pH 7.4 at room temperature (RT) with freshly prepared NNP9 (20 mM in DMSO) or bis(sulfosuccinimidyl) suberate (BS3) d0/d4 (20 mM in DMSO) using a 20:1 cross-linker:protein molar ratio. α-Syn and Hsc70 (200 and 10 µM, respectively) were incubated for 2 h at 37 °C under agitation in 50 mM Tris-HCl, pH 7.5, 150 mM KCl, before dialyzing for 2 h at 4 °C against the cross-linking buffer (40 mM HEPES-OH, pH 7.5, 75 mM KCl). Cross-linking with NNP9 and BS3 d0/d4 at a cross-linker:α-Syn or Hsc70 molar ratio of 10:1 or 200:1 was performed at RT for 30 min and stopped by addition of ammonium bicarbonate (final concentration 50 mM). The samples were immediately mixed with denaturing buffer and heated to 95 °C for 5 min before SDS-PAGE analysis on 7.5% Tris/Tricine or 12% Tris/Glycine gels for α-Syn/Hsc70 and creatine kinase, respectively. The gels were next stained using Biosafe-Coomassie (Biorad, Hercules, CA) or Coomassie blue R250 for creatine kinase and α-Syn/Hsc70, respectively. The protein bands corresponding either to monomeric, dimeric and tetrameric creatine kinase or to the α-Syn/Hsc70 complex generated upon NNP9 or BS3 d0/d4 cross-linking were excised and subjected to trypsin digestion and tryptic peptide extraction using the Progest robot (Genomic Solutions, Chemsford, MA) as described previously.25 LC/MS analysis Tryptic digests were analyzed by nanoLC-MS/MS using an Ultimate 3000 Nano HPLC system (Dionex, Thermo-Scientific, Waltham, MA) coupled to the nanoelectrospray ion source of an


6

Page 7 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


LTQ-Orbitrap Velos mass spectrometer (Thermo-Scientific, Bremen, Germany). Peptides were loaded on a C-18 µ-precolumn (C-18 PepMap100, 5 µm, 100 Å, Dionex, Thermo-Scientific, Waltham, MA) at a flow rate of 10 µL/min of solvent A and the separation was performed using an in-house packed 15 cm nano-HPLC column (75 µm inner diameter) with C-18 resin (3 µm particles, 100 Å pore size, Reprosil C-18, Dr. Maisch GmbH). Peptides were separated at a flow rate of 300 nL/min using a gradient of 2% to 55% solvent B for 95 min, followed by a 10 min washing step at 100% solvent B and a reconditioning step at 2% B for 20 min. Solvent A was 98/2/0.1 H2O/CH3CN/FA, and solvent B was 20/80/0.08 H2O/CH3CN/FA. NanoLC-MS/MS experiments were conducted in Data-Dependent Acquisition mode. A resolution of 60 000 (at m/z 400 in the Orbitrap) was used for MS scans. The 20 most intense ions, above an intensity threshold of 5 000 counts, were selected for CID fragmentation and analysis in the LTQ. The FT automatic gain control (AGC) was set at 1×106 for MS and 5×104 for MSn experiments. Data Processing NanoLC-MS/MS data were processed automatically using Mascot (version 2.4.1) search engine in Proteome Discoverer 1.4 search engine with the following chemical modifications: methionine oxidation, cysteine carbamidomethylation, and mono-link or loop-link modification of lysine, serine, threonine, and tyrosine with NNP9 or BS3. For the identification of BS3-d0, BS3-d4 or NNP9 cross-linked peptides, nanoLCMS raw files were further converted to mgf files using Proteome Discoverer before analysis using Stavrox software (2.4 Version)15 to search for BS3d0 or BS3d4 cross-links or were converted to mzXML files using MSConvert28 before xQuest analysis to search for BS3d0/d4 pairs or BS3d0 or BS3d4 cross-links.14 Parameters were set as follows: maximum 3 tryptic miscleavages, cross-linker modification on lysine, serine, threonine, tyrosine and N-terminus with a maximum tolerance of 10 ppm on the precursor ion


7


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 28

and 0.6 Da for fragment ions of cross-linked peptides. Cysteine carbamidomethylation was set as a permanent modification and methionine oxidation as a variable modification. For NNP9 crosslinker, mass modifications were set to 314.1127 Da for intra- or inter-protein cross-linked peptides. For BS3 cross-linker, mass modifications were set to 138.0681 Da and 142.0928 Da for d0 and d4 intra- or inter-protein cross-linked peptides respectively. MS/MS spectra of all crosslinked candidates were further manually checked to confirm their identification. Only fragment ion peaks with signal-to-noise ratios above 3 were labeled in the MS/MS spectra.

RESULTS AND DISCUSSION Design of a New Generation of Cross-Linkers We designed a new tri-functional cross-linker, NNP9 bearing to bear two activated carbamate moieties,29 a rigid phenyl core and an azido group (Figure 1A). Although many multifunctional scaffolds equipped with three (or more) bio-conjugates have been reported,30 only a few have been specifically designed for XL-MS applications.31 The N-succinimidyl carbamate moieties were chosen because they are much less prone to hydrolysis than conventional NHS esters.32-35 They target nucleophilic moieties and react mainly, as their ester counterparts, with primary amino groups (side-chains of lysine and N-terminus of proteins) and to a minor extent with serine, threonine and tyrosine.36,37 The phenyl core of NNP9 was intended to rigidify the structure of the spacer arm compared to the flexible aliphatic chains present in commercially available BS3 (Figure 1B) and bis(sulfosuccinimidyl) glutarate (BS2G) cross-linkers.


8

Page 9 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Finally, an azido group was added to offer the possibility to perform bio-orthogonal conjugation using "click chemistry" in order to introduce an affinity or a MS cleavable tag.38,39 This option has not been used in this paper but will be reported later. Comparison of creatine kinase cross-linking by NNP9 and BS3 NNP9 was first tested on creatine kinase (CK). CK has been shown to form a dimer40 or a tetramer41 and crystallographic structures are available.40 Moreover, cross-linking studies of CK with BS3 have already been reported.42 CK was treated by NNP9 or BS3 d0/d4 as they have similar spacer arm lengths (~10 and 11 Å, respectively) and the reaction products separated on 1D PAGE. NNP9 to protein ratio was optimized to minimize the amount of cross-linker, hence reducing unspecific interactions, while being sufficient for an efficient protein cross-linking. The 20:1 cross-linker:protein molar ratio was selected as a compromise between the intensity of the band corresponding to the crosslinked complex an a high background reflecting unspecific crosslinking on the SDS-PAGE (Figure S1). In-gel tryptic digestion of the cross-linked products was performed and the resulting peptides were analyzed by LC/MS without any enrichment step. The cross-linked candidate peptides were identified using two software tools (xQuest14, Stavrox15) and manually validated. Although most of the candidates were identical, only Stavrox offers the possibility to modify the N-terminus while only xQuest processes d0/d4 pairs of MS/MS data in the same search. Manual validation consisted in checking the quality and the annotation of MS/MS spectra for unambiguous identification of both cross-linked peptide sequences and cross-linked sites. The list of the CK cross-linked peptides identified with NNP9 and BS3 d0/d4 is given in Tables 1 and S1 A and B.


9


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 28

We identified 4 intra-molecular cross-links with NNP9 (Table 1, S1A and S1B) in monomeric CK band. Six cross-linked peptides were identified in dimeric and tetrameric CK band. Overall, 7 cross-links were found, 3 were common to monomeric, dimeric and tetrameric CK bands (K259/S239, K298/K242 and K304/K319). One cross-link was only retrieved in CK band corresponding to the monomer (K138/K265), finally 3 cross-links were observed only in the bands corresponding to dimeric and tetrameric CK (K135/S158, K015/K156 and K172/K242). These cross-links involved 11 lysines (K015, K135, K138, K156, K172, K242, K259, K265, K298, K304 and K319) and 2 serines (S158 and S239). The distance between CK residues involved within the cross-links ranges from ~ 9 to 22 Å according to CK PDB structure (PDB entry 1U6R, Figure S2 A and B) is compatible with NNP9 spacer arm together with lysine and serine residues lateral chain lengths (~ 23 Å). Based on the reported tri-dimensional structure, all cross-linked peptides corresponded to intra-molecular cross-links, except K015/K156 (Figure S3). Six cross-linked peptides were identified upon CK cross-linking with BS3 d0/d4 (Tables 1 and S1C and S1D). Only two cross-links (Y125/K242 and K298/K369), detected upon cross-linking with BS3 d0/d4 were found upon digestion of CK dimeric and tetrameric bands. Two additional cross-links were common with those observed with NNP9 (K298/K242 and K304/K319). Lysine (K015, K040, K242, K298, K304, K319 and K369) and tyrosine (Y125) residues were involved in those cross-links. In contrast to NNP9, CK treatment with BS3 d0/d4 yielded no detectable inter-molecular cross-links. CK cross-link K298/K242, identified previously using BS3 by Leitner et al11, is common to NNP9 and BS3. The fragmentation spectra of the NNP9- and BS3-mediated K298/K242 crosslink are shown in Figure 1C and 1D, respectively. Although the fragmentation patterns are


10

Page 11 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


similar, more complete b/y fragment ions series are clearly observed for NNP9-mediated CK K298/K242 cross-link. Since the precursor ions were of the same intensity in both cases, one can assume that the phenyl ring or the carbamate groups of the cross-link can play a role in favoring more complete fragment ion series. Two cross-links (K298/K242 and K304/K319) were common to BS3 and NNP9 in monomeric CK band and two additional cross-links (K015/K040 and K298/K319) were found only with BS3. The latter cross-link lies, based on CK structure (Figure S2A and B), within the region where NNP9 yielded the cross-link K304/K319. Two of the three cross-linked peptides (Y125/K242 and K298/K369) were found only with BS3 in CK dimeric and tetrameric bands. The differences we observed upon analyzing NNP9 or BS3 cross-links, primarily in the identification of an inter-molecular cross-link in CK only with NNP9, may be due either to difference in reactivity of N-succinimidyl carbamate and NHS ester moieties, the flexibility of the cross-linkers spacer arms or the fragmentation efficiency/fragment ion detection. Characterization of Hsc70/α α-Syn complex by NNP9 and BS3 cross-linking The molecular chaperone Hsc70 binds both to monomeric and fibrillar α-Syn. Hsc70 inhibits the assembly of monomeric α-Syn into fibrils that are the primary constituent of Lewy bodies, the molecular signature of Parkinson’s disease.43,44 Hsc70 also binds to fibrillar α-Syn and affects their toxicity/seeding capacities. The identification of the interaction surfaces between Hsc70 and α-Syn is thus relevant for the design of novel therapeutic strategies targeting Parkinson’s disease 45

. We previously mapped the interaction interfaces between Hsc70 and α-Syn with BS2G

d0/d4.25 We used the strategy we implemented25 with NNP9 and BS3 to strengthen our previous conclusions. Hsc70 and α-Syn were preincubated then treated by NNP9 or BS3 and the reaction products resolved on SDS-PAGE (Figure 2). The amount of Hsc70/α-Syn complex cross-linked


11


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 28

in the presence of NNP9 was significantly higher than that with BS3 (Figure 2), certainly because of a slower hydrolysis of the NHS carbamate compared to the NHS ester.. The band corresponding to Hsc70/α-Syn complex (labeled with an asterisk) was digested by trypsin and the products analyzed by nanoLC-MS/MS. Hsc70 sequence coverage was 85% within NNP9 or BS3-mediated Hsc70/α-Syn complex while α-Syn was 100% with NNP9, 78% with BS3. The numbers of inter-molecular cross-links we observed using NNP9 and BS3 were 23 and 14, respectively (Tables 2, S2). Eight inter-molecular cross-links were previously observed with BS2G.25 Only 5 cross-links were common to NNP9, BS3 and BS2G. Ten cross-links were common to NNP9 and BS3 (Table 2). Furthermore, the cross-links detected only with BS3 (K497Hsc70/K34α-Syn, K535Hsc70/K10α-Syn and K557Hsc70/K21α-Syn) were similar to those (K497Hsc70/K32α-Syn, K535Hsc70/K12α-Syn, and K557Hsc70/K23α-Syn) observed with NNP9. The cross-links that were observed with either NNP9 alone or BS3 alone are boxed (grey) in Table 2. Five out of the 25 lysines from the client proteins-binding domain of Hsc70 (K497, K507, K535, K557 and K561 with NNP9, Figure 3A; K497, K531, K535, K557 and K561 with BS3, Figure 3B) are cross-linked to α-Syn as compared to 4 (K497, K512, K557 and K561, Figure 3C) with BS2G. The two additional lysines from Hsc70 that are cross-linked to α-Syn are K507 and K535 for NNP9, K531 and K535 for BS3. These lysine residues are mapped on the crystal structure of Hsc70 client protein-binding domain (Figure 3A, 3B, 3C). The annotated MS/MS spectrum of m/z 679.355 corresponding to the cross-link K535Hsc70/K32α-Syn is shown in Figure 4A. The cross-linking of Hsc70 K512 to α-Syn was only observed with BS2G (Table 2 and Figure 3C). Similarly, Hsc70 K507 and K531 cross-linking to α-Syn were only observed with NNP9 and BS3, respectively (Table 2 and Figure 3A, B). These results stress the importance of using a variety of cross-linkers to map thoroughly surface interfaces within protein complexes.


12

Page 13 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Among the eight cross-linked peptides identified with BS2G, seven and five were identified using NNP9 and BS3, respectively. Most important are the 16 and 9 additional cross-links we observed with NNP9 and BS3, respectively (Table 2 and Figure 3A-C). Seven (K497Hsc70/K97αSyn,

K557Hsc70/M01α-Syn,

K561Hsc70/K12α-Syn,


K561Hsc70/K23α-Syn)

and

3



(K497Hsc70/K97α-Syn,


K557Hsc70/K32α-Syn) out of 16 and 9 additional cross-links obtained with NNP9 and BS3, respectively, indicate that α-Syn interacts with Hsc70 in two orientations (Figure 3D and E). The N-terminal or the C-terminal domain of α-Syn are cross-linked with K557 and K561 with NNP9 and K557 with BS3 in the lid of the client protein-binding domain of Hsc70, whereas only α-Syn C-terminal domain is cross-linked to K557 and K561 with BS2G (Figure 3). Moreover, while K497 from the bottom of the client protein binding site of Hsc70 was only cross-linked to the Nterminal domain of α-Syn with BS2G, it was cross-linked to both the N and C-terminal domains of α-Syn with NNP9 and BS3. The annotated MS/MS spectrum of m/z 701.122 corresponding to K561Hsc70/K12α-Syn illustrates the interaction between the lid of Hsc70 client proteins-binding domain with the N-terminal domain of α-Syn (Figure 4B).

CONCLUSION In this paper, a novel trifunctional cross-linking reagent has been specifically designed for XLMS experiments and its performances evaluated. The originality of this cross-linker, named NNP9, is to bear two identical N-succinimidyl carbamate moieties, a rigid core and an azido group. Thanks to the NHS carbamate moieties, which are less prone to hydrolysis than NHS esters, the cross-linking efficiency of NNP9 was shown to be higher than that of commercially available BS3 and BS2G d0/d4 reagents.


13


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 28

An inter-molecular cross-link was identified in CK only with NNP9. We identified cross-links between Hsc70 and α-Syn that are common to NNP9, BS3 and BS2G but also that are unique to each cross-linker. We finally identified an alternate cross-linking orientation between Hsc70 and α-Syn using NNP9 and BS3. Our results highlight the importance of using various cross-linkers to map thoroughly surface interfaces within protein complexes. Our study shows that the use of isotopically labeled cross-linkers and extensive enrichment of cross-linked peptides is not mandatory to achieve in depth interaction maps of protein complexes. Here, a simple and straightforward strategy was implemented without any enrichment step. In addition, the presence of an azido group, which was not used for this study, will allow the introduction of various chemical functions, such as a biotin group that could be further used to improve the overall strategy by additional cross-linked peptides enrichment, in particular for minute sample amounts and/or multimeric complexes.


14

Page 15 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figures Figure 1. Chemical structures of NNP9 (A) and BS3 (B) with spacer arm lengths. Annotated MS/MS spectra of the CK intra-molecular cross-link K298/K242 with NNP9 (C) or BS3 d4 (D) with m/z 554.696 and 653.105, respectively. The identified fragments are indicated on CK crosslinked sequences.

Figure 2. 1D SDS PAGE separation of the Hsc70/α-Syn complex before and after NNP9 or BS3 cross-linking. The red asterisks indicate the band corresponding to Hsc70/α-Syn cross-link.

Figure 3. Location of the Hsc70 residues cross-linked with α-Syn. Hsc70’s client binding domain is colored in green. Hsc70 lysines in space-fill and red were cross-linked to α-Syn with NNP9 (A, D, E), BS3 (B) or BS2G (C), respectively. Residues from α-Syn that are cross-linked to Hsc70 are indicated (in blue). (D, E) Molecular model illustrating the two orientations of αSyn in the Hsc70/α-Syn complex. The Hsc70 model was built as described previously.18 Figures were generated using PyMOL (http://www.pymol.org).

Figure 4. Identification of two cross-links between Hsc70 and α-Syn using NNP9. Annotated MS/MS spectra of (A) [K535Hsc70/K32α-Syn and (B) K561Hsc70/K12α-Syn with m/z 679.355 and 701.369, respectively. The identified fragments are indicated on the cross-linked Hsc70 (labeled α) and α-Syn (labeled ß) sequences.


15


Page 16 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment

16

Page 17 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1


17


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 28

Figure 2


18

Page 19 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3


19


Page 20 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment

20

Page 21 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4


21


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 28

Tables

Table 1. Cross-linked peptides identified for CK with NNP9 or BS3 d0/d4. For each peptide, the table gives the amino acid segment, the sequence, the cross-linked residues (XL site), the nature of CK band (monomeric, dimeric or tetrameric), the cross-linker (NNP9 or BS3), the peptides previously identified by Leitner et al.7 and the Cα/Cα distance of the cross-linked residues based on the crystal structure of CK (PDB entry 1U6R). The amino acid residues involved in the crosslink are in bold. Table 2. Hsc70/α-Syn cross-linked peptides obtained with NNP9, BS3 d0/d4 and BS2G d0/d4 18

. For each peptide the table gives the protein, the amino acid segment, the sequence and the

cross-linked residues (XL site). The cross-links that were observed with either NNP9 alone, BS3 alone or BS2G alone are boxed in grey. The amino acid residues involved in the cross-link are in bold.


22

Page 23 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


REFERENCES (1) Petrotchenko, E. V.; Borchers, C. H. Mass Spectrometry Reviews 2010, 29, 862-876. (2) Paramelle, D.; Miralles, G.; Subra, G.; Martinez, J. Proteomics 2013, 13, 438-456. (3) Sohn, C. H.; Agnew, H. D.; Lee, J. E.; Sweredoski, M. J.; Graham, R. L.; Smith, G. T.; Hess, S.; Czerwieniec, G.; Loo, J. A.; Heath, J. R.; Deshaies, R. J.; Beauchamp, J. L. Anal Chem 2012, 84, 2662-2669. (4) Fujii, N.; Jacobsen, R. B.; Wood, N. L.; Schoeniger, J. S.; Guy, R. K. Bioorganic & medicinal chemistry letters 2004, 14, 427-429. (5) Chu, F.; Mahrus, S.; Craik, C. S.; Burlingame, A. L. Journal of the American Chemical Society 2006, 128, 10362-10363. (6) Sinz, A.; Kalkhof, S.; Ihling, C. Journal of the American Society for Mass Spectrometry 2005, 16, 1921-1931. (7) Tang, X.; Bruce, J. E. Molecular BioSystems 2010, 6, 939-947. (8) Schilling, B.; Row, R. H.; Gibson, B. W.; Guo, X.; Young, M. M. J. Am. Soc. Mass Spectrom. 2003, 14, 834-850. (9) Muller, D. R.; Schindler, P.; Towbin, H.; Wirth, U.; Voshol, H.; Hoving, S.; Steinmetz, M. O. Analytical Chemistry 2001, 73, 1927-1934. (10) Ihling, C.; Schmidt, A.; Kalkhof, S.; Schulz, D. M.; Stingl, C.; Mechtler, K.; Haack, M.; Beck-Sickinger, A. G.; Cooper, D. M.; Sinz, A. J. Am. Soc. Mass Spectrom. 2006, 17, 11001113. (11) Leitner, A.; Reischl, R.; Walzthoeni, T.; Herzog, F.; Bohn, S.; Forster, F.; Aebersold, R. Molecular & Cellular Proteomics 2012, 11. (12) Fritzsche, R.; Ihling, C. H.; Goetze, M.; Sinz, A. Rapid Communications in Mass Spectrometry 2012, 26, 653-658. (13) Leitner, A.; Walzthoeni, T.; Aebersold, R. Nat. Protocols 2014, 9, 120-137. (14) Rinner, O.; Seebacher, J.; Walzthoeni, T.; Mueller, L.; Beck, M.; Schmidt, A.; Mueller, M.; Aebersold, R. Nature Methods 2008, 5, 315-318. (15) Gotze, M.; Pettelkau, J.; Schaks, S.; Bosse, K.; Ihling, C. H.; Krauth, F.; Fritzsche, R.; Kuhn, U.; Sinz, A. J. Am. Soc. Mass Spectrom. 2012, 23, 76-87. (16) Yang, B.; Wu, Y.-J.; Zhu, M.; Fan, S.-B.; Lin, J.; Zhang, K.; Li, S.; Chi, H.; Li, Y.-X.; Chen, H.-F.; Luo, S.-K.; Ding, Y.-H.; Wang, L.-H.; Hao, Z.; Xiu, L.-Y.; Chen, S.; Ye, K.; He, S.-M.; Dong, M.-Q. Nat Meth, 9, 904-906. (17) Rasmussen, M. I.; Refsgaard, J. C.; Peng, L.; Houen, G.; Hojrup, P. Journal of Proteomics 2011, 74, 1871-1883. (18) Zheng, C.; Weisbrod, C. R.; Chavez, J. D.; Eng, J. K.; Sharma, V.; Wu, X.; Bruce, J. E. J Proteome Res 2013, 12, 1989-1995. (19) Chen, Z. A.; Jawhari, A.; Fischer, L.; Buchen, C.; Tahir, S.; Kamenski, T.; Rasmussen, M.; Lariviere, L.; Bukowski-Wills, J. C.; Nilges, M.; Cramer, P.; Rappsilber, J. Embo Journal 2010, 29, 717-726. (20) Walzthoeni, T.; Claassen, M.; Leitner, A.; Herzog, F.; Bohn, S.; Forster, F.; Beck, M.; Aebersold, R. Nat Meth 2012, 9, 901-903. (21) Kao, A.; Randall, A.; Yang, Y.; Patel, V. R.; Kandur, W.; Guan, S.; Rychnovsky, S. D.; Baldi, P.; Huang, L. Molecular & cellular proteomics : MCP 2012, 11, 1566-1577. (22) Karadzic, I.; Maupin-Furlow, J.; Humbard, M.; Prunetti, L.; Singh, P.; Goodlett, D. R. Proteomics 2012, 12, 1806-1814.


23


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 28

(23) Herzog, F.; Kahraman, A.; Boehringer, D.; Mak, R.; Bracher, A.; Walzthoeni, T.; Leitner, A.; Beck, M.; Hartl, F. U.; Ban, N.; Malmstrom, L.; Aebersold, R. Science 2012, 337, 13481352. (24) Greber, B. J.; Boehringer, D.; Leitner, A.; Bieri, P.; Voigts-Hoffmann, F.; Erzberger, J. P.; Leibundgut, M.; Aebersold, R.; Ban, N. Nature 2014, 505, 515-519. (25) Redeker, V.; Pemberton, S.; Bienvenut, W.; Bousset, L.; Melki, R. The Journal of biological chemistry 2012, 287, 32630-32639. (26) Pemberton, S.; Madiona, K.; Pieri, L.; Kabani, M.; Bousset, L.; Melki, R. The Journal of biological chemistry 2011, 286, 34690-34699. (27) Melki, R.; Carlier, M. F.; Pantaloni, D. Biochemistry 1990, 29, 8921-8932. (28) Chambers, M. C.; Maclean, B.; Burke, R.; Amodei, D.; Ruderman, D. L.; Neumann, S.; Gatto, L.; Fischer, B.; Pratt, B.; Egertson, J.; Hoff, K.; Kessner, D.; Tasman, N.; Shulman, N.; Frewen, B.; Baker, T. A.; Brusniak, M.-Y.; Paulse, C.; Creasy, D.; Flashner, L.; Kani, K.; Moulding, C.; Seymour, S. L.; Nuwaysir, L. M.; Lefebvre, B.; Kuhlmann, F.; Roark, J.; Rainer, P.; Detlev, S.; Hemenway, T.; Huhmer, A.; Langridge, J.; Connolly, B.; Chadick, T.; Holly, K.; Eckels, J.; Deutsch, E. W.; Moritz, R. L.; Katz, J. E.; Agus, D. B.; MacCoss, M.; Tabb, D. L.; Mallick, P. Nat Biotech 2012, 30, 918-920. (29) Clavé, G.; Volland, H.; Flaender, M.; Gasparutto, D.; Romieu, A.; Renard, P.-Y. Org. Biomol. Chem. 2010, 8, 4329-4345. (30) Beal, D. M.; Jones, L. H. Angew. Chem. Int. Ed. 2012, 51, 6320-6326. (31) Chowdhury, S. M.; Du, X.; Tolic, N.; Wu, S.; Moore, R. J.; Mayer, M. U.; Smith, R. D.; Adkins, J. N. Anal. Chem. 2009, 81, 5524-5532. (32) Morpurgo, M.; Bayer, E. A.; Wilchek, M. J. Biochem. Biophys. Methods 1999, 38, 17-28. (33) Clavé, G.; Boutal, H.; Hoang, A.; Perraut, F.; Volland, H.; Renard, P.-Y.; Romieu, A. Org. Biomol. Chem. 2008, 6, 3065-3078. (34) Mhidia, R.; Vallin, A.; Ollivier, N.; Blanpain, A.; Shi, G.; Christiano, R.; Johannes, L.; Melnyk, O. Bioconjugate Chem. 2010, 21, 219-228. (35) Christine, C.; Koubemba, M.; Shakir, S.; Clavier, S.; Ehret-Sabatier, L.; Saupe, F.; Orend, G.; Charbonniere, L. J. Org. Biomol. Chem. 2012, 10, 9183-9190. (36) Kalkhof, S.; Sinz, A. Analytical and Bioanalytical Chemistry 2008, 392, 305-312. (37) Maedler, S.; Bich, C.; Touboul, D.; Zenobi, R. Journal of Mass Spectrometry 2009, 44, 694706. (38) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem. Int. Ed. 2001, 40, 2004-2021. (39) Tornoe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002, 67, 3057-3064. (40) Mohana Rao, J. K.; Bujacz, G.; Wlodawer, A. FEBS Letters 1998, 439, 133-137. (41) Rozanova, N. A.; Chetverikova, E. P. Biokhimiia (Moscow, Russia) 1981, 46, 2125-2135. (42) Leitner, A.; Reischl, R.; Walzthoeni, T.; Herzog, F.; Bohn, S.; Forster, F.; Aebersold, R. Mol. Cell Proteomics 2012, 11, 014126/014121-014126/014112. (43) Goedert, M. Nat Rev Neurosci 2001, 2, 492-501. (44) Spillantini, M. G.; Schmidt, M. L.; Lee, V. M. Y.; Trojanowski, J. Q.; Jakes, R.; Goedert, M. Nature 1997, 388, 839-840. (45) Pemberton, S.; Melki, R. Communicative & integrative biology 2012, 5, 94-95.


24

Page 25 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


AUTHOR INFORMATION *E-mail: [email protected] Note The authors declare no competing financial interest. ACKNOWLEDGMENT Synthesis and characterization of NNP9 were performed within the COBRA lab (Mont-SaintAignan, University of Rouen) and ICMUB (Dijon, University of Burgundy). Financial support from l'Agence Nationale de la Recherche (Programme PIRIBio 2009, ANR "CLICKMASSLINK") for a postdoctoral fellowship to S. D. and C. N. and for supporting the team from LEBS (ANR-11-BSV8-021-01) is greatly acknowledged. This work has been partially supported by INSA Rouen, Rouen University, CNRS, INSERM, Labex SynOrg (ANR-11LABX-0029) and région Haute-Normandie (CRUNCh network). Authors thank Dr. Antoine Eggenspiller for its contribution to the synthesis of NNP9, the Plateforme d'Analyse Chimique et de Synthèse Moléculaire de l'Université de Bourgogne (PACSMUB) for the access to device for NMR, IR and low resolution mass measurements respectively, and the platform “Identification and Characterization of Proteins by Mass Spectrometry” (SICaPS), a joint IMAGIF/Centre de Recherche de Gif and Federative Research Institute Genomes, Transcriptomes, Proteomes facility for help in automatic digestion and mass measurements. Prof. Anthony Romieu also thanks the Institut Universitaire de France (IUF) for financial support.

SUPPORTING INFORMATION AVAILABLE This information is available free of charge via the Internet at http://pubs.acs.org/.


25


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Page 26 of 28

Table 1

Identified with NNP9 Segment

Sequence

XL Site

Segment

Sequence

XL Site

136-148 237-242 237-247 299-307 133-151 012-025 171-177 012-025 293-304 117-130 293-304

SIKGYTLPPHCSR VISMEK VISmEKGGNmK LAHLSKHPK TGKSIKGYTLPPHCSRGER LNYKSEEEYPDLSK GKYYPLK LNYKSEEEYPDLSK GGVHVKLAHLSK GGDDLDPHYVLSSR GGVHVKLAHLSK

K138 S239 K242 K304 K135 K015 K172 K015 K298 Y125 K298

260-266 253-265 293-304 317-320 157-172 153-170 237-252 033-041 317-320 237-247 367-382

IEEIFKK FCVGLQKIEEIFKK GGVHVKLAHLSK LQKR LSVEALNSLTGEFKGK AVEKLSVEALNSLTGEFK VISMEKGGNMKEVFRR VLTPDLYKK LQKR VISmEKGGNmK LEKGQSIDDmIPAQK

K265 K259 K298 K319 S158 K156 K242 K040 K319 K242 K369

Monomer X X X X

Dimer Tetramer

Identified with BS3 Monomer

Dimer Tetramer

Found in [7] X

X X X X X X

X X

X

X

X X

X

X X

Distance (Cα/Cα) in Å 9.4 13.5 13.6 13.7 10.8 15.5* 21.9 8.7 12.2 16.5 15.7

*Intermolecular distance. Intramolecular distance: 48.2 Å


26

Page 27 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 2 Hsc70 Segment

Sequence

α-Synuclein XL site

494-500 STGKENK K497 494-500 STGKENK K497 494-500 STGKENK K497 494-500 STGKENK K497 STGKENK K497 494-500 494-500 STGKENK K497 494-500 STGKENK K497 494-500 STGKENK K497 494-500 STGKENK K497 494-500 STGKENK K497 501-509 ITITNDKGR K507 501-509 ITITNDKGR K507 501-509 ITITNDKGR K507 GRLSKEDIER 508-517 K512 525-533 YKAEDEKQR K531 534-539 DKVSSK K535 534-539 DKVSSK K535 534-539 DKVSSK K535 534-539 DKVSSK K535 551-561 ATVEDEKLQGK K557 551-561 ATVEDEKLQGK K557 551-561 ATVEDEKLQGK K557 551-561 ATVEDEKLQGK K557 551-561 ATVEDEKLQGK K557 551-561 ATVEDEKLQGK K557 558-569 LQGKINDEDKQK K561 558-569 LQGKINDEDKQK K561 558-569 LQGKINDEDKQK K561

Segment

Sequence

XL site

07-12 11-21 13-23 22-32 24-34 33-43 33-45 35-45 46-60 97-102 01-06 11-21 22-32 07-12 97-102 07-12 11-21 13-23 24-34 01-06 13-23 22-32 24-34 44-58 97-102 11-21 22-32 97-102

GLSKAK AKEGVVAAAEK EGVVAAAEKTK TKQGVAEAAGK QGVAEAAGKTK TKEGVLYVGSK TKEGVLYVGSKTK EGVLYVGSKTK EGVVHGVATVAEKTK KDQLGK MDFMK AKEGVVAAAEK TKQGVAEAAGK GLSKAK KDQLGK GLSKAK AKEGVVAAAEK EGVVAAAEKTK QGVAEAAGKTK MDFMK EGVVAAAEKTK TKQGVAEAAGK QGVAEAAGKTK TKEGVVHGVATVAEK KDQLGK AKEGVVAAAEK TKQGVAEAAGK KDQLGK

K10 K12 K21 K23 K32 K34 K43 K43 K58 K97 M01 K12 K23 K10 K97 K10 K12 K21 K32 M01 K21 K23 K32 K45 K97 K12 K23 K97

Identified with

Identified with

NNP9

BS3 d0/d4

X X X X X X X X X X X X

X X X X

Identified with BS2G d0/d4

18

X X X X X

X X

X X X X X X X

X X X

X X X X X X X

X X

X

X

X


27


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Page 28 of 28


NNP9 cross-linker

New Hsc70/α-Synuclein interaction map

Protein

Recommend Documents