Structure of prion ß-oligomers as determined by short-distance crosslinking constraint-guided discrete molecular dynamics simulations.

The conversion of the native monomeric cellular prion protein (PrPC ) into an aggregated pathological ß-oligomeric form (PrPß ) and an infectious form (PrPSc ) is the central element in the development of prion diseases. The structure of the aggregates and the molecular mechanisms of the conformational changes involved in the conversion are still unknown. We applied mass spectrometry combined with chemical crosslinking, hydrogen/deuterium exchange, limited proteolysis, and surface modification for the differential characterization of the native and the urea+acid-converted prion ß-oligomer structures to obtain insights into the mechanisms of conversion and aggregation. For the determination of the structure of the monomer and the dimer unit of the ß-oligomer, we applied a recently-developed approach for de novo protein structure determination which is based on the incorporation of zero-length and short-distance crosslinking data as intra- and inter-protein constraints in discrete molecular dynamics simulations (CL-DMD). Based on all of the structural-proteomics experimental data and the computationally predicted structures of the monomer units, we propose the potential mode of assembly of the ß-oligomer. The proposed ß-oligomer assembly provides a clue on the ß-sheet nucleation site, and how template-based conversion of the native prion molecule occurs, growth of the prion aggregates, and maturation into fibrils may occur.

The prolyl isomerase FKBP25 regulates microtubule polymerization impacting cell cycle progression and genomic stability.

FK506 binding proteins (FKBPs) catalyze the interconversion of cis-trans proline conformers in proteins. Importantly, FK506 drugs have anti-cancer and neuroprotective properties, but the effectors and mechanisms underpinning these properties are not well understood because the cellular function(s) of most FKBP proteins are unclear. FKBP25 is a nuclear prolyl isomerase that interacts directly with nucleic acids and is associated with several DNA/RNA binding proteins. Here, we show the catalytic FKBP domain binds microtubules (MTs) directly to promote their polymerization and stabilize the MT network. Furthermore, FKBP25 associates with the mitotic spindle and regulates entry into mitosis. This interaction is important for mitotic spindle dynamics, as we observe increased chromosome instability in FKBP25 knockdown cells. Finally, we provide evidence that FKBP25 association with chromatin is cell-cycle regulated by Protein Kinase C phosphorylation. This disrupts FKBP25-DNA contacts during mitosis while maintaining its interaction with the spindle apparatus. Collectively, these data support a model where FKBP25 association with chromatin and MTs is carefully choreographed to ensure faithful genome duplication. Additionally, they highlight that FKBP25 is a MT-associated FK506 receptor and potential therapeutic target in MT-associated diseases.

The basic tilted helix bundle domain of the prolyl isomerase FKBP25 is a novel double-stranded RNA binding module.

Prolyl isomerases are defined by a catalytic domain that facilitates the cis-trans interconversion of proline residues. In most cases, additional domains in these enzymes add important biological function, including recruitment to a set of protein substrates. Here, we report that the N-terminal basic tilted helix bundle (BTHB) domain of the human prolyl isomerase FKBP25 confers specific binding to double-stranded RNA (dsRNA). This binding is selective over DNA as well as single-stranded oligonucleotides. We find that FKBP25 RNA-association is required for its nucleolar localization and for the vast majority of its protein interactions, including those with 60S pre-ribosome and early ribosome biogenesis factors. An independent mobility of the BTHB and FKBP catalytic domains supports a model by which the N-terminus of FKBP25 is anchored to regions of dsRNA, whereas the FKBP domain is free to interact with neighboring proteins. Apart from the identification of the BTHB as a new dsRNA-binding module, this domain adds to the growing list of auxiliary functions used by prolyl isomerases to define their primary cellular targets.

Expression and purification of the full murine NPM2 and study of its interaction with protamines and histones.

Mouse nucleoplasmin M.NPM2 was recombinantly expressed and the protein consisting of the complete sequence was purified and characterized. Similar to its Xenopus laevis X.NPM2 counterpart, the protein forms stable pentameric complexes and exhibits an almost undistinguishable hydrodynamic ionic strength-dependent unfolding behavior. The interaction of N.PM2 with histones and mouse P1/P2 protamines revealed that these chromosomal proteins bind preferentially to the distal part of the nucleoplasmin pentamer. Moreover, the present work highlights the critical role played by histones H2B and H4 in the association of the histone H2A-H2B dimers and histone octamer with nucleoplasmin.

Comprehensive identification of disulfide bonds using non-specific proteinase K digestion and CID-cleavable crosslinking analysis methodology for Orbitrap LC/ESI-MS/MS data.

Disulfide bonds are valuable constraints in protein structure modeling. The Cys-Cys disulfide bond undergoes specific fragmentation under CID and, therefore, can be considered as a CID-cleavable crosslink. We have recently reported on the benefits of using non-specific digestion with proteinase K for inter-peptide crosslink determination. Here, we describe an updated application of our CID-cleavable crosslink analysis software and our crosslinking analysis with non-specific digestion methodology for the robust and comprehensive determination of disulfide bonds in proteins, using Orbitrap LC/ESI-MS/MS data.

(14)N(15)N DXMSMS Match program for the automated analysis of LC/ESI-MS/MS crosslinking data from experiments using (15)N metabolically labeled proteins.

Crosslinking mass spectrometric applications for the study of proteins and protein complexes benefit from using (15)N metabolically labeled proteins. Peptides, derived from crosslinked (14)N and (15)N proteins (used in a 1:1molar ratio), exhibit specific mass spectrometric signatures of doublets of peaks, reflecting the number of nitrogen atoms in the peptides. This can be used as an additional search criterion for assignment of the crosslinks. Here, we describe the further development of our ICC-CLASS software suite which is designed for automatic analysis of mass spectrometric crosslinking data, by the addition of the (14)N(15)N DXMSMS Match program. The program is designed to assist in distinguishing inter- from intra-molecular crosslinks at the interface of homodimers in protein aggregation studies. The program takes into account the number of nitrogen atoms present in (14)N(15)N-labeled crosslinked peptides and uses it as an additional parameter for the identification of crosslinks based on both the MS and MS/MS spectra. This greatly increases the confidence of the assignments, and this approach can be successfully used in other types of complicated crosslinking experiments, such as those with non-specific crosslinking sites, non-specific digestion, zero-length crosslinking, or crosslinking with unknown reaction mechanisms, by facilitating the use of (15)N metabolically labeled proteins. BIOLOGICAL SIGNIFICANCE: The new (14)N(15)N DXMSMS Match software program is a practical tool for the efficient assignment of crosslinks from LC-MS/MS experiments using an equimolar mixture of non-labeled and (15)N metabolically labeled proteins. It greatly facilitates automated data analysis from complicated crosslinking experiments, such as those using zero-length crosslinkers and those involving only a few crosslinking and digestion site restrictions.

Analysis of protein structure by cross-linking combined with mass spectrometry.

Cross-linking combined with mass spectrometry is a powerful technique to study protein structure. Here, we present an optimized protocol for the preparation, processing, and analysis of a protein sample cross-linked with isotopically coded, affinity-enrichable, and CID-cleavable cross-linker CyanurBiotinDimercaptoPropionylSuccinimide using LC/ESI-MS/MS on a Thermo Scientific Orbitrap mass spectrometer.

The prolyl isomerase, FKBP25, interacts with RNA-engaged nucleolin and the pre-60S ribosomal subunit.

Peptidyl-proline isomerases of the FK506-binding protein (FKBP) family belong to a class of enzymes that catalyze the cis-trans isomerization of prolyl-peptide bonds in proteins. A handful of FKBPs are found in the nucleus, implying that the isomerization of proline in nuclear proteins is enzymatically controlled. FKBP25 is a nuclear protein that has been shown to associate with chromatin modifiers and transcription factors. In this study, we performed the first proteomic characterization of FKBP25 and found that it interacts with numerous ribosomal proteins, ribosomal processing factors, and a small selection of chromatin modifiers. In agreement with previous reports, we found that nucleolin is a major FKBP25-interacting protein and demonstrated that this interaction is dependent on rRNA. FKBP25 interacts with the immature large ribosomal subunit in nuclear extract but does not associate with mature ribosomes, implicating this FKBP's action in ribosome biogenesis. Despite engaging nascent 60S ribosomes, FKBP25 does not affect steady-state levels of rRNAs or its pre-rRNA intermediates. We conclude that FKBP25 is likely recruited to preribosomes to chaperone one of the protein components of the ribosome large subunit.

"Out-gel" tryptic digestion procedure for chemical cross-linking studies with mass spectrometric detection.

SDS-PAGE is one of the most powerful protein separation techniques, and in-gel digestion is the leading method for converting proteins separated by SDS-PAGE into peptides suitable for mass spectrometry-based proteomic studies. In in-gel digestion, proteins are digested within the gel matrix, and the resulting peptides are extracted into an appropriate buffer. Transfer of the digested peptides to the liquid phase for subsequent mass spectrometric analysis, however, may be hampered by peptide-specific characteristics, including size, shape, poor solubility, adsorption to the polyacrylamide, and-in the case of cross-linking applications-by the branched structure of the peptides produced. This can be a limitation in cross-linking studies where efficient recoveries of the cross-linked peptides are critical. To overcome this limitation, we have developed a modification to the standard in-gel digestion procedure for SDS-PAGE-separated cross-linked proteins, based on older passive diffusion methods. By omitting the gel staining and gel fixation steps, intact proteins or cross-linked protein complexes can move through the gel and into the buffer solution. Digestion of the entire protein in the buffer outside the gel increases the probability that most of the proteolytic peptides produced will be present in the final digest solution. The resulting peptide mixture is then freed of SDS and concentrated using SCX (strong cation exchange) zip-tips and analyzed by mass spectrometry. For standard protein identification studies and the recovery of noncross-linked peptides, the in-gel procedure outperformed the out-gel procedure, but for cross-linking studies with enrichable cross-linkers (such as CBDPS), the standard out-gel procedure allowed the recoveries of cross-links not recovered via the in-gel method. Labeling experiments showed that, with an enrichable cross-linker, 93% of the cross-links showed better or equal recoveries with the out-gel procedure, as compared to the in-gel procedure. It should be noted that this method is not designed to replace in-gel digestion for most proteomics applications. However, by using the out-gel method, we were able to detect twice as many interprotein CBDPS cross-links from the histone H2A/H2B complex as were found in the in-gel digested sample.

Using isotopically-coded hydrogen peroxide as a surface modification reagent for the structural characterization of prion protein aggregates.

The conversion of the cellular prion protein (PrP(C)) into aggregated ß-oligomeric (PrP(ß)) and fibril (PrP(Sc)) forms is the central element in the development of prion diseases. Here we report the first use of isotopically-coded hydrogen peroxide surface modification combined with mass spectrometry (MS) for the differential characterization of PrP(C) and PrP(ß). (16)O and (18)O hydrogen peroxide were used to oxidize methionine and tryptophan residues in PrP(C) and PrP(ß), allowing for the relative quantitation of the extent of modification of each form of the prion protein. After modification with either light or heavy forms of hydrogen peroxide (H2(16)O2 and H2(18)O2), the PrP(C) and PrP(ß) forms of the protein were then combined, digested with trypsin, and analysed by LC-MS. The (18)O/(16)O signal intensity ratios were used to determine the relative levels of oxidation of specific amino acids in the PrP(C) and PrP(ß) forms. Using this approach we have detected several residues that are differentially-oxidized between the native and ß-oligomeric prion forms, allowing determination of the regions of PrP(C) involved in the formation of PrP(ß) aggregates. Modification of these residues in the ß-oligomeric form is compatible with a flip of the ß1-H1-ß2 loop away from amphipathic helices 2 and 3 during conversion. BIOLOGICAL SIGNIFICANCE: Surface modification using isotopically-coded hydrogen peroxide has allowed quantitative comparison of the exposure of methionine and tryptophan residues in PrP(C) and PrP(ß) forms of prion protein. Detected changes in surface exposure of a number of residues have indicated portions of the PrP structure which undergo conformational transition upon conversion. This article is part of a Special Issue entitled: Can Proteomics Fill the Gap Between Genomics and Phenotypes?

Structural and biochemical characterization of Plasmodium falciparum 12 (Pf12) reveals a unique interdomain organization and the potential for an antiparallel arrangement with Pf41.

Plasmodium falciparum is the most devastating agent of human malaria. A major contributor to its virulence is a complex lifecycle with multiple parasite forms, each presenting a different repertoire of surface antigens. Importantly, members of the 6-Cys s48/45 family of proteins are found on the surface of P. falciparum in every stage, and several of these antigens have been investigated as vaccine targets. Pf12 is the archetypal member of the 6-Cys protein family, containing just two s48/45 domains, whereas other members have up to 14 of these domains. Pf12 is strongly recognized by immune sera from naturally infected patients. Here we show that Pf12 is highly conserved and under purifying selection. Immunofluorescence data reveals a punctate staining pattern with an apical organization in late schizonts. Together, these data are consistent with an important functional role for Pf12 in parasite-host cell attachment or invasion. To infer the structural and functional diversity between Pf12 and the other 11 6-Cys domain proteins, we solved the 1.90 Å resolution crystal structure of the Pf12 ectodomain. Structural analysis reveals a unique organization between the membrane proximal and membrane distal domains and clear homology with the SRS-domain containing proteins of Toxoplasma gondii. Cross-linking and mass spectrometry confirm the previously identified Pf12-Pf41 heterodimeric complex, and analysis of individual cross-links supports an unexpected antiparallel organization. Collectively, the localization and structure of Pf12 and details of its interaction with Pf41 reveal important insight into the structural and functional properties of this archetypal member of the 6-Cys protein family.

Using multiple structural proteomics approaches for the characterization of prion proteins.

Structural proteomics approaches are valuable tools, particularly in cases where the exact mechanisms of protein conformational changes or the structures of proteins and protein complexes cannot be elucidated by traditional structural biology techniques like X-ray crystallography or NMR methods. Each structural proteomics method can provide a different set of data, all of which can be used as structural constraints for modeling the protein. We have applied a combination of limited proteolysis, surface modification, chemical crosslinking, and hydrogen/deuterium exchange for the characterization of structural differences in prion proteins in native monomeric and in the aggregated ß-oligomeric states. Data from these multiple proteomics approaches are in remarkable agreement in pointing to the rearrangement of the beta sheet 1-helix1-beta sheet 2-helix 2 (ß1-H1-ß2-H2) region as a major conformational change between the native and oligomeric prion protein forms. This data is also consistent with the ß1-H1-ß2 loop moving away from the H2-H3 core during the prion protein conversion. This is an example of how complementary data from multiple structural proteomics approaches can provide novel insights into the three-dimensional structures of proteins and protein complexes. This article is part of a Special Issue entitled: From protein structures to clinical applications.

Mass spectrometry-based structural proteomics.

Structural proteomics is the application of protein chemistry and modern mass spectrometric techniques to problems such as the characterization of protein structures and assemblies and the detailed determination of protein-protein interactions. The techniques used in structural proteomics include crosslinking, photoaffinity labeling, limited proteolysis, chemical protein modification and hydrogen/deuterium exchange, all followed by mass spectrometric analysis. None of these methods alone can provide complete structural information, but a "combination" of these complementary approaches can be used to provide enough information for answering important biological questions. Structural proteomics can help to determine, for example, the detailed structure of the interfaces between proteins that may be important drug targets and the interactions between proteins and ligands. In this review, we have tried to provide a brief overview of structural proteomics methodologies, illustrated with examples from our laboratory and from the literature.

Use of proteinase K nonspecific digestion for selective and comprehensive identification of interpeptide cross-links: application to prion proteins.

Chemical cross-linking combined with mass spectrometry is a rapidly developing technique for structural proteomics. Cross-linked proteins are usually digested with trypsin to generate cross-linked peptides, which are then analyzed by mass spectrometry. The most informative cross-links, the interpeptide cross-links, are often large in size, because they consist of two peptides that are connected by a cross-linker. In addition, trypsin targets the same residues as amino-reactive cross-linkers, and cleavage will not occur at these cross-linker-modified residues. This produces high molecular weight cross-linked peptides, which complicates their mass spectrometric analysis and identification. In this paper, we examine a nonspecific protease, proteinase K, as an alternative to trypsin for cross-linking studies. Initial tests on a model peptide that was digested by proteinase K resulted in a "family" of related cross-linked peptides, all of which contained the same cross-linking sites, thus providing additional verification of the cross-linking results, as was previously noted for other post-translational modification studies. The procedure was next applied to the native (PrP(C)) and oligomeric form of prion protein (PrPß). Using proteinase K, the affinity-purifiable CID-cleavable and isotopically coded cross-linker cyanurbiotindipropionylsuccinimide and MALDI-MS cross-links were found for all of the possible cross-linking sites. After digestion with proteinase K, we obtained a mass distribution of the cross-linked peptides that is very suitable for MALDI-MS analysis. Using this new method, we were able to detect over 60 interpeptide cross-links in the native PrP(C) and PrPß prion protein. The set of cross-links for the native form was used as distance constraints in developing a model of the native prion protein structure, which includes the 90-124-amino acid N-terminal portion of the protein. Several cross-links were unique to each form of the prion protein, including a Lys(185)-Lys(220) cross-link, which is unique to the PrPß and thus may be indicative of the conformational change involved in the formation of prion protein oligomers.

MeCP2 binds to nucleosome free (linker DNA) regions and to H3K9/H3K27 methylated nucleosomes in the brain.

Methyl-CpG-binding protein 2 (MeCP2) is a chromatin-binding protein that mediates transcriptional regulation, and is highly abundant in brain. The nature of its binding to reconstituted templates has been well characterized in vitro. However, its interactions with native chromatin are less understood. Here we show that MeCP2 displays a distinct distribution within fractionated chromatin from various tissues and cell types. Artificially induced global changes in DNA methylation by 3-aminobenzamide or 5-aza-2'-deoxycytidine, do not significantly affect the distribution or amount of MeCP2 in HeLa S3 or 3T3 cells. Most MeCP2 in brain is chromatin-bound and localized within highly nuclease-accessible regions. We also show that, while in most tissues and cell lines, MeCP2 forms stable complexes with nucleosome, in brain, a fraction of it is loosely bound to chromatin, likely to nucleosome-depleted regions. Finally, we provide evidence for novel associations of MeCP2 with mononucleosomes containing histone H2A.X, H3K9me(2) and H3K27me(3) in different chromatin fractions from brain cortex and in vitro. We postulate that the functional compartmentalization and tissue-specific distribution of MeCP2 within different chromatin types may be directed by its association with nucleosomes containing specific histone variants, and post-translational modifications.

An isotopically coded CID-cleavable biotinylated cross-linker for structural proteomics.

Successful application of cross-linking combined with mass spectrometry for structural proteomics demands specifically designed cross-linking reagents to address challenges in the detection and assignment of cross-links. A combination of affinity enrichment, isotopic coding, and cleavage of the cross-linker is beneficial for detection and identification of the peptide cross-links. Here we describe a novel cross-linker, cyanurbiotindipropionylsuccinimide (CBDPS), that allows affinity enrichment of cross-linker-containing peptides with avidin. Affinity enrichment eliminates interfering non-cross-linked peptides and allows the researcher to focus on the analysis of the cross-linked peptides. CBDPS is also isotopically coded and CID-cleavable. The cleaved fragments still contain a portion of the isotopic label and can therefore be distinguished from unlabeled fragments by their distinct isotopic signatures in the MS/MS spectra. This cleavage information has been incorporated into a program for the automatic analysis of the MS/MS spectra of the cross-links. This allows rapid determination of cross-link type in addition to facilitating identification of the individual peptides constituting the interpeptide cross-links. Thus, affinity enrichment combined with isotopic coding and CID cleavage allows in-depth mass spectrometric analysis of the peptide cross-links. We have characterized the performance of CBDPS on the 120-kDa protein heterodimer of HIV reverse transcriptase.

Use of a combination of isotopically coded cross-linkers and isotopically coded N-terminal modification reagents for selective identification of inter-peptide crosslinks.

Cross-linking combined with mass spectrometry has great potential for determining three-dimensional structures of proteins and protein assemblies. One of the main analytical challenges of this method is the specific detection and identification of the inter-peptide crosslinks in the peptide mixture after enzymatic digestion of the cross-linked protein complex. These inter-peptide crosslinks are important because they provide the critical distance information needed for structural proteomics studies. In this paper, we demonstrate the use of isotopically coded N-terminal modification (ICNTM) in combination with isotopically coded cross-linkers (ICCL) for specific detection of inter-peptide crosslinks. Inter-peptide crosslinks contain two amino termini, compared to one in the case of free peptides, dead-end crosslinks, or intra-peptide crosslinks. Therefore, N-terminal modification with a 1:1 mixture of heavy and light isotopically coded reagents produces inter-peptide crosslinks with a distinct isotopic signature (a 1:2:1 ratio). Modification also occurs at the epsilon-amino groups of non-cross-linked lysine residues, resulting in two modifications per free lysine-containing peptide. However, if ICCL and ICNTM are used together, inter-peptide crosslinks can be distinguished from free lysine-containing peptides. Specialized software has also been developed for the analysis of ICCL + ICNTM experimental data. This procedure, combined with software for data analysis, provides a simple and rapid method for specific detection of inter-peptide crosslinks.