Karl A.T. Makepeace

Structural and Biochemical Characterization of Plasmodium falciparum 12 (Pf12) Reveals a Unique Interdomain Organization and the Potential for an Antiparallel Arrangement with Pf41

Background: Pf12 is the archetypal member of the 6-Cys protein family, members of which are important Plasmodium vaccine targets. Results: Purifying selection and apical localization of Pf12, crystal structure of tandem 6-Cys domains, and mass spectrometry of cross-linked Pf12-Pf41 heterodimer are shown. Conclusion: A functionally important role for Pf12 and potential for antiparallel heterodimer is provided. Significance: First full-length 6-Cys protein structure and first details of heterodimer organization are revealed. 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.

Super Spy variants implicate flexibility in chaperone action.

Experimental study of the role of disorder in protein function is challenging. It has been proposed that proteins utilize disordered regions in the adaptive recognition of their various binding partners. However apart from a few exceptions, defining the importance of disorder in promiscuous binding interactions has proven to be difficult. In this paper, we have utilized a genetic selection that links protein stability to antibiotic resistance to isolate variants of the newly discovered chaperone Spy that show an up to 7 fold improved chaperone activity against a variety of substrates. These “Super Spy” variants show tighter binding to client proteins and are generally more unstable than is wild type Spy and show increases in apparent flexibility. We establish a good relationship between the degree of their instability and the improvement they show in their chaperone activity. Our results provide evidence for the importance of disorder and flexibility in chaperone function. DOI: http://dx.doi.org/10.7554/eLife.01584.001

Isotopically-coded short-range hetero-bifunctional photo-reactive crosslinkers for studying protein structure.

UNLABELLED The resolution and the fidelity of a protein structural model, constructed using crosslinking data, is dependent on the crosslinking distance constraints. Most of the popular amine-reactive NHS-ester crosslinkers are limited in their capacity to provide short distance constraints because of the rarity of lysine residues occurring in close proximity in the protein structure. To solve this problem, hetero-bifunctional crosslinkers containing both a photo-reactive functional group and an NHS-ester group can be used to enable non-specific crosslinking within the proximity of these lysine residues. Here we develop three such isotopically-coded hetero-bifunctional photo-reactive crosslinkers, bearing azido, diazirine or benzophenone photo-reactive groups (azido-benzoic-acid-succinimide (ABAS)-(12)C6/(13)C6, succinimidyl-diazirine (SDA)-(12)C5/(13)C5, and carboxy-benzophenone-succinimide (CBS)-(12)C6/(13)C6, respectively). These crosslinkers were validated using several model proteins/peptides and were then applied to study the structure of the native α-synuclein protein. In that case the ABAS crosslinker proved to be the most suitable, with 10 crosslinks being found in the native α-synuclein structure. BIOLOGICAL SIGNIFICANCE Structural proteomics can be used for studying protein structures which may be difficult to examine by traditional structural biology methods such as NMR or X-ray crystallography. Crosslinking in particular is used to provide distance constraints for molecular modeling of individual proteins and protein complexes. The shortest distance constraints are most valuable for the modeling process. To be able to provide such short distance constraints, non-specific photo-reactive chemistry can be used for crosslinking reactions. However, detection of such non-specific crosslinks is difficult because the signal from any particular crosslink is low due to the broad reactivity of the crosslinking reagents. To overcome this problem, we have employed isotopic labeling of these crosslinkers. In this paper, we have demonstrated their effectiveness for studying the native α-synuclein protein structure. The non-specific reactivity, in combination with isotopic coding of these crosslinkers, allowed for the formation and detection of short-range crosslinks, targeting a variety of amino acids. These reagents may prove useful for future applications to a variety of protein structural problems. This article is part of a Special Issue entitled: Protein dynamics in health and disease. Guest Editors: Pierre Thibault and Anne-Claude Gingras.

Structure of EspB from the ESX-1 Type VII Secretion System and Insights into its Export Mechanism

Mycobacterium tuberculosis (Mtb) uses the ESX-1 type VII secretion system to export virulence proteins across its lipid-rich cell wall, which helps permeabilize the hosts macrophage phagosomal membrane, facilitating the escape and cell-to-cell spread of Mtb. ESX-1 membranolytic activity depends on a set of specialized secreted Esp proteins, the structure and specific roles of which are not currently understood. Here, we report the X-ray and electron microscopic structures of the ESX-1-secreted EspB. We demonstrate that EspB adopts a PE/PPE-like fold that mediates oligomerization with apparent heptameric symmetry, generating a barrel-shaped structure with a central pore that we propose contributes to the macrophage killing functions of EspB. Our structural data also reveal unexpected direct interactions between the EspB bipartite secretion signal sequence elements that form a unified aromatic surface. These findings provide insight into how specialized proteins encoded within the ESX-1 locus are targeted for secretion, and for the first time indicate an oligomerization-dependent role for Esp virulence factors.

14N15N DXMSMS Match program for the automated analysis of LC/ESI-MS/MS crosslinking data from experiments using 15N metabolically labeled proteins

UNLABELLED 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.

DXMSMS Match Program for Automated Analysis of LC‐MS/MS Data Obtained Using Isotopically Coded CID‐Cleavable Cross‐Linking Reagents

Cross‐linking combined with mass spectrometry for the study of proteins and protein complexes is greatly facilitated by the use of isotopically coded cleavable cross‐linking reagents. The isotopic coding of the cross‐linker enables confident detection of the cross‐link signals, while cleavage of the cross‐linker provides masses of the individual peptides composing the cross‐link and, therefore, facilitates unambiguous assignment of the cross‐links. Here, we describe the DXMSMS Match program, designed for automatic analysis of LC‐MS/MS mass spectrometric data obtained with isotopically coded CID‐cleavable cross‐linkers. The program verifies the assignments of the cross‐links by precursor mass and by inspection of the MS/MS spectra for the fragments and the cleavage products of the cross‐linked peptides. The program produces nonprobabilistic scores for matching the spectra to the theoretical fragmentation of the cross‐links and a visual interface for the validation of the mass spectral matches.

Protein unfolding as a switch from self-recognition to high-affinity client binding

Stress-specific activation of the chaperone Hsp33 requires the unfolding of a central linker region. This activation mechanism suggests an intriguing functional relationship between the chaperones own partial unfolding and its ability to bind other partially folded client proteins. However, identifying where Hsp33 binds its clients has remained a major gap in our understanding of Hsp33s working mechanism. By using site-specific Fluorine-19 nuclear magnetic resonance experiments guided by in vivo crosslinking studies, we now reveal that the partial unfolding of Hsp33s linker region facilitates client binding to an amphipathic docking surface on Hsp33. Furthermore, our results provide experimental evidence for the direct involvement of conditionally disordered regions in unfolded protein binding. The observed structural similarities between Hsp33s own metastable linker region and client proteins present a possible model for how Hsp33 uses protein unfolding as a switch from self-recognition to high-affinity client binding.

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

UNLABELLED 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(β). (16)O and (18)O hydrogen peroxide were used to oxidize methionine and tryptophan residues in PrP(C) and PrP(β), 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(β) 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(β) forms. Using this approach we have detected several residues that are differentially-oxidized between the native and β-oligomeric prion forms, allowing determination of the regions of PrP(C) involved in the formation of PrP(β) aggregates. Modification of these residues in the β-oligomeric form is compatible with a flip of the β1-H1-β2 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?

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.