Realizing the AF4-UV-SAXS on-line coupling on protein and antibodies using high flux synchrotron radiation at the CoSAXS beamline, MAX IV.

In this paper, we demonstrate the coupling of synchrotron small angle X-ray scattering (SAXS) to asymmetrical flow-field flow fractionation (AF4) for protein characterization. To the best of our knowledge, this is the first time AF4 is successfully coupled to a synchrotron for on-line measurements on proteins. This coupling has potentially high impact, as it opens the possibility to characterize individual constituents of sensitive and/or complex samples, not suited for separation using other techniques, and for low electron density samples where high X-ray flux is required, e.g., biomolecules and biologics. AF4 fractionates complex samples in native or close to native environment, with low shear forces and system surface area. Many orders of magnitude in size can be fractionated in one measurement, without having to reconfigure the experimental setup. We report AF4 fractionations with correlated UV and statistically adequate SAXS data of bovine serum albumin and a monoclonal antibody and evaluate SAXS data recorded for the two protein systems.

Structural Characterization Study of a Lipid Nanocapsule Formulation Intended for Drug Delivery Applications Using Small-Angle Scattering Techniques.

Lipid nanocapsules (LNCs) are increasingly being used for various drug delivery applications due to their versatile nature and ability to carry a wide variety of therapeutic drug molecules. In the present investigation, small-angle X-ray (SAXS) and neutron scattering (SANS) techniques were used to elucidate the structure of LNCs. Overall, size measurements obtained from SAXS and SANS techniques were complemented with dynamic light scattering, zeta potential, and cryogenic transmission electron microscopy measurements. The structural aspects of LNCs can be affected by drug loading and the properties of the drug. Here, the impact of drug loading on the overall structure was evaluated using DF003 as a model drug molecule. LNCs with varying compositions were prepared using a phase inversion method. Combined analysis of SAXS and SANS measurements indicated the presence of a core-shell structure in the LNCs. Further, the drug loading did not alter the overall core-shell structure of the LNCs. SANS data revealed that the core size remained unchanged with a radius of 20.0 ± 0.9 nm for unloaded LNCs and 20.2 ± 0.6 nm for drug-loaded LNCs. Furthermore, interestingly, the shell becomes thicker in an order of ∼1 nm in presence of the drug compared to the shell thickness of unloaded LNCs as demonstrated by SAXS data. This can be correlated with the strong association of hydrophilic DF003 with Kolliphor HS 15, a polyethylene glycol-based surfactant that predominantly makes up the shell, resulting in a drug-rich hydrated shell.

Characterization of binding between model protein GA-Z and human serum albumin using asymmetrical flow field-flow fractionation and small angle X-ray scattering.

Protein-based drugs often require targeted drug delivery for optimal therapy. A successful strategy to increase the circulation time of the protein in the blood is to link the therapeutic protein with an albumin-binding domain. In this work, we characterized such a protein-based drug, GA-Z. Using asymmetrical flow field-flow fractionation coupled with multi-angle light scattering (AF4-MALS) we investigated the GA-Z monomer-dimer equilibrium as well as the molar binding ratio of GA-Z to HSA. Using small angle X-ray scattering, we studied the structure of GA-Z as well as the complex between GA-Z and HSA. The results show that GA-Z is predominantly dimeric in solution at pH 7 and that it binds to monomeric as well as dimeric HSA. Furthermore, GA-Z binds to HSA both as a monomer and a dimer, and thus, it can be expected to stay bound also upon dilution following injection in the blood stream. The results from SAXS and binding studies indicate that the GA-Z dimer is formed between two target domains (Z-domains). The results also indicate that the binding of GA-Z to HSA does not affect the ratio between HSA dimers and monomers, and that no higher order oligomers of the complex are seen other than those containing dimers of GA-Z and dimers of HSA.

Concentrated protein solutions investigated using acoustic levitation and small-angle X-ray scattering.

An acoustically levitated droplet has been used to collect synchrotron SAXS data on human serum albumin protein solutions up to a protein concentration of 400 mg ml-1. A careful selection of experiments allows for fast data collection of a large amount of data, spanning a protein concentration/solvent concentration space with limited sample consumption (down to 3 µL per experiment) and few measurements. The data analysis shows data of high quality that are reproducible and comparable with data from standard flow-through capillary-based experiments. Furthermore, using this methodology, it is possible to achieve concentrations that would not be accessible by conventional cells. The protein concentration and ionic strength parameter space diagram may be covered easily and the amount of protein sample is significantly reduced (by a factor of 100 in this work). Used in routine measurements, the benefits in terms of protein cost and time spent are very significant.

Author Correction: Active-site plasticity revealed in the asymmetric dimer of AnPrx6 the 1-Cys peroxiredoxin and molecular chaperone from Anabaena sp. PCC 7120.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Structural modelling of the DNAJB6 oligomeric chaperone shows a peptide-binding cleft lined with conserved S/T-residues at the dimer interface.

The remarkably efficient suppression of amyloid fibril formation by the DNAJB6 chaperone is dependent on a set of conserved S/T-residues and an oligomeric structure, features unusual among DNAJ chaperones. We explored the structure of DNAJB6 using a combination of structural methods. Lysine-specific crosslinking mass spectrometry provided distance constraints to select a homology model of the DNAJB6 monomer, which was subsequently used in crosslink-assisted docking to generate a dimer model. A peptide-binding cleft lined with S/T-residues is formed at the monomer-monomer interface. Mixed isotope crosslinking showed that the oligomers are dynamic entities that exchange subunits. The purified protein is well folded, soluble and composed of oligomers with a varying number of subunits according to small-angle X-ray scattering (SAXS). Elongated particles (160 × 120 Å) were detected by electron microscopy and single particle reconstruction resulted in a density map of 20 Å resolution into which the DNAJB6 dimers fit. The structure of the oligomer and the S/T-rich region is of great importance for the understanding of the function of DNAJB6 and how it can bind aggregation-prone peptides and prevent amyloid diseases.

Chaperone-client interactions between Hsp21 and client proteins monitored in solution by small angle X-ray scattering and captured by crosslinking mass spectrometry.

The small heat shock protein (sHsp) chaperones are important for stress survival, yet the molecular details of how they interact with client proteins are not understood. All sHsps share a folded middle domain to which is appended flexible N- and C-terminal regions varying in length and sequence between different sHsps which, in different ways for different sHsps, mediate recognition of client proteins. In plants there is a chloroplast-localized sHsp, Hsp21, and a structural model suggests that Hsp21 has a dodecameric arrangement with six N-terminal arms located on the outside of the dodecamer and six inwardly-facing. Here, we investigated the interactions between Hsp21 and thermosensitive model substrate client proteins in solution, by small-angle X-ray scattering (SAXS) and crosslinking mass spectrometry. The chaperone-client complexes were monitored and the Rg -values were found to increase continuously during 20 min at 45°, which could reflect binding of partially unfolded clients to the flexible N-terminal arms of the Hsp21 dodecamer. No such increase in Rg -values was observed with a mutational variant of Hsp21, which is mainly dimeric and has reduced chaperone activity. Crosslinking data suggest that the chaperone-client interactions involve the N-terminal region in Hsp21 and only certain parts in the client proteins. These parts are peripheral structural elements presumably the first to unfold under destabilizing conditions. We propose that the flexible and hydrophobic N-terminal arms of Hsp21 can trap and refold early-unfolding intermediates with or without dodecamer dissociation.

Active-site plasticity revealed in the asymmetric dimer of AnPrx6 the 1-Cys peroxiredoxin and molecular chaperone from Anabaena sp. PCC 7210.

Peroxiredoxins (Prxs) are vital regulators of intracellular reactive oxygen species levels in all living organisms. Their activity depends on one or two catalytically active cysteine residues, the peroxidatic Cys (CP) and, if present, the resolving Cys (CR). A detailed catalytic cycle has been derived for typical 2-Cys Prxs, however, little is known about the catalytic cycle of 1-Cys Prxs. We have characterized Prx6 from the cyanobacterium Anabaena sp. strain PCC7120 (AnPrx6) and found that in addition to the expected peroxidase activity, AnPrx6 can act as a molecular chaperone in its dimeric state, contrary to other Prxs. The AnPrx6 crystal structure at 2.3 Å resolution reveals different active site conformations in each monomer of the asymmetric obligate homo-dimer. Molecular dynamic simulations support the observed structural plasticity. A FSH motif, conserved in 1-Cys Prxs, precedes the active site PxxxTxxCp signature and might contribute to the 1-Cys Prx reaction cycle.

Iron-induced oligomerization of human FXN81-210 and bacterial CyaY frataxin and the effect of iron chelators.

Patients suffering from the progressive neurodegenerative disease Friedreich's ataxia have reduced expression levels of the protein frataxin. Three major isoforms of human frataxin have been identified, FXN42-210, FXN56-210 and FXN81-210, of which FXN81-210 is considered to be the mature form. Both long forms, FXN42-210 and FXN56-210, have been shown to spontaneously form oligomeric particles stabilized by the extended N-terminal sequence. The short variant FXN81-210, on other hand, has only been observed in the monomeric state. However, a highly homologous E. coli frataxin CyaY, which also lacks an N-terminal extension, has been shown to oligomerize in the presence of iron. To explore the mechanisms of stabilization of short variant frataxin oligomers we compare here the effect of iron on the oligomerization of CyaY and FXN81-210. Using dynamic light scattering, small-angle X-ray scattering, electron microscopy (EM) and cross linking mass spectrometry (MS), we show that at aerobic conditions in the presence of iron both FXN81-210 and CyaY form oligomers. However, while CyaY oligomers are stable over time, FXN81-210 oligomers are unstable and dissociate into monomers after about 24 h. EM and MS studies suggest that within the oligomers FXN81-210 and CyaY monomers are packed in a head-to-tail fashion in ring-shaped structures with potential iron-binding sites located at the interface between monomers. The higher stability of CyaY oligomers can be explained by a higher number of acidic residues at the interface between monomers, which may result in a more stable iron binding. We also show that CyaY oligomers may be dissociated by ferric iron chelators deferiprone and DFO, as well as by the ferrous iron chelator BIPY. Surprisingly, deferiprone and DFO stimulate FXN81-210 oligomerization, while BIPY does not show any effect on oligomerization in this case. The results suggest that FXN81-210 oligomerization is primarily driven by ferric iron, while both ferric and ferrous iron participate in CyaY oligomer stabilization. Analysis of the amino acid sequences of bacterial and eukaryotic frataxins suggests that variations in the position of the acidic residues in helix 1, ß-strand 1 and the loop between them may control the mode of frataxin oligomerization.

SAXS and stability studies of iron-induced oligomers of bacterial frataxin CyaY.

Frataxin is a highly conserved protein found in both prokaryotes and eukaryotes. It is involved in several central functions in cells, which include iron delivery to biochemical processes, such as heme synthesis, assembly of iron-sulfur clusters (ISC), storage of surplus iron in conditions of iron overload, and repair of ISC in aconitase. Frataxin from different organisms has been shown to undergo iron-dependent oligomerization. At least two different classes of oligomers, with different modes of oligomer packing and stabilization, have been identified. Here, we continue our efforts to explore the factors that control the oligomerization of frataxin from different organisms, and focus on E. coli frataxin CyaY. Using small-angle X-ray scattering (SAXS), we show that higher iron-to-protein ratios lead to larger oligomeric species, and that oligomerization proceeds in a linear fashion as a results of iron oxidation. Native mass spectrometry and online size-exclusion chromatography combined with SAXS show that a dimer is the most common form of CyaY in the presence of iron at atmospheric conditions. Modeling of the dimer using the SAXS data confirms the earlier proposed head-to-tail packing arrangement of monomers. This packing mode brings several conserved acidic residues into close proximity to each other, creating an environment for metal ion binding and possibly even mineralization. Together with negative-stain electron microscopy, the experiments also show that trimers, tetramers, pentamers, and presumably higher-order oligomers may exist in solution. Nano-differential scanning fluorimetry shows that the oligomers have limited stability and may easily dissociate at elevated temperatures. The factors affecting the possible oligomerization mode are discussed.

Structural model of dodecameric heat-shock protein Hsp21: Flexible N-terminal arms interact with client proteins while C-terminal tails maintain the dodecamer and chaperone activity.

Small heat-shock proteins (sHsps) prevent aggregation of thermosensitive client proteins in a first line of defense against cellular stress. The mechanisms by which they perform this function have been hard to define due to limited structural information; currently, there is only one high-resolution structure of a plant sHsp published, that of the cytosolic Hsp16.9. We took interest in Hsp21, a chloroplast-localized sHsp crucial for plant stress resistance, which has even longer N-terminal arms than Hsp16.9, with a functionally important and conserved methionine-rich motif. To provide a framework for investigating structure-function relationships of Hsp21 and understanding these sequence variations, we developed a structural model of Hsp21 based on homology modeling, cryo-EM, cross-linking mass spectrometry, NMR, and small-angle X-ray scattering. Our data suggest a dodecameric arrangement of two trimer-of-dimer discs stabilized by the C-terminal tails, possibly through tail-to-tail interactions between the discs, mediated through extended IXVXI motifs. Our model further suggests that six N-terminal arms are located on the outside of the dodecamer, accessible for interaction with client proteins, and distinct from previous undefined or inwardly facing arms. To test the importance of the IXVXI motif, we created the point mutant V181A, which, as expected, disrupts the Hsp21 dodecamer and decreases chaperone activity. Finally, our data emphasize that sHsp chaperone efficiency depends on oligomerization and that client interactions can occur both with and without oligomer dissociation. These results provide a generalizable workflow to explore sHsps, expand our understanding of sHsp structural motifs, and provide a testable Hsp21 structure model to inform future investigations.

The Structure of the Complex between Yeast Frataxin and Ferrochelatase: CHARACTERIZATION AND PRE-STEADY STATE REACTION OF FERROUS IRON DELIVERY AND HEME SYNTHESIS.

Frataxin is a mitochondrial iron-binding protein involved in iron storage, detoxification, and delivery for iron sulfur-cluster assembly and heme biosynthesis. The ability of frataxin from different organisms to populate multiple oligomeric states in the presence of metal ions, e.g. Fe(2+) and Co(2+), led to the suggestion that different oligomers contribute to the functions of frataxin. Here we report on the complex between yeast frataxin and ferrochelatase, the terminal enzyme of heme biosynthesis. Protein-protein docking and cross-linking in combination with mass spectroscopic analysis and single-particle reconstruction from negatively stained electron microscopic images were used to verify the Yfh1-ferrochelatase interactions. The model of the complex indicates that at the 2:1 Fe(2+)-to-protein ratio, when Yfh1 populates a trimeric state, there are two interaction interfaces between frataxin and the ferrochelatase dimer. Each interaction site involves one ferrochelatase monomer and one frataxin trimer, with conserved polar and charged amino acids of the two proteins positioned at hydrogen-bonding distances from each other. One of the subunits of the Yfh1 trimer interacts extensively with one subunit of the ferrochelatase dimer, contributing to the stability of the complex, whereas another trimer subunit is positioned for Fe(2+) delivery. Single-turnover stopped-flow kinetics experiments demonstrate that increased rates of heme production result from monomers, dimers, and trimers, indicating that these forms are most efficient in delivering Fe(2+) to ferrochelatase and sustaining porphyrin metalation. Furthermore, they support the proposal that frataxin-mediated delivery of this potentially toxic substrate overcomes formation of reactive oxygen species.

Architecture of the Yeast Mitochondrial Iron-Sulfur Cluster Assembly Machinery: THE SUB-COMPLEX FORMED BY THE IRON DONOR, Yfh1 PROTEIN, AND THE SCAFFOLD, Isu1 PROTEIN.

The biosynthesis of Fe-S clusters is a vital process involving the delivery of elemental iron and sulfur to scaffold proteins via molecular interactions that are still poorly defined. We reconstituted a stable, functional complex consisting of the iron donor, Yfh1 (yeast frataxin homologue 1), and the Fe-S cluster scaffold, Isu1, with 1:1 stoichiometry, [Yfh1]24·[Isu1]24 Using negative staining transmission EM and single particle analysis, we obtained a three-dimensional reconstruction of this complex at a resolution of ∼17 Å. In addition, via chemical cross-linking, limited proteolysis, and mass spectrometry, we identified protein-protein interaction surfaces within the complex. The data together reveal that [Yfh1]24·[Isu1]24 is a roughly cubic macromolecule consisting of one symmetric Isu1 trimer binding on top of one symmetric Yfh1 trimer at each of its eight vertices. Furthermore, molecular modeling suggests that two subunits of the cysteine desulfurase, Nfs1, may bind symmetrically on top of two adjacent Isu1 trimers in a manner that creates two putative [2Fe-2S] cluster assembly centers. In each center, conserved amino acids known to be involved in sulfur and iron donation by Nfs1 and Yfh1, respectively, are in close proximity to the Fe-S cluster-coordinating residues of Isu1. We suggest that this architecture is suitable to ensure concerted and protected transfer of potentially toxic iron and sulfur atoms to Isu1 during Fe-S cluster assembly.

The molecular basis of iron-induced oligomerization of frataxin and the role of the ferroxidation reaction in oligomerization.

The role of the mitochondrial protein frataxin in iron storage and detoxification, iron delivery to iron-sulfur cluster biosynthesis, heme biosynthesis, and aconitase repair has been extensively studied during the last decade. However, still no general consensus exists on the details of the mechanism of frataxin function and oligomerization. Here, using small-angle x-ray scattering and x-ray crystallography, we describe the solution structure of the oligomers formed during the iron-dependent assembly of yeast (Yfh1) and Escherichia coli (CyaY) frataxin. At an iron-to-protein ratio of 2, the initially monomeric Yfh1 is converted to a trimeric form in solution. The trimer in turn serves as the assembly unit for higher order oligomers induced at higher iron-to-protein ratios. The x-ray crystallographic structure obtained from iron-soaked crystals demonstrates that iron binds at the trimer-trimer interaction sites, presumably contributing to oligomer stabilization. For the ferroxidation-deficient D79A/D82A variant of Yfh1, iron-dependent oligomerization may still take place, although >50% of the protein is found in the monomeric state at the highest iron-to-protein ratio used. This demonstrates that the ferroxidation reaction controls frataxin assembly and presumably the iron chaperone function of frataxin and its interactions with target proteins. For E. coli CyaY, the assembly unit of higher order oligomers is a tetramer, which could be an effect of the much shorter N-terminal region of this protein. The results show that understanding of the mechanistic features of frataxin function requires detailed knowledge of the interplay between the ferroxidation reaction, iron-induced oligomerization, and the structure of oligomers formed during assembly.

Detection of crosslinks within and between proteins by LC-MALDI-TOFTOF and the software FINDX to reduce the MSMS-data to acquire for validation.

Lysine-specific chemical crosslinking in combination with mass spectrometry is emerging as a tool for the structural characterization of protein complexes and protein-protein interactions. After tryptic digestion of crosslinked proteins there are thousands of peptides amenable to MSMS, of which only very few are crosslinked peptides of interest. Here we describe how the advantage offered by off-line LC-MALDI-TOF/TOF mass spectrometry is exploited in a two-step workflow to focus the MSMS-acquisition on crosslinks mainly. In a first step, MS-data are acquired and all the peak list files from the LC-separated fractions are merged by the FINDX software and screened for presence of crosslinks which are recognized as isotope-labeled doublet peaks. Information on the isotope doublet peak mass and intensity can be used as search constraints to reduce the number of false positives that match randomly to the observed peak masses. Based on the MS-data a precursor ion inclusion list is generated and used in a second step, where a restricted number of MSMS-spectra are acquired for crosslink validation. The decoupling of MS and MSMS and the peptide sorting with FINDX based on MS-data has the advantage that MSMS can be restricted to and focused on crosslinks of Type 2, which are of highest biological interest but often lowest in abundance. The LC-MALDI TOF/TOF workflow here described is applicable to protein multisubunit complexes and using (14)N/(15)N mixed isotope strategy for the detection of inter-protein crosslinks within protein oligomers.

Structure of the cyanobacterial Magnesium Chelatase H subunit determined by single particle reconstruction and small-angle X-ray scattering.

The biosynthesis of chlorophyll, an essential cofactor for photosynthesis, requires the ATP-dependent insertion of Mg(2+) into protoporphyrin IX catalyzed by the multisubunit enzyme magnesium chelatase. This enzyme complex consists of the I subunit, an ATPase that forms a complex with the D subunit, and an H subunit that binds both the protoporphyrin substrate and the magnesium protoporphyrin product. In this study we used electron microscopy and small-angle x-ray scattering to investigate the structure of the magnesium chelatase H subunit, ChlH, from the thermophilic cyanobacterium Thermosynechococcus elongatus. Single particle reconstruction of negatively stained apo-ChlH and Chl-porphyrin proteins was used to reconstitute three-dimensional structures to a resolution of ∼30 Å. ChlH is a large, 148-kDa protein of 1326 residues, forming a cage-like assembly comprising the majority of the structure, attached to a globular N-terminal domain of ∼16 kDa by a narrow linker region. This N-terminal domain is adjacent to a 5 nm-diameter opening in the structure that allows access to a cavity. Small-angle x-ray scattering analysis of ChlH, performed on soluble, catalytically active ChlH, verifies the presence of two domains and their relative sizes. Our results provide a basis for the multiple regulatory and catalytic functions of ChlH of oxygenic photosynthetic organisms and for a chaperoning function that sequesters the enzyme-bound magnesium protoporphyrin product prior to its delivery to the next enzyme in the chlorophyll biosynthetic pathway, magnesium protoporphyrin methyltransferase.

Oligomerization propensity and flexibility of yeast frataxin studied by X-ray crystallography and small-angle X-ray scattering.

Frataxin is a mitochondrial protein with a central role in iron homeostasis. Defects in frataxin function lead to Friedreich's ataxia, a progressive neurodegenerative disease with childhood onset. The function of frataxin has been shown to be closely associated with its ability to form oligomeric species; however, the factors controlling oligomerization and the types of oligomers present in solution are a matter of debate. Using small-angle X-ray scattering, we found that Co(2+), glycerol, and a single amino acid substitution at the N-terminus, Y73A, facilitate oligomerization of yeast frataxin, resulting in a dynamic equilibrium between monomers, dimers, trimers, hexamers, and higher-order oligomers. Using X-ray crystallography, we found that Co(2+) binds inside the channel at the 3-fold axis of the trimer, which suggests that the metal has an oligomer-stabilizing role. The results reveal the types of oligomers present in solution and support our earlier suggestions that the trimer is the main building block of yeast frataxin oligomers. They also indicate that different mechanisms may control oligomer stability and oligomerization in vivo.

Thiol-exchange in DTSSP crosslinked peptides is proportional to cysteine content and precisely controlled in crosslink detection by two-step LC-MALDI MSMS.

The lysine-specific crosslinker 3,3'-dithiobis(sulfosuccinimidylpropionate) (DTSSP) is commonly used in the structural characterization of proteins by chemical crosslinking and mass spectrometry and we here describe an efficient two-step LC-MALDI-TOF/TOF procedure to detect crosslinked peptides. First MS data are acquired, and the properties of isotope-labeled DTSSP are used in data analysis to identify candidate crosslinks. MSMS data are then acquired for a restricted number of precursor ions per spot for final crosslink identification. We show that the thiol-catalyzed exchange between crosslinked peptides, which is due to the disulfide bond in DTSSP and known to possibly obscure data, can be precisely quantified using isotope-labeled DTSSP. Crosslinked peptides are recognized as 8 Da doublet peaks and a new isotopic peak with twice the intensity appears in the middle of the doublet as a consequence of the thiol-exchange. False-positive crosslinks, formed exclusively by thiol-exchange, yield a 1:2:1 isotope pattern, whereas true crosslinks, formed by two lysine residues within crosslinkable distance in the native protein structure, yield a 1:0:1 isotope pattern. Peaks with a 1:X:1 isotope pattern, where 0 < X < 2, can be trusted as true crosslinks, with a defined proportion of the signal [2X/(2 + X)] being noise from the thiol-exchange. The thiol-exchange was correlated with the protein cysteine content and was minimized by shortening the trypsin incubation time, and for two molecular chaperone proteins with known structure all crosslinks fitted well to the structure data. The thiol-exchange can thus be controlled and isotope-labeled DTSSP safely used to detect true crosslinks between lysine residues in proteins.

Bacterial ferrochelatase turns human: Tyr13 determines the apparent metal specificity of Bacillus subtilis ferrochelatase.

Ferrochelatase catalyzes the insertion of Fe(2+) into protoporphyrin IX. The enzymatic product heme (protoheme IX) is a well-known cofactor in a wide range of proteins. The insertion of metal ions other than Fe(2+) occurs rarely in vivo, but all ferrochelatases that have been studied can insert Zn(2+) at a good rate in vitro. Co(2+), but not Cu(2+), is known to be a good substrate of the mammalian and Saccharomyces cerevisiae ferrochelatases. In contrast, Cu(2+), but not Co(2+), has been found to be a good substrate of bacterial Bacillus subtilis ferrochelatase. It is not known how ferrochelatase discriminates between different metal ion substrates. Structural analysis of B. subtilis ferrochelatase has shown that Tyr13 is an indirect ligand of Fe(2+) and a direct ligand of a copper mesoporphyrin product. A structure-based comparison revealed that Tyr13 aligns with a Met residue in the S. cerevisiae and human ferrochelatases. Tyr13 was changed to Met in the B. subtilis enzyme by site-directed mutagenesis. Enzymatic measurements showed that the modified enzyme inserted Co(2+) at a higher rate than the wild-type B. subtilis ferrochelatase, but it had lost the ability to use Cu(2+) as a substrate. Thus, the B. subtilis Tyr13Met ferrochelatase showed the same metal specificity as that of the ferrochelatases from S. cerevisiae and human.

Penicillin-binding protein SpoVD disulphide is a target for StoA in Bacillus subtilis forespores.

The bacterial endospore is a dormant and heat-resistant form of life. StoA (SpoIVH) in Bacillus subtilis is a membrane-bound thioredoxin-like protein involved in endospore cortex synthesis. It is proposed to reduce disulphide bonds in hitherto unknown proteins in the intermembrane compartment of developing forespores. Starting with a bioinformatic analysis combined with mutant studies we identified the sporulation-specific, high-molecular-weight, class B penicillin-binding protein SpoVD as a putative target for StoA. We then demonstrate that SpoVD is a membrane-bound protein with two exposed redox-active cysteine residues. Structural modelling of SpoVD, based on the well characterized orthologue PBP2x of Streptococcus pneumoniae, confirmed that a disulphide bond can form close to the active site of the penicillin-binding domain restricting access of enzyme substrate or functional association with other cortex biogenic proteins. Finally, by exploiting combinations of mutations in the spoVD, stoA and ccdA genes in B. subtilis cells, we present strong in vivo evidence that supports the conclusion that StoA functions to specifically break the disulphide bond in the SpoVD protein in the forespore envelope. The findings contribute to our understanding of endospore biogenesis and open a new angle to regulation of cell wall synthesis and penicillin-binding protein activity.