Resolving the zinc binding capacity of honey bee vitellogenin and locating its putative binding sites.

The protein vitellogenin (Vg) plays a central role in lipid transportation in most egg-laying animals. High Vg levels correlate with stress resistance and lifespan potential in honey bees (Apis mellifera). Vg is the primary circulating zinc-carrying protein in honey bees. Zinc is an essential metal ion in numerous biological processes, including the function and structure of many proteins. Measurements of Zn2+ suggest a variable number of ions per Vg molecule in different animal species, but the molecular implications of zinc-binding by this protein are not well-understood. We used inductively coupled plasma mass spectrometry to determine that, on average, each honey bee Vg molecule binds 3 Zn2+ -ions. Our full-length protein structure and sequence analysis revealed seven potential zinc-binding sites. These are located in the ß-barrel and α-helical subdomains of the N-terminal domain, the lipid binding site, and the cysteine-rich C-terminal region of unknown function. Interestingly, two potential zinc-binding sites in the ß-barrel can support a proposed role for this structure in DNA-binding. Overall, our findings suggest that honey bee Vg bind zinc at several functional regions, indicating that Zn2+ -ions are important for many of the activities of this protein. In addition to being potentially relevant for other egg-laying species, these insights provide a platform for studies of metal ions in bee health, which is of global interest due to recent declines in pollinator numbers.

Investigating the Disordered and Membrane-Active Peptide A-Cage-C Using Conformational Ensembles.

The driving forces and conformational pathways leading to amphitropic protein-membrane binding and in some cases also to protein misfolding and aggregation is the subject of intensive research. In this study, a chimeric polypeptide, A-Cage-C, derived from α-Lactalbumin is investigated with the aim of elucidating conformational changes promoting interaction with bilayers. From previous studies, it is known that A-Cage-C causes membrane leakages associated with the sporadic formation of amorphous aggregates on solid-supported bilayers. Here we express and purify double-labelled A-Cage-C and prepare partially deuterated bicelles as a membrane mimicking system. We investigate A-Cage-C in the presence and absence of these bicelles at non-binding (pH 7.0) and binding (pH 4.5) conditions. Using in silico analyses, NMR, conformational clustering, and Molecular Dynamics, we provide tentative insights into the conformations of bound and unbound A-Cage-C. The conformation of each state is dynamic and samples a large amount of overlapping conformational space. We identify one of the clusters as likely representing the binding conformation and conclude tentatively that the unfolding around the central W23 segment and its reorientation may be necessary for full intercalation at binding conditions (pH 4.5). We also see evidence for an overall elongation of A-Cage-C in the presence of model bilayers.

A polybasic motif in ErbB3-binding protein 1 (EBP1) has key functions in nucleolar localization and polyphosphoinositide interaction.

Polyphosphoinositides (PPIns) are present in the nucleus where they participate in crucial nuclear processes, such as chromatin remodelling, transcription and mRNA processing. In a previous interactomics study, aimed to gain further insight into nuclear PPIns functions, we identified ErbB3 binding protein 1 (EBP1) as a potential nuclear PPIn-binding protein in a lipid pull-down screen. EBP1 is a ubiquitous and conserved protein, located in both the cytoplasm and nucleolus, and associated with cell proliferation and survival. In the present study, we show that EBP1 binds directly to several PPIns via two distinct PPIn-binding sites consisting of clusters of lysine residues and positioned at the N- and C-termini of the protein. Using interaction mutants, we show that the C-terminal PPIn-binding motif contributes the most to the localization of EBP1 in the nucleolus. Importantly, a K372N point mutation, located within the C-terminal motif and found in endometrial tumours, is sufficient to alter the nucleolar targeting of EBP1. Our study reveals also the presence of the class I phosphoinositide 3-kinase (PI3K) catalytic subunit p110ß and its product PtdIns(3,4,5)P3 together with EBP1 in the nucleolus. Using NMR, we further demonstrate an association between EBP1 and PtdIns(3,4,5)P3 via both electrostatic and hydrophobic interactions. Taken together, these results show that EBP1 interacts directly with PPIns and associate with PtdIns(3,4,5)P3 in the nucleolus. The presence of p110ß and PtdIns(3,4,5)P3 in the nucleolus indicates their potential role in regulating nucleolar processes, at least via EBP1.

Molecular landscape of the interaction between the urease accessory proteins UreE and UreG.

Urease, the most efficient enzyme so far discovered, depends on the presence of nickel ions in the catalytic site for its activity. The transformation of inactive apo-urease into active holo-urease requires the insertion of two Ni(II) ions in the substrate binding site, a process that involves the interaction of four accessory proteins named UreD, UreF, UreG and UreE. This study, carried out using calorimetric and NMR-based structural analysis, is focused on the interaction between UreE and UreG from Sporosarcina pasteurii, a highly ureolytic bacterium. Isothermal calorimetric protein-protein titrations revealed the occurrence of a binding event between SpUreE and SpUreG, entailing two independent steps with positive cooperativity (Kd1=42±9µM; Kd2=1.7±0.3µM). This was interpreted as indicating the formation of the (UreE)2(UreG)2 hetero-oligomer upon binding of two UreG monomers onto the pre-formed UreE dimer. The molecular details of this interaction were elucidated using high-resolution NMR spectroscopy. The occurrence of SpUreE chemical shift perturbations upon addition of SpUreG was investigated and analyzed to establish the protein-protein interaction site. The latter appears to involve the Ni(II) binding site as well as mobile portions on the C-terminal and the N-terminal domains. Docking calculations based on the information obtained from NMR provided a structural basis for the protein-protein contact site. The high sequence and structural similarity within these protein classes suggests a generality of the interaction mode among homologous proteins. The implications of these results on the molecular details of the urease activation process are considered and analyzed.

On the interaction of Helicobacter pylori NikR, a Ni(II)-responsive transcription factor, with the urease operator: in solution and in silico studies.

Helicobacter pylori (Hp) is a carcinogen that relies on Ni(II) to survive in the extreme pH conditions of the human guts. The regulation of genes coding for Ni(II) enzymes and proteins is effected by the nickel-responsive transcription factor NikR, composed of a DNA-binding domain (DBD) and a metal-binding domain (MBD). The scope of this study is to obtain the molecular details of the HpNikR interaction with the urease operator OP ureA , in solution. The size of the full-length protein prevents the characterization of the HpNikR-OP ureA interaction using NMR. We thus investigated the two separate domains of HpNikR. The conservation of their oligomeric state was established by multiple-angle light scattering. Isothermal calorimetric titrations indicated that the thermodynamics of Ni(II) binding to the isolated MBD is independent of the presence of the adjacent DBDs. The NMR spectra of the isolated DBD support considerable conservation of its structural properties. The spectral perturbations induced on the DBD by OP ureA provided information useful to calculate a structural model of the HpNikR-OP ureA complex using a docking computational protocol. The NMR assignment of the residues involved in the protein-DNA interaction represents a starting point for the development of drugs potentially able to eradicate H. pylori infections. All evidences so far collected, in this and previous studies, consistently indicate that binding of Ni(II) to the MBD increases the HpNikR-DNA affinity by modulating the dynamic, and not the structural, properties of the protein, suggesting that the formation of a stable complex relies upon an induced fit mechanism.

Competitive cobalt for zinc substitution in mammalian methionine sulfoxide reductase B1 overexpressed in E. coli: structural and functional insight.

Expression of the mammalian enzyme methionine sulfoxide reductase B1 (MsrB1) in Escherichia coli growing in cobalt-containing media resulted in the reproducible appearance of the stable cobalt-containing protein MsrB1-Co. NMR studies and biocomputing using the programs AnisoFit and Amber allowed us to generate a structure of MsrB1-Co sharing the overall fold with the native zinc-containing protein MsrB1-Zn. Our data suggest that the N-terminus containing resolving cysteine tends to be closer to the protein's catalytic center than was previously reported. It is argued that this proximity supports the proposed catalytic mechanism and ensures high catalytic efficiency of MsrB1. Functional studies showed that both MsrB1-Zn and MsrB1-Co exhibit similar levels of activity, in agreement with the structural studies performed. The proposed metal ion substitution approach may have a methodological significance in determining whether methionine sulfoxide reductase B proteins contain a metal ion.

Structural dynamics of human deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase).

Structural- and functional heterogeneity, as well as allosteric regulation, in homo-monomeric enzymes is a highly active area of research. One such enzyme is human nuclear-associated deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase), which has emerged as an interesting drug target in combination therapy with traditional nucleotide analogue treatment of cancer. We report, for the first time, a full structural dynamics study of human dUTPase by NMR. dUTPase has been investigated in terms of structural dynamics in its apo form, in complex with the modified substrate resistant to hydrolysis, 2'-deoxyuridine 5'-α,ß-imido-triphosphate (dUpNHpp), as well as the product, 2'-deoxy-uridine-monophosphate (dUMP). The apo form of the enzyme displayed slow dynamics in the milli- to microsecond regime in relaxation dispersion experiments, which was further slowed down to observable heterogeneity upon substrate-analogue binding. The results suggest that the non-hydrolysable substrate-analogue traps the enzyme in the conformational isomerization step that has been previously suggested to be part of the enzyme catalysis kinetics cycle. The observed heterogeneity fits well with the pattern expected to emerge from the suggested kinetic model, and no evidence for homotropic allosterism was found. The heatmaps of the slow dynamics, chemical shift perturbation upon substrate binding and conserved regions of the enzyme sequence all displayed a similar pattern, which suggests that the structural dynamics is finely tuned and important for the biological function of the enzyme for binding, conformational shift, catalysis and substrate release.

Structural insights into interaction between mammalian methionine sulfoxide reductase B1 and thioredoxin.

Maintenance of the cellular redox balance has vital importance for correcting organism functioning. Methionine sulfoxide reductases (Msrs) are among the key members of the cellular antioxidant defence system. To work properly, methionine sulfoxide reductases need to be reduced by their biological partner, thioredoxin (Trx). This process, according to the available kinetic data, represents the slowest step in the Msrs catalytic cycle. In the present paper, we investigated structural aspects of the intermolecular complex formation between mammalian MsrB1 and Trx. NMR spectroscopy and biocomputing were the two mostly used through the research approaches. The formation of NMR detectable MsrB1/Trx complex was monitored and studied in attempt to understand MsrB1 reduction mechanism. Using NMR data, molecular mechanics, protein docking, and molecular dynamics simulations, it was found that intermediate MsrB1/Trx complex is stabilized by interprotein ß-layer. The complex formation accompanied by distortion of disulfide bond within MsrB1 facilitates the reduction of oxidized MsrB1 as it is evidenced by the obtained data.

Binding Specificity of ASHH2 CW Domain Toward H3K4me1 Ligand Is Coupled to Its Structural Stability Through Its α1-Helix.

The CW domain binds to histone tail modifications found in different protein families involved in epigenetic regulation and chromatin remodeling. CW domains recognize the methylation state of the fourth lysine on histone 3 and could, therefore, be viewed as a reader of epigenetic information. The specificity toward different methylation states such as me1, me2, or me3 depends on the particular CW subtype. For example, the CW domain of ASHH2 methyltransferase binds preferentially to H3K4me1, and MORC3 binds to both H3K4me2 and me3 modifications, while ZCWPW1 is more specific to H3K4me3. The structural basis for these preferential bindings is not well understood, and recent research suggests that a more complete picture will emerge if dynamical and energetic assessments are included in the analysis of interactions. This study uses fold assessment by NMR in combination with mutagenesis, ITC affinity measurements, and thermal denaturation studies to investigate possible couplings between ASHH2 CW selectivity toward H3K4me1 and the stabilization of the domain and loops implicated in binding. The key elements of the binding site-the two tryptophans and the α1-helix form and maintain the binding pocket- were perturbed by mutagenesis and investigated. Results show that the α1-helix maintains the overall stability of the fold via the I915 and L919 residues and that the correct binding consolidates the loops designated as η1 and η3, as well as the C-terminal. This consolidation is incomplete for H3K4me3 binding to CW, which experiences a decrease in overall thermal stability on binding. Loop mutations not directly involved in the binding site, nonetheless, affect the equilibrium positions of the key residues.

The Arabidopsis (ASHH2) CW domain binds monomethylated K4 of the histone H3 tail through conformational selection.

Chromatin post-translational modifications are thought to be important for epigenetic effects on gene expression. Methylation of histone N-terminal tail lysine residues constitutes one of many such modifications, executed by families of histone lysine methyltransferase (HKMTase). One such protein is ASHH2 from the flowering plant Arabidopsis thaliana, equipped with the interaction domain, CW, and the HKMTase domain, SET. The CW domain of ASHH2 is a selective binder of monomethylation at lysine 4 on histone H3 (H3K4me1) and likely helps the enzyme dock correctly onto chromatin sites. The study of CW and related interaction domains has so far been emphasizing lock-key models, missing important aspects of histone-tail CW interactions. We here present an analysis of the ASHH2 CW-H3K4me1 complex using NMR and molecular dynamics, as well as mutation and affinity studies of flexible coils. ß-augmentation and rearrangement of coils coincide with changes in the flexibility of the complex, in particular the η1, η3 and C-terminal coils, but also in the ß1 and ß2 strands and the C-terminal part of the ligand. Furthermore, we show that mutating residues with outlier dynamic behaviour affect the complex binding affinity despite these not being in direct contact with the ligand. Overall, the binding process is consistent with conformational selection. We propose that this binding mechanism presents an advantage when searching for the correct post-translational modification state among the highly modified and flexible histone tails, and also that the binding shifts the catalytic SET domain towards the nucleosome. DATABASES: Structural data are available in the PDB database under the accession code 6QXZ. Resonance assignments for CW42 in its apo- and holo-forms are available in the BMRB database under the accession code 27251.

1H, 13C, and 15N resonance assignments of CW domain of the N-methyltransferase ASHH2 free and bound to the mono-, di- and tri-methylated histone H3 tail peptides.

The ASHH2 CW domain is responsible for recognizing the methylation state at lysine 4 of histone 3 N-terminal tails and implicated in the recruitment of the ASHH2 methyltransferase enzyme correctly to the histones. The ASHH2 CW domain binds H3 lysine motifs that can be either mono-, di-, or tri-methylated [ARTK(meX)QTAR, where X denotes the number of methylations], but binds strongest to monomethylated instances (Kd values reported in the range of 1 µm to 500 nM). Hoppmann et al. published the uncomplexed NMR structure of an ASHH2 CW domain in 2011. Here we document the assignment of a shortened ASHH2 CW construct, CW42, with similar binding affinity and better expression yields than the one used to solve the uncomplexed structure. We also perform 1H-15N HSQC-monitored titrations that document at which protein-peptide ratios the complex is saturated. Backbone resonance assignments are presented for this shortened ASHH2 CW domain alone and bound to an H3 histone tail mimicking peptide monomethylated on lysine 4 (ARTK(me1)QTAR). Likewise, the assignment was also performed for the protein in complex with the dimethylated (ARTK(me2)QTAR) and trimethylated (ARTK(me3)QTAR) peptide. Overall, these two latter situations displayed a similar perturbation of shifts as the mono-methylated instance. In the case of the monomethylated histone tail mimic, side-chain assignment of CW42 in this complex was performed and reported in addition to backbone assignment, in preparation of a future solution structure determination and dynamics characterization of the CW42-ARTK(me1)QTAR complex.

Structural analysis of the interaction between Jaburetox, an intrinsically disordered protein, and membrane models.

Jack bean urease is entomotoxic to insects with cathepsin-like digestive enzymes, and its toxicity is mainly caused by a polypeptide called Jaburetox (Jbtx), released by cathepsin-dependent hydrolysis of the enzyme. Jbtx is intrinsically disordered in aqueous solution, as shown by CD and NMR. Jbtx is able to alter the permeability of membranes, hinting to a role of Jbtx-membrane interaction as the basis for its toxicity. The present study addresses the structural aspects of this interaction by investigating the behaviour of Jbtx when in contact with membrane models, using nuclear magnetic resonance and circular dichroism spectroscopies in the absence or presence of micelles, large unilamellar vesicles, and bicelles. Fluorescence microscopy was also used to detect protein-insect membrane interaction. Significant differences were observed depending on the type of membrane model used. The interaction with negatively charged SDS micelles increases the secondary and tertiary structure content of the polypeptide, while, in the case of large unilamellar vesicles and bicelles, conformational changes were observed at the terminal regions, with no significant acquisition of secondary structure motifs. These results were interpreted as suggesting that the Jbtx-lipids interaction anchors the polypeptide to the cellular membrane through the terminal portions of the polypeptide and that, following this interaction, Jbtx undergoes conformational changes to achieve a more ordered structure that could facilitate its interaction with membrane-bound proteins. Consistently with this hypothesis, the presence of these membrane models decreases the ability of Jbtx to bind cellular membranes of insect nerve cord. The collected evidence from these studies implies that the biological activity of Jbtx is due to protein-phospholipid interactions.

Pliable natural biocide: Jaburetox is an intrinsically disordered insecticidal and fungicidal polypeptide derived from jack bean urease.

Jaburetox is a polypeptide derived from jack bean (Canavalia ensiformis) urease and toxic to a broad spectrum of insects, phytopathogenic filamentous fungi and yeasts of medical importance. The elucidation of the structural basis for the mode of action of Jaburetox is the focus of this multifaceted study. Jaburetox in solution is a monomer of 11.0 kDa featuring a large hydrodynamic radius, suggestive of a disordered polypeptide. The intrinsically disordered nature of Jaburetox was theoretically predicted by a comprehensive bioinformatics analysis and experimentally confirmed by light scattering as well as by circular dichroism and NMR spectroscopy. NMR signal assignment provided backbone secondary chemical shifts that indicated that Jaburetox has a low propensity to assume a stable secondary structure. (15)N relaxation studies revealed significant backbone mobility, especially in the N-terminal portion of the polypeptide. The solution structure of Jaburetox shows the presence of an α-helical motif close to the N terminus, together with two turn-like structures situated in the central portion of the protein and close to the C terminus. Similar regions were predicted as potential protein-protein interaction sites using computational tools. The knowledge of the structural properties of Jaburetox in solution is a key step to correlate its structural and biological activities.

Structural analysis of glutaredoxin domain of Mus musculus thioredoxin glutathione reductase.

Thioredoxin glutathione reductase (TGR) is a member of the mammalian thioredoxin reductase family that has a monothiol glutaredoxin (Grx) domain attached to the thioredoxin reductase module. Here, we report a structure of the Grx domain of mouse TGR, determined through high resolution NMR spectroscopy to the final backbone RMSD value of 0.48 ± 0.10 Å. The structure represents a sandwich-like molecule composed of a four stranded ß-sheet flanked by five α-helixes, with the CxxS active motif located on the catalytic loop. We structurally characterized the glutathione-binding site in the protein and describe sequence and structural relationships of the domain with glutaredoxins. The structure illuminates a key functional center that evolved in mammalian TGRs to act in thiol-disulfide reactions. Our study allows us to hypothesize that Cys105 might be functionally relevant for TGR catalysis. In addition, the data suggest that the N-terminus of Grx acts as a possible regulatory signal also protecting the protein active site from unwanted interactions in cellular cytosol.