Abundant neuroprotective chaperone Lipocalin-type prostaglandin D synthase (L-PGDS) disassembles the Amyloid-ß fibrils.

Misfolding of Amyloid ß (Aß) peptides leads to the formation of extracellular amyloid plaques. Molecular chaperones can facilitate the refolding or degradation of such misfolded proteins. Here, for the first time, we report the unique ability of Lipocalin-type Prostaglandin D synthase (L-PGDS) protein to act as a disaggregase on the pre-formed fibrils of Aß(1-40), abbreviated as Aß40, and Aß(25-35) peptides, in addition to inhibiting the aggregation of Aß monomers. Furthermore, our proteomics results indicate that L-PGDS can facilitate extraction of several other proteins from the insoluble aggregates extracted from the brain of an Alzheimer's disease patient. In this study, we have established the mode of binding of L-PGDS with monomeric and fibrillar Aß using Nuclear Magnetic Resonance (NMR) Spectroscopy, Small Angle X-ray Scattering (SAXS), and Transmission Electron Microscopy (TEM). Our results confirm a direct interaction between L-PGDS and monomeric Aß40 and Aß(25-35), thereby inhibiting their spontaneous aggregation. The monomeric unstructured Aß40 binds to L-PGDS via its C-terminus, while the N-terminus remains free which is observed as a new domain in the L-PGDS-Aß40 complex model.

Targeting the Mycobacterium ulcerans cytochrome bc1:aa3 for the treatment of Buruli ulcer.

Mycobacterium ulcerans is the causative agent of Buruli ulcer, a neglected tropical skin disease that is most commonly found in children from West and Central Africa. Despite the severity of the infection, therapeutic options are limited to antibiotics with severe side effects. Here, we show that M. ulcerans is susceptible to the anti-tubercular drug Q203 and related compounds targeting the respiratory cytochrome bc1:aa3. While the cytochrome bc1:aa3 is the primary terminal oxidase in Mycobacterium tuberculosis, the presence of an alternate bd-type terminal oxidase limits the bactericidal and sterilizing potency of Q203 against this bacterium. M. ulcerans strains found in Buruli ulcer patients from Africa and Australia lost all alternate terminal electron acceptors and rely exclusively on the cytochrome bc1:aa3 to respire. As a result, Q203 is bactericidal at low dose against M. ulcerans replicating in vitro and in mice, making the drug a promising candidate for Buruli ulcer treatment.

The NMR solution structure of Mycobacterium tuberculosis F-ATP synthase subunit ε provides new insight into energy coupling inside the rotary engine.

Mycobacterium tuberculosis (Mt) F1 F0 ATP synthase (α3 :ß3 :γ:δ:ε:a:b:b':c9 ) is essential for the viability of growing and nongrowing persister cells of the pathogen. Here, we present the first NMR solution structure of Mtε, revealing an N-terminal ß-barrel domain (NTD) and a C-terminal domain (CTD) composed of a helix-loop-helix with helix 1 and -2 being shorter compared to their counterparts in other bacteria. The C-terminal amino acids are oriented toward the NTD, forming a domain-domain interface between the NTD and CTD. The Mtε structure provides a novel mechanistic model of coupling c-ring- and ε rotation via a patch of hydrophobic residues in the NTD and residues of the CTD to the bottom of the catalytic α3 ß3 -headpiece. To test our model, genome site-directed mutagenesis was employed to introduce amino acid changes in these two parts of the epsilon subunit. Inverted vesicle assays show that these mutations caused an increase in ATP hydrolysis activity and a reduction in ATP synthesis. The structural and enzymatic data are discussed in light of the transition mechanism of a compact and extended state of Mtε, which provides the inhibitory effects of this coupling subunit inside the rotary engine. Finally, the employment of these data with molecular docking shed light into the second binding site of the drug Bedaquiline. DATABASE: Structural data are available in the PDB under the accession number 5YIO.

Insights into Biofilm Dispersal Regulation from the Crystal Structure of the PAS-GGDEF-EAL Region of RbdA from Pseudomonas aeruginosa.

RbdA is a positive regulator of biofilm dispersal of Pseudomonas aeruginosa Its cytoplasmic region (cRbdA) comprises an N-terminal Per-ARNT-Sim (PAS) domain followed by a diguanylate cyclase (GGDEF) domain and an EAL domain, whose phosphodiesterase activity is allosterically stimulated by GTP binding to the GGDEF domain. We report crystal structures of cRbdA and of two binary complexes: one with GTP/Mg2+ bound to the GGDEF active site and one with the EAL domain bound to the c-di-GMP substrate. These structures unveil a 2-fold symmetric dimer stabilized by a closely packed N-terminal PAS domain and a noncanonical EAL dimer. The autoinhibitory switch is formed by an α-helix (S-helix) immediately N-terminal to the GGDEF domain that interacts with the EAL dimerization helix (α6-E) of the other EAL monomer and maintains the protein in a locked conformation. We propose that local conformational changes in cRbdA upon GTP binding lead to a structure with the PAS domain and S-helix shifted away from the GGDEF-EAL domains, as suggested by small-angle X-ray scattering (SAXS) experiments. Domain reorientation should be facilitated by the presence of an α-helical lever (H-helix) that tethers the GGDEF and EAL regions, allowing the EAL domain to rearrange into an active dimeric conformation.IMPORTANCE Biofilm formation by bacterial pathogens increases resistance to antibiotics. RbdA positively regulates biofilm dispersal of Pseudomonas aeruginosa The crystal structures of the cytoplasmic region of the RbdA protein presented here reveal that two evolutionarily conserved helices play an important role in regulating the activity of RbdA, with implications for other GGDEF-EAL dual domains that are abundant in the proteomes of several bacterial pathogens. Thus, this work may assist in the development of small molecules that promote bacterial biofilm dispersal.

Self-coacervation of modular squid beak proteins - a comparative study.

The beak of the Humboldt squid is a biocomposite material made solely of organic components - chitin and proteins - which exhibits 200-fold stiffness and hardness gradients from the soft base to the exceptionally hard tip (rostrum). The outstanding mechanical properties of the squid beak are achieved via controlled hydration and impregnation of the chitin-based scaffold by protein coacervates. Molecular-based understanding of these proteins is essential to mimic the natural beak material. Here, we present detailed studies of two histidine-rich beak proteins (HBP-1 and -2) that play central roles during beak bio-fabrication. We show that both proteins have the ability to self-coacervate, which is governed intrinsically by the sequence modularity of their C-terminus and extrinsically by pH and ionic strength. We demonstrate that HBPs possess dynamic structures in solution and achieve maximum folding in the coacervate state, and propose that their self-coacervation is driven by hydrophobic interactions following charge neutralization through salt-screening. Finally, we show that subtle differences in the modular repeats of HBPs result in significant changes in the rheological response of the coacervates. This knowledge may be exploited to design self-coacervating polypeptides for a wide range of engineering and biomedical applications, for example bio-inspired composite materials, smart hydrogels and adhesives, and biomedical implants.

NMR studies reveal a novel grab and release mechanism for efficient catalysis of the bacterial 2-Cys peroxiredoxin machinery.

In bacteria, an ensemble of alkyl hydroperoxide reductase subunits C (AhpC) and F (AhpF) is responsible for scavenging H2O2. AhpC donates electrons for the reduction of H2O2, which are provided after NADH oxidation by AhpF. The latter contains an N-terminal domain (NTD), catalyzing the electron transfer from NADH via a FAD of the C-terminal domain (CTD) into AhpC. The NADH-bound Escherichia coli AhpF structure revealed that NADH binding brings the substrate to the re-face of the FAD, making the Cys-Cys center of the CTD accessible to the NTD disulfide center for electron transfer (Kamariah et al. (2015) Biochim Biophys Acta 1847, 1139-1152). So far insight into the epitope and mechanism of AhpF and AhpC interaction as well as the electron transfer from the NTD to AhpC have been lacking. Here using NMR spectroscopy, we glean insight into the interaction of the NTD of AhpF with AhpC from E. coli. A coordinated disappearance of EcAhpF NTD peaks was observed in the presence of full length EcAhpC, indicating a long-lived AhpC-AhpF complex. C-terminal truncated EcAhpC resulted in a more dynamic interaction, revealing specific residue chemical shift perturbation and hence the binding epitope of the complex. Combined with docking studies, we have suggested that the C terminus of AhpC binds to the backside groove of the NTD. In addition, AhpC-AhpF formation is abolished under reducing conditions. We propose for the first time a binding mechanism in which the C terminus of AhpC wraps around the NTD, slowing the dissociation rate for an efficient electron transfer process, and a release mechanism mediated by the conformational change of the C terminus of AhpC upon reduction.

Structural architecture and interplay of the nucleotide- and erythrocyte binding domain of the reticulocyte binding protein Py235 from Plasmodium yoelii.

Human malaria is caused by the cyclical invasion of the host's red blood cells (RBCs) by the invasive form of the parasite, the merozoite. The invasion of the RBC involves a range of parasite ligand receptor interactions, a process which is under intensive investigation. Two protein families are known to be important in the recognition and invasion of the human erythrocyte, the erythrocyte-binding like (EBL) proteins and the reticulocyte binding like proteins, of which the Py235 family in Plasmodium yoelii is a member. Recently the nucleotide binding domain (NBD94), that plays a role in ATP sensing, and the erythrocyte binding domain (EBD) of Py235, called EBD(1-194), have been identified. Binding of ATP leads to conformational changes within Py235 from P. yoelli and results in enhanced binding of the protein to the RBC. Structural features of these domains have been obtained, providing the platform to discuss how the structural architecture creates the basis for an interplay of the sensing NBD and the EBD domain in Py235. In analogy to the receptor-mediated ligand-dimerization model of the EBL proteins PvDBP and PfEBA-175 from Plasmodium vivax and Plasmodium falciparum, respectively, we hypothesise that Py235 of P. yoelii binds via its EBD(1-194) domain to the RBC receptor, thereby inducing dimerization of the Py235-receptor complex.

Structural characterization of the erythrocyte binding domain of the reticulocyte binding protein homologue family of Plasmodium yoelii.

Invasion of the host cell by the malaria parasite is a key step for parasite survival and the only stage of its life cycle where the parasite is extracellular, and it is therefore a target for an antimalaria intervention strategy. Multiple members of the reticulocyte binding protein homologues (RH) family are found in all plasmodia and have been shown to bind to host red blood cells directly. In the study described here, we delineated the erythrocyte binding domain (EBD) of one member of the RH family, termed Py235, from Plasmodium yoelii. Moreover, we have obtained the low-resolution structure of the EBD using small-angle X-ray scattering. Comparison of the EDB structure to other characterized Plasmodium receptor binding domains suggests that there may be an overall structural conservation. These findings may help in developing new approaches to target receptor ligand interactions mediated by parasite proteins.

Solution structure of subunit F (Vma7p) of the eukaryotic V(1)V(O) ATPase from Saccharomyces cerevisiae derived from SAXS and NMR spectroscopy.

Vacuolar ATPases use the energy derived from ATP hydrolysis, catalyzed in the A(3)B(3) sector of the V(1) ATPase to pump protons via the membrane-embedded V(O) sector. The energy coupling between the two sectors occurs via the so-called central stalk, to which subunit F does belong. Here we present the first low resolution structure of recombinant subunit F (Vma7p) of a eukaryotic V-ATPase from Saccharomyces cerevisiae, analyzed by small angle X-ray scattering (SAXS). The protein is divided into a 5.5nm long egg-like shaped region, connected via a 1.5nm linker to a hook-like segment at one end. Circular dichroism spectroscopy revealed that subunit F comprises of 43% α-helix, 32% ß-sheet and a 25% random coil arrangement. To determine the localization of the N- and C-termini in the protein, the C-terminal truncated form of F, F(1-94) was produced and analyzed by SAXS. Comparison of the F(1-94) shape with the one of subunit F showed the missing hook-like region in F(1-94), supported by the decreased D(max) value of F(1-94) (7.0nm), and indicating that the hook-like region consists of the C-terminal residues. The NMR solution structure of the C-terminal peptide, F(90-116), was solved, displaying an α-helical region between residues 103 and 113. The F(90-116) solution structure fitted well in the hook-like region of subunit F. Finally, the arrangement of subunit F within the V(1) ATPase is discussed.

Crystallographic studies of the coupling segment NBD94(674-781) of the nucleotide-binding domain of the Plasmodium yoelii reticulocyte-binding protein Py235.

The Plasmodium yoelii reticulocyte-binding protein Py235 has a role as an ATP/ADP sensor. The sensor domain of Py235 is called NBD94; it consists of at least three functional regions, the nucleotide-binding region (NBD94(444-547)), hinge region (NBD94(566-663)) and C-terminal coupling region (NBD94(674-781)), and has been proposed to link ATP/ADP binding to the interaction of Py235 with the red blood cell. Here, NBD94(674-781) was cloned, expressed and purified to high purity. The monodisperse protein was crystallized by vapour diffusion. A diffraction data set was collected to 2.9 Šresolution with 97.2% completeness using a synchrotron-radiation source. The crystals belonged to space group C2, with unit-cell parameters a=65.08, b=82.71, c=114.27 Å, ß=94.72°, and contained four molecules in the asymmetric unit.

The NMR solution structure of subunit G (G(61)(-)(101)) of the eukaryotic V1VO ATPase from Saccharomyces cerevisiae.

Subunit G is an essential stalk subunit of the eukaryotic proton pump V(1)V(O) ATPase. Previously the structure of the N-terminal region, G(1)(-)(59), of the 13kDa subunit G was solved at higher resolution. Here solution NMR was performed to determine the structure of the recombinant C-terminal region (G(61)(-)(101)) of subunit G of the Saccharomyces cerevisiae V(1)V(O) ATPase. The protein forms an extended alpha-helix between residues 64 and 100, whereby the first five- and the last residues of G(61)(-)(101) are flexible. The surface charge distribution of G(61)(-)(101) reveals an amphiphilic character at the C-terminus due to positive and negative charge distribution at one side and a hydrophobic surface on the opposite side of the structure. The hydrophobic surface pattern is mainly formed by alanine residues. The alanine residues 72, 74 and 81 were exchanged by a single cysteine in the entire subunit G. Cysteines at positions 72 and 81 showed disulfide formation. In contrast, no crosslink could be formed for the mutant Ala74Cys. Together with the recently determined NMR solution structure of G(1)(-)(59), the presented solution structure of G(61)(-)(101) enabled us to present a first structural model of the entire subunit G of the S. cerevisiae V(1)V(O) ATPase.

Structural determination of functional units of the nucleotide binding domain (NBD94) of the reticulocyte binding protein Py235 of Plasmodium yoelii.

BACKGROUND: Invasion of the red blood cells (RBC) by the merozoite of malaria parasites involves a large number of receptor ligand interactions. The reticulocyte binding protein homologue family (RH) plays an important role in erythrocyte recognition as well as virulence. Recently, it has been shown that members of RH in addition to receptor binding may also have a role as ATP/ADP sensor. A 94 kDa region named Nucleotide-Binding Domain 94 (NBD94) of Plasmodium yoelii YM, representative of the putative nucleotide binding region of RH, has been demonstrated to bind ATP and ADP selectively. Binding of ATP or ADP induced nucleotide-dependent structural changes in the C-terminal hinge-region of NBD94, and directly impacted on the RBC binding ability of RH. METHODOLOGY/PRINCIPAL FINDINGS: In order to find the smallest structural unit, able to bind nucleotides, and its coupling module, the hinge region, three truncated domains of NBD94 have been generated, termed NBD94(444-547), NBD94(566-663) and NBD94(674-793), respectively. Using fluorescence correlation spectroscopy NBD94(444-547) has been identified to form the smallest nucleotide binding segment, sensitive for ATP and ADP, which became inhibited by 4-Chloro-7-nitrobenzofurazan. The shape of NBD94(444-547) in solution was calculated from small-angle X-ray scattering data, revealing an elongated molecule, comprised of two globular domains, connected by a spiral segment of about 73.1 A in length. The high quality of the constructs, forming the hinge-region, NBD94(566-663) and NBD94(674-793) enabled to determine the first crystallographic and solution structure, respectively. The crystal structure of NBD94(566-663) consists of two helices with 97.8 A and 48.6 A in length, linked by a loop. By comparison, the low resolution structure of NBD94(674-793) in solution represents a chair-like shape with three architectural segments. CONCLUSIONS: These structures give the first insight into how nucleotide binding impacts on the overall structure of RH and demonstrates the potential use of this region as a novel drug target.

Assembly of subunit d (Vma6p) and G (Vma10p) and the NMR solution structure of subunit G (G(1-59)) of the Saccharomyces cerevisiae V(1)V(O) ATPase.

Understanding the structural traits of subunit G is essential, as it is needed for V(1)V(O) assembly and function. Here solution NMR of the recombinant N- (G(1-59)) and C-terminal segment (G(61-114)) of subunit G, has been performed in the absence and presence of subunit d of the yeast V-ATPase. The data show that G does bind to subunit d via its N-terminal part, G(1-59) only. The residues of G(1-59) involved in d binding are Gly7 to Lys34. The structure of G(1-59) has been solved, revealing an alpha-helix between residues 10 and 56, whereby the first nine- and the last three residues of G(1-59) are flexible. The surface charge distribution of G(1-59) reveals an amphiphilic character at the N-terminus due to positive and negative charge distribution at one side and a hydrophobic surface on the opposite side of the structure. The C-terminus exhibits a strip of negative residues. The data imply that G(1-59)-d assembly is accomplished by hydrophobic interactions and salt-bridges of the polar residues. Based on the recently determined NMR structure of segment E(18-38) of subunit E of yeast V-ATPase and the presently solved structure of G(1-59), both proteins have been docked and binding epitopes have been analyzed.

Spectroscopical identification of residues of subunit G of the yeast V-ATPase in its connection with subunit E.

A critical point in the V(1) sector and entire V(1)V(O) complex is the interaction of stalk subunits G (Vma10p) and E (Vma4p). Previous work, using precipitation assays, has shown that both subunits form a complex. In this work, we have analysed the N-terminal segment of subunit G (G(1-59)) of the V(1)V(O) ATPase from Saccharomyces cerevisiae by using nuclear magnetic resonance (NMR) spectroscopy. Analyses of (1)H-(15)N heteronuclear single quantum coherence (HSQC) spectra of G(1-59) in the absence and presence of the N-terminal peptides E(1-18) and E(18-38) as well as the produced and purified C-terminal segment (E(39-233)) shows specific interactions only with the peptide fragment E(18-38). The binding of this peptide occurs via the residues M(1), V(2), S(3), and K(5) as well for V(22), S(23), K(24), A(25) and R(26) of G(1-59). The specific E(18-38)/G(1-59) binding has been confirmed by fluorescence correlation spectroscopy data. The E(18-38) peptide has been studied by CD spectroscopy and NMR. The 3D structure of this peptide adopts a stable helix-hinge-helix formation in solution. A model structure of the E(18-38)/G(1-59) complex reveals the orientation of E(18-38) relative to G(1-59) via salt-bridges of the polar residues and van der Waals forces at the very N-terminus of both segments.