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.

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.

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.

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.

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.