Goran Biuković

Recombination between the linear plasmid pPZG101 and the linear chromosome of Streptomyces rimosus can lead to exchange of ends

The 387 kb linear plasmid pPZG101 of Streptomyces rimosus R6 can integrate into the chromosome or form a prime plasmid carrying the oxytetracycline biosynthesis cluster. The integration of plasmid pPZG101 into the linear chromosome of S. rimosus R6‐501 in mutant MV25 was shown to be due to a single cross‐over at a 4 bp common sequence. pPZG101 had integrated into a 250 kb DNA sequence that was reiterated at a low level. This sequence includes the oxytetracycline biosynthesis cluster, so that homologous recombination generated a mixed population carrying different copy numbers of the region. The 1 Mb linear plasmid pPZG103 in mutant MV17 had also arisen from a cross‐over between pPZG101 and the chromosome, so that one end of pPZG103 consists of c. 850 kb of chromosomal sequence including the oxytetracycline biosynthesis cluster. The plasmid pPZG101 was shown to consist of a unique central region of about 30 kb flanked by terminal inverted repeats of about 180 kb. Analysis of a presumed ancestor plasmid pPZG102 suggested that the long terminal repeats had arisen by a recombination event during the strain development programme.

Variations of Subunit ε of the Mycobacterium tuberculosis F1Fo ATP Synthase and a Novel Model for Mechanism of Action of the Tuberculosis Drug TMC207

ABSTRACT The subunit ε of bacterial F1FO ATP synthases plays an important regulatory role in coupling and catalysis via conformational transitions of its C-terminal domain. Here we present the first low-resolution solution structure of ε of Mycobacterium tuberculosis (Mtε) F1FO ATP synthase and the nuclear magnetic resonance (NMR) structure of its C-terminal segment (Mtε103–120). Mtε is significantly shorter (61.6 Å) than forms of the subunit in other bacteria, reflecting a shorter C-terminal sequence, proposed to be important in coupling processes via the catalytic β subunit. The C-terminal segment displays an α-helical structure and a highly positive surface charge due to the presence of arginine residues. Using NMR spectroscopy, fluorescence spectroscopy, and mutagenesis, we demonstrate that the new tuberculosis (TB) drug candidate TMC207, proposed to bind to the proton translocating c-ring, also binds to Mtε. A model for the interaction of TMC207 with both ε and the c-ring is presented, suggesting that TMC207 forms a wedge between the two rotating subunits by interacting with the residues W15 and F50 of ε and the c-ring, respectively. T19 and R37 of ε provide the necessary polar interactions with the drug molecule. This new model of the mechanism of TMC207 provides the basis for the design of new drugs targeting the F1FO ATP synthase in M. tuberculosis.

The Stem Region of Premembrane Protein Plays an Important Role in the Virus Surface Protein Rearrangement during Dengue Maturation

Background: Dengue virus surface proteins, envelope (E) and pre-membrane (prM), undergo rearrangement during the maturation process at acidic condition. Results: prM-stem region binds tighter to both E protein and lipid membrane when environment becomes acidic. Conclusion: At acidic condition, E proteins are attracted to the membrane-associated prM-stem. Significance: prM-stem region induces virus structural changes during maturation. Newly assembled dengue viruses (DENV) undergo maturation to become infectious particles. The maturation process involves major rearrangement of virus surface premembrane (prM) and envelope (E) proteins. The prM-E complexes on immature viruses are first assembled as trimeric spikes in the neutral pH environment of the endoplasmic reticulum. When the virus is transported to the low pH environment of the exosomes, these spikes rearrange into dimeric structures, which lie parallel to the virus lipid envelope. The proteins involved in driving this process are unknown. Previous cryoelectron microscopy studies of the mature DENV showed that the prM-stem region (residues 111–131) is membrane-associated and may interact with the E proteins. Here we investigated the prM-stem region in modulating the virus maturation process. The binding of the prM-stem region to the E protein was shown to increase significantly at low pH compared with neutral pH in ELISAs and surface plasmon resonance studies. In addition, the affinity of the prM-stem region for the liposome, as measured by fluorescence correlation spectroscopy, was also increased when pH is lowered. These results suggest that the prM-stem region forms a tight association with the virus membrane and attracts the associated E protein in the low pH environment of exosomes. This will lead to the surface protein rearrangement observed during maturation.

Characteristics of a Streptomyces coelicolor A3(2) Extracellular Protein Targeting Chitin and Chitosan

ABSTRACT Upstream of the Streptomyces coelicolor A3(2) chitinase G gene, a small gene (named chb3) is located whose deduced product shares 37% identical amino acids with the previously described CHB1 protein from Streptomyces olivaceoviridis. Thechb3 gene and its upstream region were cloned in a multicopy vector and transformed into the plasmid-freeStreptomyces lividans TK21 strain. The CHB3 protein (14.9 kDa) was secreted by the S. lividans TK21 transformant during growth in the presence of glucose,N-acetylglucosamine, yeast extract, and chitin. The protein was purified to homogeneity using anionic exchange, hydrophobic interaction chromatographies, and gel filtration. In contrast to CHB1, CHB3 targets α-chitin, β-chitin, and chitosan at pH 6.0 but does so relatively loosely. The ecological implications of the divergence of substrate specificity of various types of chitin-binding proteins are described.

Solution structure, determined by nuclear magnetic resonance, of the b30-82 domain of subunit b of Escherichia coli F1Fo ATP synthase

Subunit b, the peripheral stalk of bacterial F(1)F(o) ATP synthases, is composed of a membrane-spanning and a soluble part. The soluble part is divided into tether, dimerization, and delta-binding domains. The first solution structure of b30-82, including the tether region and part of the dimerization domain, has been solved by nuclear magnetic resonance, revealing an alpha-helix between residues 39 and 72. In the solution structure, b30-82 has a length of 48.07 A. The surface charge distribution of b30-82 shows one side with a hydrophobic surface pattern, formed by alanine residues. Alanine residues 61, 68, 70, and 72 were replaced by single cysteines in the soluble part of subunit b, b22-156. The cysteines at positions 61, 68, and 72 showed disulfide formation. In contrast, no cross-link could be formed for the A70C mutant. The patterns of disulfide bonding, together with the circular dichroism spectroscopy data, are indicative of an adjacent arrangement of residues 61, 68, and 72 in both alpha-helices in b22-156.

Identification of critical residues of subunit H in its interaction with subunit E of the A‐ATP synthase from Methanocaldococcus jannaschii

The boomerang‐like H subunit of A1A0 ATP synthase forms one of the peripheral stalks connecting the A1 and A0 sections. Structural analyses of the N‐terminal part (H1–47) of subunit H of the A1A0 ATP synthase from Methanocaldococcus jannaschii have been performed by NMR spectroscopy. Our initial NMR structural calculations for H1–47 indicate that amino acid residues 7–44 fold into a single α‐helical structure. Using the purified N‐ (E1–100) and C‐terminal domains (E101–206) of subunit E, NMR titration experiments revealed that the N‐terminal residues Met1–6, Lys10, Glu11, Ala15, Val20 and Glu24 of H1–47 interact specifically with the N‐terminal domain E1–100 of subunit E. A more detailed picture regarding the residues of E1–100 involved in this association was obtained by titration studies using the N‐terminal peptides E1–20, E21–40 and E41–60. These data indicate that the N‐terminal tail E41–60 interacts with the N‐terminal amino acids of H1–47, and this has been confirmed by fluorescence correlation spectroscopy results. Analysis of 1H–15N heteronuclear single quantum coherence (HSQC) spectra of the central stalk subunit F in the presence and absence of E101–206 show no obvious interaction between the C‐terminal domain of E and subunit F. The data presented provide, for the first time, structural insights into the interaction of subunits E and H, and their arrangement within A1A0 ATP synthase.

Power Stroke Angular Velocity Profiles of Archaeal A-ATP Synthase Versus Thermophilic and Mesophilic F-ATP Synthase Molecular Motors

The angular velocities of ATPase-dependent power strokes as a function of the rotational position for the A-type molecular motor A3B3DF, from the Methanosarcina mazei Gö1 A-ATP synthase, and the thermophilic motor α3β3γ, from Geobacillus stearothermophilus (formerly known as Bacillus PS3) F-ATP synthase, are resolved at 5 μs resolution for the first time. Unexpectedly, the angular velocity profile of the A-type was closely similar in the angular positions of accelerations and decelerations to the profiles of the evolutionarily distant F-type motors of thermophilic and mesophilic origins, and they differ only in the magnitude of their velocities. M. mazei A3B3DF power strokes occurred in 120° steps at saturating ATP concentrations like the F-type motors. However, because ATP-binding dwells did not interrupt the 120° steps at limiting ATP, ATP binding to A3B3DF must occur during the catalytic dwell. Elevated concentrations of ADP did not increase dwells occurring 40° after the catalytic dwell. In F-type motors, elevated ADP induces dwells 40° after the catalytic dwell and slows the overall velocity. The similarities in these power stroke profiles are consistent with a common rotational mechanism for A-type and F-type rotary motors, in which the angular velocity is limited by the rotary position at which ATP binding occurs and by the drag imposed on the axle as it rotates within the ring of stator subunits.

Deletion of a unique loop in the mycobacterial F-ATP synthase γ subunit sheds light on its inhibitory role in ATP hydrolysis-driven H(+) pumping.

The F1FO‐ATP synthase is one of the enzymes that is essential to meet the energy requirement of both the proliferating aerobic and hypoxic dormant stages of the life cycle of mycobacteria. Most F‐ATP synthases consume ATP in the α3:β3 headpiece to drive the γ subunit, which couples ATP cleavage with proton pumping in the c ring of FO via the bottom of the γ subunit. ATPase‐driven H+ pumping is latent in mycobacteria. The presence of a unique 14 amino acid residue loop of the mycobacterial γ subunit has been described and aligned in close vicinity to the c‐ring loop Priya R et al. (2013) J Bioenerg Biomembr 45, 121‐129 Here, we used inverted membrane vesicles (IMVs) of fast‐growing Mycobacterium smegmatis and a variety of covalent and non‐covalent inhibitors to characterize the ATP hydrolysis activity of the F‐ATP synthase inside IMVs. These vesicles formed a platform to investigate the function of the unique mycobaterial γ loop by deleting the respective loop‐encoding sequence (γ166–179) in the genome of M. smegmatis. ATP hydrolysis‐driven H+ pumping was observed in IMVs containing the Δγ166–179 mutant protein but not for IMVs containing the wild‐type F‐ATP synthase. In addition, when compared to the wild‐type enzyme, IMVs containing the Δγ166–179 mutant protein showed increased ATP cleavage and lower levels of ATP synthesis, demonstrating that the loop affects ATPase activity, ATPase‐driven H+ pumping and ATP synthesis. These results further indicate that the loop may affect coupling of ATP hydrolysis and synthesis in a different mode.

Domain Features of the Peripheral Stalk Subunit H of the Methanogenic A1AO ATP Synthase and the NMR Solution Structure of H1-47

A series of truncated forms of subunit H were generated to establish the domain features of that protein. Circular dichroism analysis demonstrated that H is divided at least into a C-terminal coiled-coil domain within residues 54-104, and an N-terminal domain formed by adjacent alpha-helices. With a cysteine at the C-terminus of each of the truncated proteins (H(1-47), H(1-54), H(1-59), H(1-61), H(1-67), H(1-69), H(1-71), H(1-78), H(1-80), H(1-91), and H(47-105)), the residues involved in formation of the coiled-coil interface were determined. Proteins H(1-54), H(1-61), H(1-69), and H(1-80) showed strong cross-link formation, which was weaker in H(1-47), H(1-59), H(1-71), and H(1-91). A shift in disulfide formation between cysteines at positions 71 and 80 reflected an interruption in the periodicity of hydrophobic residues in the region 71AEKILEETEKE81. To understand how the N-terminal domain of H is formed, we determined for the first time, to our knowledge, the solution NMR structure of H(1-47), which revealed an alpha-helix between residues 15-42 and a flexible N-terminal stretch. The alpha-helix includes a kink that would bring the two helices of the C-terminus into the coiled-coil arrangement. H(1-47) revealed a strip of alanines involved in dimerization, which were tested by exchange to single cysteines in subunit H mutants.

The uniqueness of subunit α of mycobacterial F-ATP synthases: An evolutionary variant for niche adaptation

The F1F0 -ATP (F-ATP) synthase is essential for growth of Mycobacterium tuberculosis, the causative agent of tuberculosis (TB). In addition to their synthase function most F-ATP synthases possess an ATP-hydrolase activity, which is coupled to proton-pumping activity. However, the mycobacterial enzyme lacks this reverse activity, but the reason for this deficiency is unclear. Here, we report that a Mycobacterium-specific, 36-amino acid long C-terminal domain in the nucleotide-binding subunit α (Mtα) of F-ATP synthase suppresses its ATPase activity and determined the mechanism of suppression. First, we employed vesicles to show that in intact membrane-embedded mycobacterial F-ATP synthases deletion of the C-terminal domain enabled ATPase and proton-pumping activity. We then generated a heterologous F-ATP synthase model system, which demonstrated that transfer of the mycobacterial C-terminal domain to a standard F-ATP synthase α subunit suppresses ATPase activity. Single-molecule rotation assays indicated that the introduction of this Mycobacterium-specific domain decreased the angular velocity of the power-stroke after ATP binding. Solution X-ray scattering data and NMR results revealed the solution shape of Mtα and the 3D structure of the subunit α C-terminal peptide 521PDEHVEALDEDKLAKEAVKV540 of M. tubercolosis (Mtα(521–540)), respectively. Together with cross-linking studies, the solution structural data lead to a model, in which Mtα(521–540) comes in close proximity with subunit γ residues 104–109, whose interaction may influence the rotation of the camshaft-like subunit γ. Finally, we propose that the unique segment Mtα(514–549), which is accessible at the C terminus of mycobacterial subunit α, is a promising drug epitope.