Asha Manikkoth Balakrishna

Structure, mechanism and ensemble formation of the alkylhydroperoxide reductase subunits AhpC and AhpF from Escherichia coli

Hydroperoxides are reactive oxygen species (ROS) that are toxic to all cells and must be converted into the corresponding alcohols to alleviate oxidative stress. In Escherichia coli, the enzyme primarily responsible for this reaction is alkylhydroperoxide reductase (AhpR). Here, the crystal structures of both of the subunits of EcAhpR, EcAhpF (57 kDa) and EcAhpC (21 kDa), have been solved. The EcAhpF structures (2.0 and 2.65 Å resolution) reveal an open and elongated conformation, while that of EcAhpC (3.3 Å resolution) forms a decameric ring. Solution X-ray scattering analysis of EcAhpF unravels the flexibility of its N-terminal domain, and its binding to EcAhpC was demonstrated by isothermal titration calorimetry. These studies suggest a novel overall mechanistic model of AhpR as a hydroperoxide scavenger, in which the dimeric, extended AhpF prefers complex formation with the AhpC ring to accelerate the catalytic activity and thus to increase the chance of rescuing the cell from ROS.

Crystal Structure of Subunits D and F in Complex Gives Insight into Energy Transmission of the Eukaryotic V-ATPase from Saccharomyces cerevisiae.

Background: Subunits D and F are essential in ATP hydrolysis and reversible disassembly of V1VO-ATPases. Results: First crystallographic structure of eukaryotic V-ATPase subunit D and entire subunit F is presented. Conclusion: Structural elements in subunits D and F enable the DF assembly to become a central motor element in the engine V-ATPase. Significance: Mechanistic understanding of the subunit DF assembly and its key roles in enzyme catalysis and regulation are presented. Eukaryotic V1VO-ATPases hydrolyze ATP in the V1 domain coupled to ion pumping in VO. A unique mode of regulation of V-ATPases is the reversible disassembly of V1 and VO, which reduces ATPase activity and causes silencing of ion conduction. The subunits D and F are proposed to be key in these enzymatic processes. Here, we describe the structures of two conformations of the subunit DF assembly of Saccharomyces cerevisiae (ScDF) V-ATPase at 3.1 Å resolution. Subunit D (ScD) consists of a long pair of α-helices connected by a short helix (79IGYQVQE85) as well as a β-hairpin region, which is flanked by two flexible loops. The long pair of helices is composed of the N-terminal α-helix and the C-terminal helix, showing structural alterations in the two ScDF structures. The entire subunit F (ScF) consists of an N-terminal domain of four β-strands (β1–β4) connected by four α-helices (α1–α4). α1 and β2 are connected via the loop 26GQITPETQEK35, which is unique in eukaryotic V-ATPases. Adjacent to the N-terminal domain is a flexible loop, followed by a C-terminal α-helix (α5). A perpendicular and extended conformation of helix α5 was observed in the two crystal structures and in solution x-ray scattering experiments, respectively. Fitted into the nucleotide-bound A3B3 structure of the related A-ATP synthase from Enterococcus hirae, the arrangements of the ScDF molecules reflect their central function in ATPase-coupled ion conduction. Furthermore, the flexibility of the terminal helices of both subunits as well as the loop 26GQITPETQEK35 provides information about the regulatory step of reversible V1VO disassembly.

Structural Insights into Substrate Binding by PvFKBP35, a Peptidylprolyl cis-trans Isomerase from the Human Malarial Parasite Plasmodium vivax

ABSTRACT The immunosuppressive drug FK506 binding proteins (FKBPs), an immunophilin family with the immunosuppressive drug FK506 binding property, exhibit peptidylprolyl cis-trans isomerase (PPIase) activity. While the cyclophilin-catalyzed peptidylprolyl isomerization of X-Pro peptide bonds has been extensively studied, the mechanism of the FKBP-mediated peptidylprolyl isomerization remains uncharacterized. Thus, to investigate the binding of FKBP with its substrate and the underlying catalytic mechanism of the FKBP-mediated proline isomerization, here we employed the FK506 binding domain (FKBD) of the human malarial parasite Plasmodium vivax FK506 binding protein 35 (PvFKBP35) and examined the details of the molecular interaction between the isomerase and a peptide substrate. The crystallographic structures of apo PvFKBD35 and its complex with the tetrapeptide substrate succinyl-Ala-Leu-Pro-Phe-p-nitroanilide (sALPFp) determined at 1.4 Å and 1.65 Å resolutions, respectively, showed that the substrate binds to PvFKBD35 in a cis conformation. Nuclear magnetic resonance (NMR) studies demonstrated the chemical shift perturbations of D55, H67, V73, and I74 residues upon the substrate binding. In addition, the X-ray crystal structure, along with the mutational studies, shows that Y100 is a key residue for the catalytic activity. Taken together, our results provide insights into the catalytic mechanism of PvFKBP35-mediated cis-trans isomerization of substrate and ultimately might aid designing substrate mimetic inhibitors targeting the malarial parasite FKBPs.

Spectroscopic and crystallographic studies of the mutant R416W give insight into the nucleotide binding traits of subunit B of the A1Ao ATP synthase

A strategically placed tryptophan in position of Arg416 was used as an optical probe to monitor adenosine triphosphate and adenosine‐diphosphate binding to subunit B of the A1AO adenosine triphosphate (ATP) synthase from Methanosarcina mazei Gö1. Tryptophan fluorescence and fluorescence correlation spectroscopy gave binding constants indicating a preferred binding of ATP over ADP to the protein. The X‐ray crystal structure of the R416W mutant protein in the presence of ATP was solved to 2.1 Å resolution, showing the substituted Trp‐residue inside the predicted adenine‐binding pocket. The cocrystallized ATP molecule could be trapped in a so‐called transition nucleotide‐binding state. The high resolution structure shows the phosphate residues of the ATP near the P‐loop region (S150‐E158) and its adenine ring forms π–π interaction with Phe149. This transition binding position of ATP could be confirmed by tryptophan emission spectra using the subunit B mutant F149W. The trapped ATP position, similar to the one of the binding region of the antibiotic efrapeptin in F1FO ATP synthases, is discussed in light of a transition nucleotide‐binding state of ATP while on its way to the final binding pocket. Finally, the inhibitory effect of efrapeptin C in ATPase activity of a reconstituted A3B3‐ and A3B(R416W)3‐subcomplex, composed of subunit A and the B subunit mutant R416W, of the A1AO ATP synthase is shown. Proteins 2009.

Redox chemistry of Mycobacterium tuberculosis alkylhydroperoxide reductase E (AhpE): Structural and mechanistic insight into a mycoredoxin-1 independent reductive pathway of AhpE via mycothiol

Mycobacterium tuberculosis (Mtb) has the ability to persist within the human host for a long time in a dormant stage and re-merges when the immune system is compromised. The pathogenic bacterium employs an elaborate antioxidant defence machinery composed of the mycothiol- and thioredoxin system in addition to a superoxide dismutase, a catalase, and peroxiredoxins (Prxs). Among the family of Peroxiredoxins, Mtb expresses a 1-cysteine peroxiredoxin, known as alkylhydroperoxide reductase E (MtAhpE), and defined as a potential tuberculosis drug target. The reduced MtAhpE (MtAhpE-SH) scavenges peroxides to become converted to MtAhpE-SOH. To provide continuous availability of MtAhpE-SH, MtAhpE-SOH has to become reduced. Here, we used NMR spectroscopy to delineate the reduced (MtAhpE-SH), sulphenic (MtAhpE-SOH) and sulphinic (MtAhpE-SO2H) states of MtAhpE through cysteinyl-labelling, and provide for the first time evidence of a mycothiol-dependent mechanism of MtAhpE reduction. This is confirmed by crystallographic studies, wherein MtAhpE was crystallized in the presence of mycothiol and the structure was solved at 2.43Å resolution. Combined with NMR-studies, the crystallographic structures reveal conformational changes of important residues during the catalytic cycle of MtAhpE. In addition, alterations of the overall protein in solution due to redox modulation are observed by small angle X-ray scattering (SAXS) studies. Finally, by employing SAXS and dynamic light scattering, insight is provided into the most probable physiological oligomeric state of MtAhpE necessary for activity, being also discussed in the context of concerted substrate binding inside the dimeric MtAhpE.

Structural basis of typhoid: Salmonella typhi type IVb pilin (PilS) and cystic fibrosis transmembrane conductance regulator interaction

The type IVb pilus of the enteropathogenic bacteria Salmonella typhi is a major adhesion factor during the entry of this pathogen into gastrointestinal epithelial cells. Its target of adhesion is a stretch of 10 residues from the first extracellular domain of cystic fibrosis transmembrane conductance regulator (CFTR). The crystal structure of the N‐terminal 25 amino acid deleted S. typhi native PilS protein (ΔPilS), which makes the pilus, was determined at 1.9 Å resolution by the multiwavelength anomalous dispersion method. Also, the structure of the complex of ΔPilS and a target CFTR peptide, determined at 1.8 Å, confirms that residues 113–117 (NKEER) of CFTR are involved in binding with the pilin protein and gives us insight on the amino acids that are essential for binding. Furthermore, we have also explored the role of a conserved disulfide bridge in pilus formation. The subunit structure and assembly architecture are crucial for understanding pilus functions and designing suitable therapeutics against typhoid. Proteins 2009.

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.

A second transient position of ATP on its trail to the nucleotide-binding site of subunit B of the motor protein A1AO ATP synthase

The adenosine triphosphate (ATP) entrance into the nucleotide-binding subunits of ATP synthases is a puzzle. In the previously determined structure of subunit B mutant R416W of the Methanosarcina mazei Gö1 A-ATP synthase one ATP could be trapped at a transition position, close to the phosphate-binding loop. Using defined parameters for co-crystallization of an ATP-bound B-subunit, a unique transition position of ATP could be found in the crystallographic structure of this complex, solved at 3.4 A resolution. The nucleotide is found near the helix-turn-helix motif in the C-terminal domain of the protein; the location occupied by the gamma-subunit to interact with the empty beta-subunit in the thermoalkaliphilic Bacillus sp. TA2.A1 of the related F-ATP synthase. When compared with the determined structure of the ATP-transition position, close to the P-loop, and the nucleotide-free form of subunit B, the C-terminal domain of the B mutant is rotated by around 6 degrees, implicating an ATP moving pathway. We propose that, in the nucleotide empty state the central stalk subunit D is in close contact with subunit B and when the ATP molecule enters, D moves slightly, paving way for it to interact with the subunit B, which makes the C-terminal domain rotate by 6 degrees.

Crystal- and NMR structures give insights into the role and dynamics of subunit F of the eukaryotic V-ATPase from Saccharomyces cerevisiae

Background: Subunit F is a stalk subunit in V-ATPases. Results: This work is the first crystallographic and NMR structure of eukaryotic V-ATPase subunit F from Saccharomyces cerevisiae. Conclusion: Subunit F plays a central role during reversible disassembly and ATP hydrolysis by transmitting movements of subunit d and H. Significance: Insights into the structure and dynamics of subunit F are essential for the understanding of V1VO disassembly and ATP hydrolysis regulation. Subunit F of V-ATPases is proposed to undergo structural alterations during catalysis and reversible dissociation from the V1VO complex. Recently, we determined the low resolution structure of F from Saccharomyces cerevisiae V-ATPase, showing an N-terminal egg shape, connected to a C-terminal hook-like segment via a linker region. To understand the mechanistic role of subunit F of S. cerevisiae V-ATPase, composed of 118 amino acids, the crystal structure of the major part of F, F(1–94), was solved at 2.3 Å resolution. The structural features were confirmed by solution NMR spectroscopy using the entire F subunit. The eukaryotic F subunit consists of the N-terminal F(1–94) domain with four-parallel β-strands, which are intermittently surrounded by four α-helices, and the C terminus, including the α5-helix encompassing residues 103 to 113. Two loops 26GQITPETQEK35 and 60ERDDI64 are described to be essential in mechanistic processes of the V-ATPase enzyme. The 26GQITPETQEK35 loop becomes exposed when fitted into the recently determined EM structure of the yeast V1VO-ATPase. A mechanism is proposed in which the 26GQITPETQEK35 loop of subunit F and the flexible C-terminal domain of subunit H move in proximity, leading to an inhibitory effect of ATPase activity in V1. Subunits D and F are demonstrated to interact with subunit d. Together with NMR dynamics, the role of subunit F has been discussed in the light of its interactions in the processes of reversible disassembly and ATP hydrolysis of V-ATPases by transmitting movements of subunit d and H of the VO and V1 sector, respectively.

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 NBD94444–547, NBD94566–663 and NBD94674–793, respectively. Using fluorescence correlation spectroscopy NBD94444–547 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 NBD94444–547 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 Å in length. The high quality of the constructs, forming the hinge-region, NBD94566–663 and NBD94674–793 enabled to determine the first crystallographic and solution structure, respectively. The crystal structure of NBD94566–663 consists of two helices with 97.8 Å and 48.6 Å in length, linked by a loop. By comparison, the low resolution structure of NBD94674–793 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.