Malathy Sony Subramanian Manimekalai

ATP synthases from archaea: the beauty of a molecular motor.

Archaea live under different environmental conditions, such as high salinity, extreme pHs and cold or hot temperatures. How energy is conserved under such harsh environmental conditions is a major question in cellular bioenergetics of archaea. The key enzymes in energy conservation are the archaeal A1AO ATP synthases, a class of ATP synthases distinct from the F1FO ATP synthase ATP synthase found in bacteria, mitochondria and chloroplasts and the V1VO ATPases of eukaryotes. A1AO ATP synthases have distinct structural features such as a collar-like structure, an extended central stalk, and two peripheral stalks possibly stabilizing the A1AO ATP synthase during rotation in ATP synthesis/hydrolysis at high temperatures as well as to provide the storage of transient elastic energy during ion-pumping and ATP synthesis/-hydrolysis. High resolution structures of individual subunits and subcomplexes have been obtained in recent years that shed new light on the function and mechanism of this unique class of ATP synthases. An outstanding feature of archaeal A1AO ATP synthases is their diversity in size of rotor subunits and the coupling ion used for ATP synthesis with H(+), Na(+) or even H(+) and Na(+) using enzymes. The evolution of the H(+) binding site to a Na(+) binding site and its implications for the energy metabolism and physiology of the cell are discussed.

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.

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 insight and flexible features of NS5 proteins from all four serotypes of Dengue virus in solution

Infection by the four serotypes of Dengue virus (DENV-1 to DENV-4) causes an important arthropod-borne viral disease in humans. The multifunctional DENV nonstructural protein 5 (NS5) is essential for capping and replication of the viral RNA and harbours a methyltransferase (MTase) domain and an RNA-dependent RNA polymerase (RdRp) domain. In this study, insights into the overall structure and flexibility of the entire NS5 of all four Dengue virus serotypes in solution are presented for the first time. The solution models derived revealed an arrangement of the full-length NS5 (NS5FL) proteins with the MTase domain positioned at the top of the RdRP domain. The DENV-1 to DENV-4 NS5 forms are elongated and flexible in solution, with DENV-4 NS5 being more compact relative to NS5 from DENV-1, DENV-2 and DENV-3. Solution studies of the individual MTase and RdRp domains show the compactness of the RdRp domain as well as the contribution of the MTase domain and the ten-residue linker region to the flexibility of the entire NS5. Swapping the ten-residue linker between DENV-4 NS5FL and DENV-3 NS5FL demonstrated its importance in MTase-RdRp communication and in concerted interaction with viral and host proteins, as probed by amide hydrogen/deuterium mass spectrometry. Conformational alterations owing to RNA binding are presented.

Low resolution solution structure of HAMLET and the importance of its alpha-domains in tumoricidal activity.

HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) is the first member in a new family of protein-lipid complexes with broad tumoricidal activity. Elucidating the molecular structure and the domains crucial for HAMLET formation is fundamental for understanding its tumoricidal function. Here we present the low-resolution solution structure of the complex of oleic acid bound HAMLET, derived from small angle X-ray scattering data. HAMLET shows a two-domain conformation with a large globular domain and an extended part of about 2.22 nm in length and 1.29 nm width. The structure has been superimposed into the related crystallographic structure of human α-lactalbumin, revealing that the major part of α-lactalbumin accommodates well in the shape of HAMLET. However, the C-terminal residues from L105 to L123 of the crystal structure of the human α-lactalbumin do not fit well into the HAMLET structure, resulting in an extended conformation in HAMLET, proposed to be required to form the tumoricidal active HAMLET complex with oleic acid. Consistent with this low resolution structure, we identified biologically active peptide epitopes in the globular as well as the extended domains of HAMLET. Peptides covering the alpha1 and alpha2 domains of the protein triggered rapid ion fluxes in the presence of sodium oleate and were internalized by tumor cells, causing rapid and sustained changes in cell morphology. The alpha peptide-oleate bound forms also triggered tumor cell death with comparable efficiency as HAMLET. In addition, shorter peptides corresponding to those domains are biologically active. These findings provide novel insights into the structural prerequisites for the dramatic effects of HAMLET on tumor cells.

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.

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.

Structure of the nucleotide‐binding subunit B of the energy producer A1A0 ATP synthase in complex with adenosine diphosphate

A1A0 ATP synthases are the major energy producers in archaea. Like the related prokaryotic and eukaryotic F1F0 ATP synthases, they are responsible for most of the synthesis of adenosine triphosphate. The catalytic events of A1A0 ATP synthases take place inside the A3B3 hexamer of the A1 domain. Recently, the crystallographic structure of the nucleotide-free subunit B of Methanosarcina mazei Gö1 A1A0 ATP synthase has been determined at 1.5 A resolution. To understand more about the nucleotide-binding mechanism, a protocol has been developed to crystallize the subunit B-ADP complex. The crystallographic structure of this complex has been solved at 2.7 A resolution. The ADP occupies a position between the essential phosphate-binding loop and amino-acid residue Phe149, which are involved in the binding of the antibiotic efrapeptin in the related F1F0 ATP synthases. This trapped ADP location is about 13 A distant from its final binding site and is therefore called the transition ADP-binding position. In the trapped ADP position the structure of subunit B adopts a different conformation, mainly in its C-terminal domain and also in the final nucleotide-binding site of the central alphabeta-domain. This atomic model provides insight into how the substrate enters into the nucleotide-binding protein and thereby into the catalytic A3B3 domain.

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 Characterization of the Erythrocyte Binding Domain of the Reticulocyte Binding Protein Homologue Family of Plasmodium yoelii

ABSTRACT 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.