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

Substrate‐induced structural alterations of Mycobacterial mycothione reductase and critical residues involved

Redox homeostasis is a prerequisite for survival of the pathogen Mycobacterium tuberculosis (Mtb) which employs the low molecular weight thiol mycothiol (MSH). The Mycobacterial NADPH-dependent mycothione reductase (MtMtr), composed of an NADPH-, FAD-, and a dimerization-domain connected by linkers, regulates the balance of oxidized-reduced MSH. Here, we demonstrate by small-angle X-ray scattering, that NADPH-binding alters the oligomeric state equilibrium of the protein with no significant overall structural change after MSH-binding. Mutation of critical residues in the linker regions of MtMtr eliminate partially or totally the NADPH-induced oligomerization effect with simultaneous effect on enzyme activity. The data provide insight into the MtMtr linker regions involved in the novel oligomerization equilibrium of the Mycobacterial enzyme.Ministry of Education (MOE

Features and functional importance of key residues of the Mycobacterium tuberculosis cytochrome bd oxidase

Cytochrome bd (cyt-bd) oxygen reductases have a high affinity to oxygen and use the two electrons provided by ubiquinol or menaquinol, like in mycobacteria, to reduce oxygen to water. Although they do not pump protons from the cytoplasmic to the periplasmic side, they generate a proton motive force due to the release of protons after quinol oxidation. Here, we show that the mycobacterial cyt-bd has a number of specific features, including a 17-residue stretch (307SGVTLQGIRDLQQEYQQ323) near the Q-loop of the Mycobacterium tuberculosis subunit CydA and a 412QLVRLTVKA420 region on the periplasmic side. Site directed mutagenesis and whole-bacteria assays demonstrated that these mycobacteria-specific stretches are essential for the oxidase’s function. Single amino acid substitutions around the 307SGVTLQGIRDLQQEYQQ323 stretch revealed the importance of the aromatic residue Y330 in oxygen consumption and consequently in ATP synthesis. A moderate reduction and no effect was observed for mutants F325 and Y321, respectively, while the double mutant CydAY321/F325 drastically reduced enzyme activity. In addition, single mutants of the mycobacterial cyt-bd were generated to probe the role of proposed critical residues for proton shuffling. Further data demonstrate that amino acids W64 and F18 in the CydB subunit might be important as any slight destabilization of the hydrophobic environment near them makes the enzyme inactive. Finally, the potential of the mycobacterial cyt-bd as a drug target is discussed.National Research Foundation (NRF)Accepted versionThis research and the PhD scholarship of Ekaterina Sviriaeva were supported by the National Research Foundation (NRF) Singapore, NRF Competitive Research Programme (CRP), Grant Award Number NRF–CRP18–2017–01 to G.G. and K.P

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 EBD1–194, 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 EBD1–194 domain to the RBC receptor, thereby inducing dimerization of the Py235-receptor complex

Crystal structure of subunits D and F in complex gives insight into energy transmission of the eukaryotic V-ATPase from Saccharomyces cerevisiae

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.MOE (Min. of Education, S’pore)Published versio

Crystallization and preliminary X-ray crystallographic analysis of subunit F (F1-94), an essential coupling subunit of the eukaryotic V1VO-ATPase from Saccharomyces cerevisiae

V-ATPases are very complex multi-subunit enzymes which function as proton-pumping rotary nanomotors. The rotary and coupling subunit F (F1-94) was crystallized by the hanging-drop vapour-diffusion method. The native crystals diffracted to a resolution of 2.64 Å and belonged to space group C2221, with unit-cell parameters a = 47.21, b = 160.26, c = 102.49 Å. The selenomethionyl form of the F1-94 I69M mutant diffracted to a resolution of 2.3 Å and belonged to space group C2221, with unit-cell parameters a = 47.22, b = 160.83, c = 102.74 Å. Initial phasing and model building suggested the presence of four molecules in the asymmetric unit.Published versio

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.MOE (Min. of Education, S’pore)Accepted versio

Structure and flexibility of non-structural proteins 3 and -5 of Dengue- and Zika viruses in solution

Dengue- (DENV) and Zika viruses (ZIKV) rely on their non-structural protein 5 (NS5) including a methyl-transferase (MTase) and a RNA-dependent RNA polymerase (RdRp) for capping and synthesis of the viral RNA, and the non-structural protein 3 (NS3) with its protease and helicase domain for polyprotein possessing, unwinding dsRNA proceeding replication, and NTPase/RTPase activities. Accumulation of data for DENV- and ZIKV NS3 and NS5 in solution during recent years provides information about their overall shape, substrate-induced alterations, oligomeric forms and flexibility, with the latter being essential for domain-domain crosstalk. The importance and differences of the linker regions that connect the two domains of NS3 or NS5 are highlighted in particular with respect to the different DENV serotypes (DENV-1 to −4) as well as to the sequence diversities between the DENV and ZIKV proteins. Novel mutants of the French Polynesia ZIKV NS3 linker presented, identify critical residues in protein stability and enzymatic activity.Ministry of Education (MOE)Accepted versionThis work as well as the research scholarship of Ankita Pan was supported by the Ministry of Education MOE Tier 3 (MOE2012-T3- 1-008), Singapore to G.G

Relevance of the conserved histidine and asparagine residues in the phosphate-binding loop of the nucleotide binding subunit B of A1AO ATP synthases

The nucleotide binding sites in A-ATP synthases are located at the interfaces of subunit A and B, which is proposed to play a regulatory role. Differential binding of MgATP and -ADP to subunit B has been described, which does not exist in the related α and B subunits of F-ATP synthases and V-ATPases, respectively. The conserved phosphate loop residues, histidine and asparagine, of the A-ATP synthase subunit B have been proposed to be essential for γ-phosphate interaction. To investigate the role of these conserved P-loop residues in nucleotide-binding, subunit B residues H156 and N157 of the Methanosarcina mazei Gö1 A-ATP synthase were separately substituted with alanine. In addition, N157 was mutated to threonine, because it is the corresponding amino acid in the P-loop of F-ATP synthase subunit α. The structures of the subunit B mutants H156A, N157A/T were solved up to a resolution of 1.75 and 1.7 Å. The binding constants for MgATP and -ADP were determined, demonstrating that the H156A and N157A mutants have a preference to the nucleotide over the wild type and N157T proteins. Importantly, the ability to distinguish MgATP or -ADP was lost, demonstrating that the histidine and asparagine residues are crucial for nucleotide differentiation in subunit B. The structures reveal that the enhanced binding of the alanine mutants is attributed to the increased accessibility of the nucleotide binding cavity, explaining that the structural arrangement of the conserved H156 and N157 define the nucleotide-binding characteristics of the regulatory subunit B of A-ATP synthases