Svetlana Antonyuk - Academia.edu (original) (raw)

Papers by Svetlana Antonyuk

Acta Crystallographica Section D Structural Biology

Methionine adenosyltransferase (MAT) deficiency, characterized by isolated persistent hypermethio... more Methionine adenosyltransferase (MAT) deficiency, characterized by isolated persistent hypermethioninemia (IPH), is caused by mutations in the MAT1A gene encoding MATαl, one of the major hepatic enzymes. Most of the associated hypermethioninemic conditions are inherited as autosomal recessive traits; however, dominant inheritance of hypermethioninemia is caused by an Arg264His (R264H) mutation. This mutation has been confirmed in a screening programme of newborns as the most common mutation in babies with IPH. Arg264 makes an inter-subunit salt bridge located at the dimer interface where the active site assembles. Here, it is demonstrated that the R264H mutation results in greatly reduced MAT activity, while retaining its ability to dimerize, indicating that the lower activity arises from alteration at the active site. The first crystallographic structure of the apo form of the wild-type MATαl enzyme is provided, which shows a tetrameric assembly in which two compact dimers combine t...

Journal of Cell Science

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited condition that can c... more Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited condition that can cause fatal cardiac arrhythmia. Human mutations in the Ca2+ sensor calmodulin (CaM) have been associated with CPVT susceptibility, suggesting that CaM dysfunction is a key driver of the disease. However, the detailed molecular mechanism remains unclear. Focusing on the interaction with the cardiac ryanodine receptor (RyR2), we determined the effect of CPVT-associated variants N53I and A102V on the structural characteristics of CaM and on Ca2+ fluxes in live cells. We provide novel data showing that binding of both Ca2+/CaM-N53I and Ca2+/CaM-A102V to RyR23583-3603 is decreased. Ca2+/CaM:RyR23583-3603 high-resolution crystal structures highlight subtle conformational changes for the N53I variant, with A102V being similar to wild-type. We show that co-expression of CaM-N53I or CaM-A102V with RyR2 in HEK293 cells significantly increased the duration of Ca2+ events, CaM-A102V exhibited a lower...

Acta Crystallographica. Section D, Structural Biology, 2020

Dominant inheritance of hypermethioninemia is caused by an Arg264His mutation in methionine adeno... more Dominant inheritance of hypermethioninemia is caused by an Arg264His mutation in methionine adenosyltransferase. The mutation lowers the affinity for dimer–dimer interaction and enzymatic activity, which can be restored by chemical intervention.

F1000Research, 2011

ABSTRACT Background / Purpose: The combination of single crystal spectroscopies (e.g. optical, Ra... more ABSTRACT Background / Purpose: The combination of single crystal spectroscopies (e.g. optical, Raman and X-ray absorption) with high-resolution protein crystallography, working in tandem to probe the same protein crystal in situ at the X-ray beamline, can yield a complete and accurate identification of the oxidation and ligation states of redox centres and/or active sites (Hough et al 2008 (1); Antonyuk and Hough 2011 (2)). With this approach, the functional properties of the active site in the protein are directly related to its contemporaneously measured crystal structure(s). Importantly, controlled X-ray radiolysis of protein crystals provides a powerful means to access transient states such as catalytic intermediates (Hough et al 2008 (1)). Main conclusion: Here, we describe (i) a systematic application of these combined tools in order to gain access to functionally relevant transient catalytic states and to generate a high-resolution ‘structural movie’; through the catalytic cycle of a Cu-containing nitrite reductase; (ii) the fingerprinting of redox-ligand states in crystals of haem containing cytochromes.

European Journal of Medicinal Chemistry Reports

Chemical Science

Observation of side-on copper-nitrosyl intermediate and its confirmation by DFT during catalysis ... more Observation of side-on copper-nitrosyl intermediate and its confirmation by DFT during catalysis of copper nitrite reductases.

FEMS Microbiology Letters

This study investigated the genetic basis of multidrug resistance in two strains of Achromobacter... more This study investigated the genetic basis of multidrug resistance in two strains of Achromobacter xylosoxidans isolated from patients attending a hospital in Thailand in 2012. These isolates were highly resistant to cephalosporins, aminoglycosides, fluoroquinolones, co-trimoxazole and carbapenems. Whole genome sequencing revealed that the two isolates were not clonally related and identified a carbapenem resistance gene-habouring integron (In687), residing in a novel genomic island, AcGI1. This In687 shares 100% identical nucleotide sequence with ones found in Acinetobacter baumannii Aci 16, isolated from the same hospital in 2007. We report the first analysis of multidrug-resistant A. xylosoxidans isolated in Thailand, and the first example of this island in A. xylosoxidans. Our data support the idea that resistance has spread in Thailand via horizontal gene transfer between species and suggest the possibility of A. xylosoxidans may serve as a reservoir of antibiotic resistance, es...

Acta Crystallographica Section A Foundations and Advances

Frontiers in Cellular and Infection Microbiology

Apicomplexan infections cause substantial morbidity and mortality, worldwide. New, improved thera... more Apicomplexan infections cause substantial morbidity and mortality, worldwide. New, improved therapies are needed. Herein, we create a next generation anti-apicomplexan lead compound, JAG21, a tetrahydroquinolone, with increased sp3-character to improve parasite selectivity. Relative to other cytochrome b inhibitors, JAG21 has improved solubility and ADMET properties, without need for pro-drug. JAG21 significantly reduces Toxoplasma gondii tachyzoites and encysted bradyzoites in vitro, and in primary and established chronic murine infections. Moreover, JAG21 treatment leads to 100% survival. Further, JAG21 is efficacious against drug-resistant Plasmodium falciparum in vitro. Causal prophylaxis and radical cure are achieved after P. berghei sporozoite infection with oral administration of a single dose (2.5 mg/kg) or 3 days treatment at reduced dose (0.625 mg/kg/day), eliminating parasitemia, and leading to 100% survival. Enzymatic, binding, and co-crystallography/pharmacophore studies demonstrate selectivity for apicomplexan relative to mammalian enzymes. JAG21 has significant promise as a pre-clinical candidate for prevention, treatment, and cure of toxoplasmosis and malaria.

Neisseria meningitidis is carried by nearly a billion humans, causing developmental impairment an... more Neisseria meningitidis is carried by nearly a billion humans, causing developmental impairment and over 100 000 deaths a year. A quinol-dependent nitric oxide reductase (qNOR) plays a critical role in the survival of the bacterium in the human host. X-ray crystallographic analyses of qNOR, including that from N. meningitidis (NmqNOR) reported here at 3.15 Å resolution, show monomeric assemblies, despite the more active dimeric sample being used for crystallization. Cryo-electron microscopic analysis of the same chromatographic fraction of NmqNOR, however, revealed a dimeric assembly at 3.06 Å resolution. It is shown that zinc (which is used in crystallization) binding near the dimer-stabilizing TMII region contributes to the disruption of the dimer. A similar destabilization is observed in the monomeric ($85 kDa) cryo-EM structure of a mutant (Glu494Ala) qNOR from the opportunistic pathogen Alcaligenes (Achromobacter) xylosoxidans, which primarily migrates as a monomer. The monomer-dimer transition of qNORs seen in the cryo-EM and crystallographic structures has wider implications for structural studies of multimeric membrane proteins. X-ray crystallographic and cryo-EM structural analyses have been performed on the same chromatographic fraction of NmqNOR to high resolution. This represents one of the first examples in which the two approaches have been used to reveal a monomeric assembly in crystallo and a dimeric assembly in vitrified cryo-EM grids. A number of factors have been identified that may trigger the destabilization of helices that are necessary to preserve the integrity of the dimer. These include zinc binding near the entry of the putative proton-transfer channel and the preservation of the conformational integrity of the active site. The mutation near the active site results in disruption of the active site, causing an additional destabilization of helices (TMIX and TMX) that flank the proton-transfer channel helices, creating an inert monomeric enzyme. research papers IUCrJ (2020). 7, 404-415 M. Arif M. Jamali et al. Quinol-dependent nitric oxide reductase 405

Biochemical Journal

As part of the infective process, Porphyromonas gingivalis must acquire heme which is indispensab... more As part of the infective process, Porphyromonas gingivalis must acquire heme which is indispensable for life and enables the microorganism to survive and multiply at the infection site. This oral pathogenic bacterium uses a newly discovered novel hmu heme uptake system with a leading role played by the HmuY hemophore-like protein, responsible for acquiring heme and increasing virulence of this periodontopathogen. We demonstrated that Prevotella intermedia produces two HmuY homologs, termed PinO and PinA. Both proteins were produced at higher mRNA and protein levels when the bacterium grew under low-iron/heme conditions. PinO and PinA bound heme, but preferentially under reducing conditions, and in a manner different from that of the P. gingivalis HmuY. The analysis of the three-dimensional structures confirmed differences between apo-PinO and apo-HmuY, mainly in the fold forming the heme-binding pocket. Instead of two histidine residues coordinating heme iron in P. gingivalis HmuY, ...

Bioscience Reports

Porphyromonas gingivalis is considered the principal etiologic agent and keystone pathogen of chr... more Porphyromonas gingivalis is considered the principal etiologic agent and keystone pathogen of chronic periodontitis. As an auxotrophic bacterium, it must acquire heme to survive and multiply at the infection site. P. gingivalis HmuY is the first member of a novel family of hemophore-like proteins. Bacterial heme-binding proteins usually use histidine-methionine or histidine-tyrosine residues to ligate heme-iron, whereas P. gingivalis HmuY uses two histidine residues. We hypothesized that other ‘red complex’ members, i.e. Tannerella forsythia and Treponema denticola might utilize similar heme uptake mechanisms to the P. gingivalis HmuY. Comparative and phylogenetic analyses suggested differentiation of HmuY homologs and low conservation of heme-coordinating histidine residues present in HmuY. The homologs were subjected to duplication before divergence of Bacteroidetes lineages, which could facilitate evolution of functional diversification. We found that T. denticola does not code a...

Acta Crystallographica Section A Foundations and Advances

ACS Medicinal Chemistry Letters

A series of 2-pyrazolyl quinolones has been designed and synthesized in 5−7 steps to optimize for... more A series of 2-pyrazolyl quinolones has been designed and synthesized in 5−7 steps to optimize for both in vitro antimalarial potency and various in vitro drug metabolism and pharmacokinetics (DMPK) features. The most potent compounds display no cross-resistance with multidrug resistant parasite strains (W2) compared to drug sensitive strains (3D7), with IC 50 (concentration of drug required to achieve half maximal growth suppression) values in the range of 15−33 nM. Furthermore, members of the series retain moderate activity against the atovaquone-resistant parasite isolate (TM90C2B). The described 2-pyrazoyl series displays improved DMPK properties, including improved aqueous solubility compared to previously reported quinolone series and acceptable safety margin through in vitro cytotoxicity assessment. The 2-pyrazolyl quinolones are believed to bind to the ubiquinone-reducing Q i site of the parasite bc 1 complex, which is supported by crystallographic studies of bovine cytochrome bc 1 complex.

IUCrJ, 2018

High-resolution crystal structures of enzymes in relevant redox states have transformed our under... more High-resolution crystal structures of enzymes in relevant redox states have transformed our understanding of enzyme catalysis. Recent developments have demonstrated that X-rays can be used, the generation of solvated electrons, to drive reactions in crystals at cryogenic temperatures (100 K) to generate 'structural movies' of enzyme reactions. However, a serious limitation at these temperatures is that protein conformational motion can be significantly supressed. Here, the recently developed MSOX (multiple serial structures from one crystal) approach has been applied to nitrite-bound copper nitrite reductase at room temperature and at 190 K, close to the glass transition. During both series of multiple structures, nitrite was initially observed in a 'top-hat' geometry, which was rapidly transformed to a 'side-on' configuration before conversion to side-on NO, followed by dissociation of NO and substitution by water to reform the resting state. Density functio...

Journal of virology, Apr 1, 2018

Japanese encephalitis virus (JEV) is a mosquito-transmitted flavivirus that is closely related to... more Japanese encephalitis virus (JEV) is a mosquito-transmitted flavivirus that is closely related to other emerging viral pathogens, including dengue virus (DENV), West Nile virus (WNV), and Zika virus (ZIKV). JEV infection can result in meningitis and encephalitis, which in severe cases cause permanent brain damage and death. JEV occurs predominantly in rural areas throughout Southeast Asia, the Pacific Islands, and the Far East, causing around 68,000 cases of infection worldwide each year. In this report, we present a 2.1-Å-resolution crystal structure of the C-terminal β-ladder domain of JEV nonstructural protein 1 (NS1-C). The surface charge distribution of JEV NS1-C is similar to those of WNV and ZIKV but differs from that of DENV. Analysis of the JEV NS1-C structure, with molecular dynamics simulation and experimental solution small-angle X-ray scattering, indicates extensive loop flexibility on the exterior of the protein. This, together with the surface charge distribution, ind...

Proceedings of the National Academy of Sciences of the United States of America, Jan 18, 2018

Cyanobacteria are important photosynthetic organisms inhabiting a range of dynamic environments. ... more Cyanobacteria are important photosynthetic organisms inhabiting a range of dynamic environments. This phylum is distinctive among photosynthetic organisms in containing genes encoding uncharacterized cystathionine β-synthase (CBS)-chloroplast protein (CP12) fusion proteins. These consist of two domains, each recognized as stand-alone photosynthetic regulators with different functions described in cyanobacteria (CP12) and plants (CP12 and CBSX). Here we show that CBS-CP12 fusion proteins are encoded in distinct gene neighborhoods, several unrelated to photosynthesis. Most frequently, CBS-CP12 genes are in a gene cluster with thioredoxin A (TrxA), which is prevalent in bloom-forming, marine symbiotic, and benthic mat cyanobacteria. Focusing on a CBS-CP12 from PCC 7806 encoded in a gene cluster with TrxA, we reveal that the domain fusion led to the formation of a hexameric protein. We show that the CP12 domain is essential for hexamerization and contains an ordered, previously structur...

Scientific reports, Jan 26, 2018

Bacterial nitric oxide reductases (NORs) catalyse the reduction of NO to NO and HO. NORs are foun... more Bacterial nitric oxide reductases (NORs) catalyse the reduction of NO to NO and HO. NORs are found either in denitrification chains, or in pathogens where their primary role is detoxification of NO produced by the immune defense of the host. Although NORs belong to the heme-copper oxidase superfamily, comprising proton-pumping O-reducing enzymes, the best studied NORs, cNORs (cytochrome c-dependent), are non-electrogenic. Here, we focus on another type of NOR, qNOR (quinol-dependent). Recombinant qNOR from Neisseria meningitidis, a human pathogen, purified from Escherichia coli, showed high catalytic activity and spectroscopic properties largely similar to cNORs. However, in contrast to cNOR, liposome-reconstituted qNOR showed respiratory control ratios above two, indicating that NO reduction by qNOR was electrogenic. Further, we determined a 4.5 Å crystal structure of the N. meningitidis qNOR, allowing exploration of a potential proton transfer pathway from the cytoplasm by mutagen...

Acta Crystallographica Section A Foundations of Crystallography

Microsymposia fold increase in dose. Comparing structural results from EXAFS to those from crysta... more Microsymposia fold increase in dose. Comparing structural results from EXAFS to those from crystallography on this and similar proteins, show that x-ray induced photoreduction has impacted the crystallographic data and subsequent structure solutions. These results indicate the importance of using LHe-based cooling for metalloprotein crystallography in order to limit changes at the metalloprotein active sites. The study also illustrates the need for direct measurement of redox states of the metals, through XAS, simultaneously with the crystallographic measurements. The work was performed at SSRL with support from the NIH NCRR BTP program and the US DOE BER. SSRL operations are funded by the US DOE BES.

Acta Crystallographica Section D Structural Biology

Methionine adenosyltransferase (MAT) deficiency, characterized by isolated persistent hypermethio... more Methionine adenosyltransferase (MAT) deficiency, characterized by isolated persistent hypermethioninemia (IPH), is caused by mutations in the MAT1A gene encoding MATαl, one of the major hepatic enzymes. Most of the associated hypermethioninemic conditions are inherited as autosomal recessive traits; however, dominant inheritance of hypermethioninemia is caused by an Arg264His (R264H) mutation. This mutation has been confirmed in a screening programme of newborns as the most common mutation in babies with IPH. Arg264 makes an inter-subunit salt bridge located at the dimer interface where the active site assembles. Here, it is demonstrated that the R264H mutation results in greatly reduced MAT activity, while retaining its ability to dimerize, indicating that the lower activity arises from alteration at the active site. The first crystallographic structure of the apo form of the wild-type MATαl enzyme is provided, which shows a tetrameric assembly in which two compact dimers combine t...

Journal of Cell Science

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited condition that can c... more Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited condition that can cause fatal cardiac arrhythmia. Human mutations in the Ca2+ sensor calmodulin (CaM) have been associated with CPVT susceptibility, suggesting that CaM dysfunction is a key driver of the disease. However, the detailed molecular mechanism remains unclear. Focusing on the interaction with the cardiac ryanodine receptor (RyR2), we determined the effect of CPVT-associated variants N53I and A102V on the structural characteristics of CaM and on Ca2+ fluxes in live cells. We provide novel data showing that binding of both Ca2+/CaM-N53I and Ca2+/CaM-A102V to RyR23583-3603 is decreased. Ca2+/CaM:RyR23583-3603 high-resolution crystal structures highlight subtle conformational changes for the N53I variant, with A102V being similar to wild-type. We show that co-expression of CaM-N53I or CaM-A102V with RyR2 in HEK293 cells significantly increased the duration of Ca2+ events, CaM-A102V exhibited a lower...

Acta Crystallographica. Section D, Structural Biology, 2020

Dominant inheritance of hypermethioninemia is caused by an Arg264His mutation in methionine adeno... more Dominant inheritance of hypermethioninemia is caused by an Arg264His mutation in methionine adenosyltransferase. The mutation lowers the affinity for dimer–dimer interaction and enzymatic activity, which can be restored by chemical intervention.

F1000Research, 2011

ABSTRACT Background / Purpose: The combination of single crystal spectroscopies (e.g. optical, Ra... more ABSTRACT Background / Purpose: The combination of single crystal spectroscopies (e.g. optical, Raman and X-ray absorption) with high-resolution protein crystallography, working in tandem to probe the same protein crystal in situ at the X-ray beamline, can yield a complete and accurate identification of the oxidation and ligation states of redox centres and/or active sites (Hough et al 2008 (1); Antonyuk and Hough 2011 (2)). With this approach, the functional properties of the active site in the protein are directly related to its contemporaneously measured crystal structure(s). Importantly, controlled X-ray radiolysis of protein crystals provides a powerful means to access transient states such as catalytic intermediates (Hough et al 2008 (1)). Main conclusion: Here, we describe (i) a systematic application of these combined tools in order to gain access to functionally relevant transient catalytic states and to generate a high-resolution ‘structural movie’; through the catalytic cycle of a Cu-containing nitrite reductase; (ii) the fingerprinting of redox-ligand states in crystals of haem containing cytochromes.

European Journal of Medicinal Chemistry Reports

Chemical Science

Observation of side-on copper-nitrosyl intermediate and its confirmation by DFT during catalysis ... more Observation of side-on copper-nitrosyl intermediate and its confirmation by DFT during catalysis of copper nitrite reductases.

FEMS Microbiology Letters

This study investigated the genetic basis of multidrug resistance in two strains of Achromobacter... more This study investigated the genetic basis of multidrug resistance in two strains of Achromobacter xylosoxidans isolated from patients attending a hospital in Thailand in 2012. These isolates were highly resistant to cephalosporins, aminoglycosides, fluoroquinolones, co-trimoxazole and carbapenems. Whole genome sequencing revealed that the two isolates were not clonally related and identified a carbapenem resistance gene-habouring integron (In687), residing in a novel genomic island, AcGI1. This In687 shares 100% identical nucleotide sequence with ones found in Acinetobacter baumannii Aci 16, isolated from the same hospital in 2007. We report the first analysis of multidrug-resistant A. xylosoxidans isolated in Thailand, and the first example of this island in A. xylosoxidans. Our data support the idea that resistance has spread in Thailand via horizontal gene transfer between species and suggest the possibility of A. xylosoxidans may serve as a reservoir of antibiotic resistance, es...

Acta Crystallographica Section A Foundations and Advances

Frontiers in Cellular and Infection Microbiology

Apicomplexan infections cause substantial morbidity and mortality, worldwide. New, improved thera... more Apicomplexan infections cause substantial morbidity and mortality, worldwide. New, improved therapies are needed. Herein, we create a next generation anti-apicomplexan lead compound, JAG21, a tetrahydroquinolone, with increased sp3-character to improve parasite selectivity. Relative to other cytochrome b inhibitors, JAG21 has improved solubility and ADMET properties, without need for pro-drug. JAG21 significantly reduces Toxoplasma gondii tachyzoites and encysted bradyzoites in vitro, and in primary and established chronic murine infections. Moreover, JAG21 treatment leads to 100% survival. Further, JAG21 is efficacious against drug-resistant Plasmodium falciparum in vitro. Causal prophylaxis and radical cure are achieved after P. berghei sporozoite infection with oral administration of a single dose (2.5 mg/kg) or 3 days treatment at reduced dose (0.625 mg/kg/day), eliminating parasitemia, and leading to 100% survival. Enzymatic, binding, and co-crystallography/pharmacophore studies demonstrate selectivity for apicomplexan relative to mammalian enzymes. JAG21 has significant promise as a pre-clinical candidate for prevention, treatment, and cure of toxoplasmosis and malaria.

Neisseria meningitidis is carried by nearly a billion humans, causing developmental impairment an... more Neisseria meningitidis is carried by nearly a billion humans, causing developmental impairment and over 100 000 deaths a year. A quinol-dependent nitric oxide reductase (qNOR) plays a critical role in the survival of the bacterium in the human host. X-ray crystallographic analyses of qNOR, including that from N. meningitidis (NmqNOR) reported here at 3.15 Å resolution, show monomeric assemblies, despite the more active dimeric sample being used for crystallization. Cryo-electron microscopic analysis of the same chromatographic fraction of NmqNOR, however, revealed a dimeric assembly at 3.06 Å resolution. It is shown that zinc (which is used in crystallization) binding near the dimer-stabilizing TMII region contributes to the disruption of the dimer. A similar destabilization is observed in the monomeric ($85 kDa) cryo-EM structure of a mutant (Glu494Ala) qNOR from the opportunistic pathogen Alcaligenes (Achromobacter) xylosoxidans, which primarily migrates as a monomer. The monomer-dimer transition of qNORs seen in the cryo-EM and crystallographic structures has wider implications for structural studies of multimeric membrane proteins. X-ray crystallographic and cryo-EM structural analyses have been performed on the same chromatographic fraction of NmqNOR to high resolution. This represents one of the first examples in which the two approaches have been used to reveal a monomeric assembly in crystallo and a dimeric assembly in vitrified cryo-EM grids. A number of factors have been identified that may trigger the destabilization of helices that are necessary to preserve the integrity of the dimer. These include zinc binding near the entry of the putative proton-transfer channel and the preservation of the conformational integrity of the active site. The mutation near the active site results in disruption of the active site, causing an additional destabilization of helices (TMIX and TMX) that flank the proton-transfer channel helices, creating an inert monomeric enzyme. research papers IUCrJ (2020). 7, 404-415 M. Arif M. Jamali et al. Quinol-dependent nitric oxide reductase 405

Biochemical Journal

As part of the infective process, Porphyromonas gingivalis must acquire heme which is indispensab... more As part of the infective process, Porphyromonas gingivalis must acquire heme which is indispensable for life and enables the microorganism to survive and multiply at the infection site. This oral pathogenic bacterium uses a newly discovered novel hmu heme uptake system with a leading role played by the HmuY hemophore-like protein, responsible for acquiring heme and increasing virulence of this periodontopathogen. We demonstrated that Prevotella intermedia produces two HmuY homologs, termed PinO and PinA. Both proteins were produced at higher mRNA and protein levels when the bacterium grew under low-iron/heme conditions. PinO and PinA bound heme, but preferentially under reducing conditions, and in a manner different from that of the P. gingivalis HmuY. The analysis of the three-dimensional structures confirmed differences between apo-PinO and apo-HmuY, mainly in the fold forming the heme-binding pocket. Instead of two histidine residues coordinating heme iron in P. gingivalis HmuY, ...

Bioscience Reports

Porphyromonas gingivalis is considered the principal etiologic agent and keystone pathogen of chr... more Porphyromonas gingivalis is considered the principal etiologic agent and keystone pathogen of chronic periodontitis. As an auxotrophic bacterium, it must acquire heme to survive and multiply at the infection site. P. gingivalis HmuY is the first member of a novel family of hemophore-like proteins. Bacterial heme-binding proteins usually use histidine-methionine or histidine-tyrosine residues to ligate heme-iron, whereas P. gingivalis HmuY uses two histidine residues. We hypothesized that other ‘red complex’ members, i.e. Tannerella forsythia and Treponema denticola might utilize similar heme uptake mechanisms to the P. gingivalis HmuY. Comparative and phylogenetic analyses suggested differentiation of HmuY homologs and low conservation of heme-coordinating histidine residues present in HmuY. The homologs were subjected to duplication before divergence of Bacteroidetes lineages, which could facilitate evolution of functional diversification. We found that T. denticola does not code a...

Acta Crystallographica Section A Foundations and Advances

ACS Medicinal Chemistry Letters

A series of 2-pyrazolyl quinolones has been designed and synthesized in 5−7 steps to optimize for... more A series of 2-pyrazolyl quinolones has been designed and synthesized in 5−7 steps to optimize for both in vitro antimalarial potency and various in vitro drug metabolism and pharmacokinetics (DMPK) features. The most potent compounds display no cross-resistance with multidrug resistant parasite strains (W2) compared to drug sensitive strains (3D7), with IC 50 (concentration of drug required to achieve half maximal growth suppression) values in the range of 15−33 nM. Furthermore, members of the series retain moderate activity against the atovaquone-resistant parasite isolate (TM90C2B). The described 2-pyrazoyl series displays improved DMPK properties, including improved aqueous solubility compared to previously reported quinolone series and acceptable safety margin through in vitro cytotoxicity assessment. The 2-pyrazolyl quinolones are believed to bind to the ubiquinone-reducing Q i site of the parasite bc 1 complex, which is supported by crystallographic studies of bovine cytochrome bc 1 complex.

IUCrJ, 2018

High-resolution crystal structures of enzymes in relevant redox states have transformed our under... more High-resolution crystal structures of enzymes in relevant redox states have transformed our understanding of enzyme catalysis. Recent developments have demonstrated that X-rays can be used, the generation of solvated electrons, to drive reactions in crystals at cryogenic temperatures (100 K) to generate 'structural movies' of enzyme reactions. However, a serious limitation at these temperatures is that protein conformational motion can be significantly supressed. Here, the recently developed MSOX (multiple serial structures from one crystal) approach has been applied to nitrite-bound copper nitrite reductase at room temperature and at 190 K, close to the glass transition. During both series of multiple structures, nitrite was initially observed in a 'top-hat' geometry, which was rapidly transformed to a 'side-on' configuration before conversion to side-on NO, followed by dissociation of NO and substitution by water to reform the resting state. Density functio...

Journal of virology, Apr 1, 2018

Japanese encephalitis virus (JEV) is a mosquito-transmitted flavivirus that is closely related to... more Japanese encephalitis virus (JEV) is a mosquito-transmitted flavivirus that is closely related to other emerging viral pathogens, including dengue virus (DENV), West Nile virus (WNV), and Zika virus (ZIKV). JEV infection can result in meningitis and encephalitis, which in severe cases cause permanent brain damage and death. JEV occurs predominantly in rural areas throughout Southeast Asia, the Pacific Islands, and the Far East, causing around 68,000 cases of infection worldwide each year. In this report, we present a 2.1-Å-resolution crystal structure of the C-terminal β-ladder domain of JEV nonstructural protein 1 (NS1-C). The surface charge distribution of JEV NS1-C is similar to those of WNV and ZIKV but differs from that of DENV. Analysis of the JEV NS1-C structure, with molecular dynamics simulation and experimental solution small-angle X-ray scattering, indicates extensive loop flexibility on the exterior of the protein. This, together with the surface charge distribution, ind...

Proceedings of the National Academy of Sciences of the United States of America, Jan 18, 2018

Cyanobacteria are important photosynthetic organisms inhabiting a range of dynamic environments. ... more Cyanobacteria are important photosynthetic organisms inhabiting a range of dynamic environments. This phylum is distinctive among photosynthetic organisms in containing genes encoding uncharacterized cystathionine β-synthase (CBS)-chloroplast protein (CP12) fusion proteins. These consist of two domains, each recognized as stand-alone photosynthetic regulators with different functions described in cyanobacteria (CP12) and plants (CP12 and CBSX). Here we show that CBS-CP12 fusion proteins are encoded in distinct gene neighborhoods, several unrelated to photosynthesis. Most frequently, CBS-CP12 genes are in a gene cluster with thioredoxin A (TrxA), which is prevalent in bloom-forming, marine symbiotic, and benthic mat cyanobacteria. Focusing on a CBS-CP12 from PCC 7806 encoded in a gene cluster with TrxA, we reveal that the domain fusion led to the formation of a hexameric protein. We show that the CP12 domain is essential for hexamerization and contains an ordered, previously structur...

Scientific reports, Jan 26, 2018

Bacterial nitric oxide reductases (NORs) catalyse the reduction of NO to NO and HO. NORs are foun... more Bacterial nitric oxide reductases (NORs) catalyse the reduction of NO to NO and HO. NORs are found either in denitrification chains, or in pathogens where their primary role is detoxification of NO produced by the immune defense of the host. Although NORs belong to the heme-copper oxidase superfamily, comprising proton-pumping O-reducing enzymes, the best studied NORs, cNORs (cytochrome c-dependent), are non-electrogenic. Here, we focus on another type of NOR, qNOR (quinol-dependent). Recombinant qNOR from Neisseria meningitidis, a human pathogen, purified from Escherichia coli, showed high catalytic activity and spectroscopic properties largely similar to cNORs. However, in contrast to cNOR, liposome-reconstituted qNOR showed respiratory control ratios above two, indicating that NO reduction by qNOR was electrogenic. Further, we determined a 4.5 Å crystal structure of the N. meningitidis qNOR, allowing exploration of a potential proton transfer pathway from the cytoplasm by mutagen...

Acta Crystallographica Section A Foundations of Crystallography

Microsymposia fold increase in dose. Comparing structural results from EXAFS to those from crysta... more Microsymposia fold increase in dose. Comparing structural results from EXAFS to those from crystallography on this and similar proteins, show that x-ray induced photoreduction has impacted the crystallographic data and subsequent structure solutions. These results indicate the importance of using LHe-based cooling for metalloprotein crystallography in order to limit changes at the metalloprotein active sites. The study also illustrates the need for direct measurement of redox states of the metals, through XAS, simultaneously with the crystallographic measurements. The work was performed at SSRL with support from the NIH NCRR BTP program and the US DOE BER. SSRL operations are funded by the US DOE BES.