Bacterial heterodimeric tryptophan-containing diketopiperazines (HTDKPs) are a growing family of ... more Bacterial heterodimeric tryptophan-containing diketopiperazines (HTDKPs) are a growing family of bioactive natural products. They are challenging to prepare by chemical routes due to the polycyclic and densely functionalized backbone. Through functional characterization and investigation, we herein identify a family of three related HTDKP-forming cytochrome P450s (NasbB, NasS1868 and NasF5053) and reveal four critical residues (Qln65, Ala86, Ser284 and Val288) that control their regio- and stereo-selectivity to generate diverse dimeric DKP frameworks. Engineering these residues can alter the specificities of the enzymes to produce diverse frameworks. Determining the crystal structures (1.70–1.47 Å) of NasF5053 (ligand-free and substrate-bound NasF5053 and its Q65I-A86G and S284A-V288A mutants) and molecular dynamics simulation finally elucidate the specificity-conferring mechanism of these residues. Our results provide a clear molecular and mechanistic basis into this family of HTDK...
During infection, plant pathogens secrete effector proteins to facilitate colonization. Compared ... more During infection, plant pathogens secrete effector proteins to facilitate colonization. Compared to our knowledge of bacterial effectors, the current understanding of how fungal effectors function is limited. In this study we show that the effector AvrL567-A from the flax rust fungus Melampsora lini interacts with a flax cytosolic cytokinin oxidase, LuCKX1.1 using both yeast-two-hybrid and in planta bimolecular fluorescence assays. Purified LuCKX1.1 protein shows catalytic activity against both N6-(Δ2-isopentenyl)-adenine (iP) and trans-zeatin (tZ) substrates. Incubation of LuCKX1.1 with AvrL567-A results in increased catalytic activity against both substrates. The crystal structure of LuCKX1.1 and docking studies with AvrL567-A indicate that the AvrL567 binding site involves a flexible surface exposed region that surrounds the cytokinin substrate access site, which may explain its effect in modulating LuCKX1.1 activity. Expression of AvrL567-A in transgenic flax plants gave rise to...
UDP-glucose pyrophosphorylase (UGPase) is found in all organisms and catalyses the formation of U... more UDP-glucose pyrophosphorylase (UGPase) is found in all organisms and catalyses the formation of UDP-glucose. In sugarcane, UDP-glucose is a branch-point in the carbon channelling into other carbohydrates, such as sucrose and cellulose, which are the major factors for sugarcane productivity. In most plants, UGPase has been described to be enzymatically active in the monomeric form, while in human and yeast, homo-octamers represent the active form of the protein. Here, we present the crystal structure of UGPase from sugarcane (ScUGPase-1) at resolution of 2.0 Å. The crystals of ScUGPase-1 reveal the presence of two molecules in the asymmetric unit and the multi-angle light scattering analysis shows that ScUGPase-1 forms a mixture of species ranging from monomers to larger oligomers in solution, suggesting similarities with the orthologs from yeast and human.
The recent development of X-ray free electron lasers (XFELs) has spurred the development of seria... more The recent development of X-ray free electron lasers (XFELs) has spurred the development of serial femtosecond nanocrystallography (SFX) which, for the first time, is enabling structure retrieval from sub-micron protein crystals. Although there are already a growing number of structures published using SFX, the technology is still very new and presents a number of unique challenges as well as opportunities for structural biologists. One of the biggest barriers to the success of SFX experiments is the preparation and selection of suitable protein crystal samples. Here we outline a protocol for preparing and screening for suitable XFEL targets. X-ray free electron lasers (XFELs) are the latest cutting-edge tool for structural biologists. The highly intense, femtosecond pulse structure make them ideally suited to the study of nanocrystals and for investigating the dynamics of proteins. Currently, there are two hard X-ray free electron lasers in operation that can be used for serial femtosecond crystallography (SFX); these are the Linac Coherent Light Source (LCLS) in the USA and the SPring-8 Angstrom Compact Free Electron Laser (SACLA) source in Japan. The unique capability of these sources and their scarcity mean that there is a constant high-demand for access to these facilities. Recent XFEL experiments have resulted in a number of high-impact biological structures, which include but not limited to the photosystem I and the serotonin receptor 1-8. This technology, however, is still in its infancy and little has been published regarding the preparation and selection of suitable XFEL crystalline samples, which differ, in many cases significantly, from the types of crystals that are routinely measured at synchrotron sources. Significant advancements in micro-focus crystallography beamlines at third-generation synchrotrons have seen the size of crystals that can be measured at such sources become ever smaller. The minimisation of background signal and the introduction of serial synchrotron crystallography (SSX) has extended the limit from which single crystal data can be collected at the synchrotron to the micron scale 9,10. For protein crystals well-below the 1 micron scale, (i.e. those where growth is difficult to optimise beyond the nanometre scale), SFX is the only viable option for obtaining high-resolution single crystal diffraction data due to the rapid degradation of the measured diffracted intensities by onset radiation damage 11. Even with the extremely intense beams available at XFELs, measuring samples with the correct characteristics in terms of size, quality and crystalline order is essential for a successful experiment. Nanocrystals that are poorly ordered will likely not yield high-resolution at the XFEL, whereas those that are highly ordered will still be too small for single crystal synchrotron measurement but may be perfect candidates for XFEL analysis. The key questions that need to be answered in screening for suitable XFEL targets therefore are: (1) Is the crystal too small for single crystal measurements at the synchrotron? (2) Is the crystal protein or salt? (3) Is the crystal quality sufficient for high-resolution structural information to be collected at the XFEL?
NMR spectroscopy is a powerful method in structural and functional analysis of macromolecules and... more NMR spectroscopy is a powerful method in structural and functional analysis of macromolecules and has become particularly prevalent in studies of protein structure, function and dynamics. Unique to NMR spectroscopy is the relatively low constraints on sample preparation and the high level of control of sample conditions. Proteins can be studied in a wide range of buffer conditions, e.g. different pHs and variable temperatures, allowing studies of proteins under conditions that are closer to their native environment compared to other structural methods such as X-ray crystallography and electron microscopy. The key disadvantage of NMR is the relatively low sensitivity of the method, requiring either concentrated samples or very lengthy data-acquisition times. Thus, proteins that are unstable or can only be studied in dilute solutions are often considered practically unfeasible for NMR studies. Here, we describe a general method, where non-uniform sampling (NUS) allows for signal avera...
Motivation: Genome-wide association studies are identifying single nucleotide variants (SNVs) lin... more Motivation: Genome-wide association studies are identifying single nucleotide variants (SNVs) linked to various diseases, however the functional effect caused by these variants is often unknown. One potential functional effect, the loss or gain of protein phosphorylation sites, can be induced through variations in key amino acids that disrupt or introduce valid kinase binding patterns. Current methods for predicting the effect of SNVs on phosphorylation operate on the sequence content of reference and variant proteins. However, consideration of the amino acid sequence alone is insufficient for predicting phosphorylation change, as context factors determine kinase-substrate selection. Results: We present here a method for quantifying the effect of SNVs on protein phosphorylation through an integrated system of motif analysis and context-based assessment of kinase targets. By predicting the effect that known variants across the proteome have on phosphorylation, we are able to use this background of proteome-wide variant effects to quantify the significance of novel variants for modifying phosphorylation. We validate our method on a manually curated set of phosphorylation change-causing variants from the primary literature, showing that the method predicts known examples of phosphorylation change at high levels of specificity. We apply our approach to data-sets of variants in phosphorylation site regions, showing that variants causing predicted phosphorylation loss are over-represented among disease-associated variants.
The plant immune system employs intracellular NLRs (nucleotide binding [NB], leucine-rich repeat ... more The plant immune system employs intracellular NLRs (nucleotide binding [NB], leucine-rich repeat [LRR]/nucleotide-binding oligomerization domain [NOD]-like receptors) to detect effector proteins secreted into the plant cell by potential pathogens. Activated plant NLRs trigger a range of immune responses, collectively known as the hypersensitive response (HR), which culminates in death of the infected cell. Plant NLRs show structural and functional resemblance to animal NLRs involved in inflammatory and innate immune responses. Therefore, knowledge of the activation and regulation of animal NLRs can help us understand the mechanism of action of plant NLRs, and vice versa. This review provides an overview of the innate immune pathways in plants and animals, focusing on the available structural and biochemical information available for both plant and animal NLRs. We highlight the gap in knowledge between the animal and plant systems, in particular the lack of structural information for...
The Plant journal : for cell and molecular biology, Oct 3, 2016
Recent studies have identified that proteinaceous effectors secreted by Parastagonospora nodorum ... more Recent studies have identified that proteinaceous effectors secreted by Parastagonospora nodorum are required to cause disease on wheat. These effectors interact in a gene-for-gene manner with host dominant susceptibilty loci, resulting in disease. However, whilst the requirement of these effectors for infection is clear, their mechanisms of action remain poorly understood. A yeast-two-hybrid library approach was used to search for wheat proteins that interacted with the necrotrophic effector SnTox3. Using this strategy we indentified an interaction between SnTox3 and the wheat pathogenicity-related protein TaPR-1-1, and confirmed it by in-planta co-immunprecipitation. PR-1 proteins represent a large family (23 in wheat) of proteins that are up-regulated early in the defence response, however their function remains ellusive. Interestingly, the P. nodorum effector SnToxA has recently been shown to interact specifically with TaPR-1-5. Our analysis of the SnTox3-TaPR-1 interaction demo...
Principles of Kinase Regulation Bostjan Kobe1 and Bruce E. Kemp2 1 School of Chemistry Molecular ... more Principles of Kinase Regulation Bostjan Kobe1 and Bruce E. Kemp2 1 School of Chemistry Molecular Biosciences, University of Queensland, Brisbane ... Sessa F, Mapelli M, Ciferri C, Tarricone C, Areces LB, Schneider TR, Stukenberg PT, Musacchio A. Mechanism of Aurora B ...
The biosynthetic pathway for the branched-chain amino acids is present in plants, fungi and bacte... more The biosynthetic pathway for the branched-chain amino acids is present in plants, fungi and bacteria, but not in animals, making it an attractive target for herbicidal and antimicrobial drug discovery. Ketol-acid reductoisomerase (KARI; EC 1.1.1.86) is the second enzyme in this pathway, converting in a Mg(2+) - and NADPH-dependent reaction either 2-acetolactate or 2-aceto-2-hydroxybutyrate to their corresponding 2,3-dihydroxy-3-alkylbutyrate products. Here, we have determined the crystal structure of Mycobacterium tuberculosis KARI (Mt KARI), a class I KARI, with two magnesium ions bound in the active site. X-ray data were obtained to 1.0 Å resolution and the final model has an Rfree of 0.174. The structure shows that the active site is solvent-accessible with the two metal ions separated by 4.7 Å. A comparison of this structure with that of Mg(2+) -free Pseudomonas aeruginosa KARI suggests that upon magnesium binding no movement of the N-domain relative to the C-domain occurs. Howe...
Bacterial heterodimeric tryptophan-containing diketopiperazines (HTDKPs) are a growing family of ... more Bacterial heterodimeric tryptophan-containing diketopiperazines (HTDKPs) are a growing family of bioactive natural products. They are challenging to prepare by chemical routes due to the polycyclic and densely functionalized backbone. Through functional characterization and investigation, we herein identify a family of three related HTDKP-forming cytochrome P450s (NasbB, NasS1868 and NasF5053) and reveal four critical residues (Qln65, Ala86, Ser284 and Val288) that control their regio- and stereo-selectivity to generate diverse dimeric DKP frameworks. Engineering these residues can alter the specificities of the enzymes to produce diverse frameworks. Determining the crystal structures (1.70–1.47 Å) of NasF5053 (ligand-free and substrate-bound NasF5053 and its Q65I-A86G and S284A-V288A mutants) and molecular dynamics simulation finally elucidate the specificity-conferring mechanism of these residues. Our results provide a clear molecular and mechanistic basis into this family of HTDK...
During infection, plant pathogens secrete effector proteins to facilitate colonization. Compared ... more During infection, plant pathogens secrete effector proteins to facilitate colonization. Compared to our knowledge of bacterial effectors, the current understanding of how fungal effectors function is limited. In this study we show that the effector AvrL567-A from the flax rust fungus Melampsora lini interacts with a flax cytosolic cytokinin oxidase, LuCKX1.1 using both yeast-two-hybrid and in planta bimolecular fluorescence assays. Purified LuCKX1.1 protein shows catalytic activity against both N6-(Δ2-isopentenyl)-adenine (iP) and trans-zeatin (tZ) substrates. Incubation of LuCKX1.1 with AvrL567-A results in increased catalytic activity against both substrates. The crystal structure of LuCKX1.1 and docking studies with AvrL567-A indicate that the AvrL567 binding site involves a flexible surface exposed region that surrounds the cytokinin substrate access site, which may explain its effect in modulating LuCKX1.1 activity. Expression of AvrL567-A in transgenic flax plants gave rise to...
UDP-glucose pyrophosphorylase (UGPase) is found in all organisms and catalyses the formation of U... more UDP-glucose pyrophosphorylase (UGPase) is found in all organisms and catalyses the formation of UDP-glucose. In sugarcane, UDP-glucose is a branch-point in the carbon channelling into other carbohydrates, such as sucrose and cellulose, which are the major factors for sugarcane productivity. In most plants, UGPase has been described to be enzymatically active in the monomeric form, while in human and yeast, homo-octamers represent the active form of the protein. Here, we present the crystal structure of UGPase from sugarcane (ScUGPase-1) at resolution of 2.0 Å. The crystals of ScUGPase-1 reveal the presence of two molecules in the asymmetric unit and the multi-angle light scattering analysis shows that ScUGPase-1 forms a mixture of species ranging from monomers to larger oligomers in solution, suggesting similarities with the orthologs from yeast and human.
The recent development of X-ray free electron lasers (XFELs) has spurred the development of seria... more The recent development of X-ray free electron lasers (XFELs) has spurred the development of serial femtosecond nanocrystallography (SFX) which, for the first time, is enabling structure retrieval from sub-micron protein crystals. Although there are already a growing number of structures published using SFX, the technology is still very new and presents a number of unique challenges as well as opportunities for structural biologists. One of the biggest barriers to the success of SFX experiments is the preparation and selection of suitable protein crystal samples. Here we outline a protocol for preparing and screening for suitable XFEL targets. X-ray free electron lasers (XFELs) are the latest cutting-edge tool for structural biologists. The highly intense, femtosecond pulse structure make them ideally suited to the study of nanocrystals and for investigating the dynamics of proteins. Currently, there are two hard X-ray free electron lasers in operation that can be used for serial femtosecond crystallography (SFX); these are the Linac Coherent Light Source (LCLS) in the USA and the SPring-8 Angstrom Compact Free Electron Laser (SACLA) source in Japan. The unique capability of these sources and their scarcity mean that there is a constant high-demand for access to these facilities. Recent XFEL experiments have resulted in a number of high-impact biological structures, which include but not limited to the photosystem I and the serotonin receptor 1-8. This technology, however, is still in its infancy and little has been published regarding the preparation and selection of suitable XFEL crystalline samples, which differ, in many cases significantly, from the types of crystals that are routinely measured at synchrotron sources. Significant advancements in micro-focus crystallography beamlines at third-generation synchrotrons have seen the size of crystals that can be measured at such sources become ever smaller. The minimisation of background signal and the introduction of serial synchrotron crystallography (SSX) has extended the limit from which single crystal data can be collected at the synchrotron to the micron scale 9,10. For protein crystals well-below the 1 micron scale, (i.e. those where growth is difficult to optimise beyond the nanometre scale), SFX is the only viable option for obtaining high-resolution single crystal diffraction data due to the rapid degradation of the measured diffracted intensities by onset radiation damage 11. Even with the extremely intense beams available at XFELs, measuring samples with the correct characteristics in terms of size, quality and crystalline order is essential for a successful experiment. Nanocrystals that are poorly ordered will likely not yield high-resolution at the XFEL, whereas those that are highly ordered will still be too small for single crystal synchrotron measurement but may be perfect candidates for XFEL analysis. The key questions that need to be answered in screening for suitable XFEL targets therefore are: (1) Is the crystal too small for single crystal measurements at the synchrotron? (2) Is the crystal protein or salt? (3) Is the crystal quality sufficient for high-resolution structural information to be collected at the XFEL?
NMR spectroscopy is a powerful method in structural and functional analysis of macromolecules and... more NMR spectroscopy is a powerful method in structural and functional analysis of macromolecules and has become particularly prevalent in studies of protein structure, function and dynamics. Unique to NMR spectroscopy is the relatively low constraints on sample preparation and the high level of control of sample conditions. Proteins can be studied in a wide range of buffer conditions, e.g. different pHs and variable temperatures, allowing studies of proteins under conditions that are closer to their native environment compared to other structural methods such as X-ray crystallography and electron microscopy. The key disadvantage of NMR is the relatively low sensitivity of the method, requiring either concentrated samples or very lengthy data-acquisition times. Thus, proteins that are unstable or can only be studied in dilute solutions are often considered practically unfeasible for NMR studies. Here, we describe a general method, where non-uniform sampling (NUS) allows for signal avera...
Motivation: Genome-wide association studies are identifying single nucleotide variants (SNVs) lin... more Motivation: Genome-wide association studies are identifying single nucleotide variants (SNVs) linked to various diseases, however the functional effect caused by these variants is often unknown. One potential functional effect, the loss or gain of protein phosphorylation sites, can be induced through variations in key amino acids that disrupt or introduce valid kinase binding patterns. Current methods for predicting the effect of SNVs on phosphorylation operate on the sequence content of reference and variant proteins. However, consideration of the amino acid sequence alone is insufficient for predicting phosphorylation change, as context factors determine kinase-substrate selection. Results: We present here a method for quantifying the effect of SNVs on protein phosphorylation through an integrated system of motif analysis and context-based assessment of kinase targets. By predicting the effect that known variants across the proteome have on phosphorylation, we are able to use this background of proteome-wide variant effects to quantify the significance of novel variants for modifying phosphorylation. We validate our method on a manually curated set of phosphorylation change-causing variants from the primary literature, showing that the method predicts known examples of phosphorylation change at high levels of specificity. We apply our approach to data-sets of variants in phosphorylation site regions, showing that variants causing predicted phosphorylation loss are over-represented among disease-associated variants.
The plant immune system employs intracellular NLRs (nucleotide binding [NB], leucine-rich repeat ... more The plant immune system employs intracellular NLRs (nucleotide binding [NB], leucine-rich repeat [LRR]/nucleotide-binding oligomerization domain [NOD]-like receptors) to detect effector proteins secreted into the plant cell by potential pathogens. Activated plant NLRs trigger a range of immune responses, collectively known as the hypersensitive response (HR), which culminates in death of the infected cell. Plant NLRs show structural and functional resemblance to animal NLRs involved in inflammatory and innate immune responses. Therefore, knowledge of the activation and regulation of animal NLRs can help us understand the mechanism of action of plant NLRs, and vice versa. This review provides an overview of the innate immune pathways in plants and animals, focusing on the available structural and biochemical information available for both plant and animal NLRs. We highlight the gap in knowledge between the animal and plant systems, in particular the lack of structural information for...
The Plant journal : for cell and molecular biology, Oct 3, 2016
Recent studies have identified that proteinaceous effectors secreted by Parastagonospora nodorum ... more Recent studies have identified that proteinaceous effectors secreted by Parastagonospora nodorum are required to cause disease on wheat. These effectors interact in a gene-for-gene manner with host dominant susceptibilty loci, resulting in disease. However, whilst the requirement of these effectors for infection is clear, their mechanisms of action remain poorly understood. A yeast-two-hybrid library approach was used to search for wheat proteins that interacted with the necrotrophic effector SnTox3. Using this strategy we indentified an interaction between SnTox3 and the wheat pathogenicity-related protein TaPR-1-1, and confirmed it by in-planta co-immunprecipitation. PR-1 proteins represent a large family (23 in wheat) of proteins that are up-regulated early in the defence response, however their function remains ellusive. Interestingly, the P. nodorum effector SnToxA has recently been shown to interact specifically with TaPR-1-5. Our analysis of the SnTox3-TaPR-1 interaction demo...
Principles of Kinase Regulation Bostjan Kobe1 and Bruce E. Kemp2 1 School of Chemistry Molecular ... more Principles of Kinase Regulation Bostjan Kobe1 and Bruce E. Kemp2 1 School of Chemistry Molecular Biosciences, University of Queensland, Brisbane ... Sessa F, Mapelli M, Ciferri C, Tarricone C, Areces LB, Schneider TR, Stukenberg PT, Musacchio A. Mechanism of Aurora B ...
The biosynthetic pathway for the branched-chain amino acids is present in plants, fungi and bacte... more The biosynthetic pathway for the branched-chain amino acids is present in plants, fungi and bacteria, but not in animals, making it an attractive target for herbicidal and antimicrobial drug discovery. Ketol-acid reductoisomerase (KARI; EC 1.1.1.86) is the second enzyme in this pathway, converting in a Mg(2+) - and NADPH-dependent reaction either 2-acetolactate or 2-aceto-2-hydroxybutyrate to their corresponding 2,3-dihydroxy-3-alkylbutyrate products. Here, we have determined the crystal structure of Mycobacterium tuberculosis KARI (Mt KARI), a class I KARI, with two magnesium ions bound in the active site. X-ray data were obtained to 1.0 Å resolution and the final model has an Rfree of 0.174. The structure shows that the active site is solvent-accessible with the two metal ions separated by 4.7 Å. A comparison of this structure with that of Mg(2+) -free Pseudomonas aeruginosa KARI suggests that upon magnesium binding no movement of the N-domain relative to the C-domain occurs. Howe...
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