Members of the Gcn5-related N-acetyltransferase (GNAT) superfamily catalyze the acetylation of a ... more Members of the Gcn5-related N-acetyltransferase (GNAT) superfamily catalyze the acetylation of a wide range of small molecule and protein substrates. Due to their abundance in all kingdoms of life and diversity of their functions, they are implicated in many aspects of eukaryotic and prokaryotic physiology. Although numerous GNATs have been identified thus far, many remain structurally and functionally uncharacterized. The elucidation of their structures and functions is critical for broadening our knowledge of this diverse and important superfamily. In this work, we present the structural and kinetic analyses of two previously uncharacterized bacterial acetyltransferases - SACOL1063 from Staphylococcus aureus strain COL and CD1211 from Clostridium difficile strain 630. Our structures of SACOL1063 show substantial flexibility of a loop that is likely responsible for substrate recognition and binding compared to structures of other homologs. In the CoA complex structure, we found two...
Sperm Lysozyme-Like Protein 1 (SLLP1) is one of the lysozyme-like proteins predominantly expresse... more Sperm Lysozyme-Like Protein 1 (SLLP1) is one of the lysozyme-like proteins predominantly expressed in mammalian testes that lacks bacteriolytic activity, localizes in the sperm acrosome, and exhibits high affinity for an oolemmal receptor, SAS1B. The crystal structure of mouse SLLP1 (mSLLP1) was determined at 2.15Å resolution. mSLLP1 monomer adopts a structural fold similar to that of chicken/mouse lysozymes retaining all four canonical disulfide bonds. mSLLP1 is distinct from c-lysozyme by substituting two essential catalytic residues (E35T/D52N), exhibiting different surface charge distribution, and by forming helical filaments approximately 75Å in diameter with a 25Å central pore comprised of six monomers per helix turn repeating every 33Å. Cross-species alignment of all reported SLLP1 sequences revealed a set of invariant surface regions comprising a characteristic fingerprint uniquely identifying SLLP1 from other clysozyme family members. The fingerprint surface regions reside around the lips of the putative glycan binding groove including three polar residues (Y33/E46/H113). A flexible salt bridge (E46-R61) was observed covering the glycan binding groove. The conservation of these regions may be linked to their involvement in oolemmal protein binding. Interaction between SLLP1 monomer and its oolemmal receptor SAS1B was modeled using protein-protein docking algorithms, utilizing the SLLP1 fingerprint regions along with the SAS1B conserved surface regions. This computational model revealed complementarity between the conserved SLLP1/SAS1B interacting
Journal of structural and functional genomics, 2014
This study describes the structure of the putative ABC-type 2 transporter TM0543 from Thermotoga ... more This study describes the structure of the putative ABC-type 2 transporter TM0543 from Thermotoga maritima MSB8 determined at a resolution of 2.3 Å. In comparative sequence-clustering analysis, TM0543 displays similarity to NatAB-like proteins, which are components of the ABC-type Na(+) efflux pump permease. However, the overall structure fold of the predicted nucleotide-binding domain reveals that it is different from any known structure of ABC-type efflux transporters solved to date. The structure of the putative TM0543 domain also exhibits different dimer architecture and topology of its presumed ATP binding pocket, which may indicate that it does not bind nucleotide at all. Structural analysis of calcium ion binding sites found at the interface between TM0543 dimer subunits suggests that protein may be involved in ion-transporting activity. A detailed analysis of the protein sequence and structure is presented and discussed.
Oncogenic fusion proteins, such as EWS-FLI1, are excellent therapeutic targets as they are only l... more Oncogenic fusion proteins, such as EWS-FLI1, are excellent therapeutic targets as they are only located within the tumor. However, there are currently no agents targeted toward transcription factors, which are often considered to be 'undruggable.' A considerable body of evidence is accruing that refutes this claim based upon the intrinsic disorder of transcription factors. Our previous studies show that RNA Helicase A (RHA) enhances the oncogenesis of EWS-FLI1, a putative intrinsically disordered protein. Interruption of this protein-protein complex by small molecule inhibitors validates this interaction as a unique therapeutic target. Single enantiomer activity from a chiral compound has been recognized as strong evidence for specificity in a small molecule-protein interaction. Our compound, YK-4-279, has a chiral center and can be separated into two enantiomers by chiral HPLC. We show that there is a significant difference in activity between the two enantiomers. (S)-YK-4-...
Background: EWS-FLI1 requires the protein binding of RNA Helicase A (RHA) to enable oncogenic tra... more Background: EWS-FLI1 requires the protein binding of RNA Helicase A (RHA) to enable oncogenic transformation. RHA is a DExH box RNA helicase family member. RHA has critical roles modulating transcription, splicing and translation. RHA also functions as a scaffolding protein in multi-protein complexes including BRCA1, DICER, EGFR, POL2R, TOPO2. Our previous work demonstrated that YK-4-279 inhibited the protein-protein interaction of RHA, with EWS-FLI1 and led to cellular apoptosis. The biochemical mechanism remained cryptic, so we investigated the effect of EWS-FLI1 upon the helicase activity of RHA. Methods: Full-length purified recombinant RHA from insect cells. Infrared-labeled double stranded RNA was used as a substrate to measure helicase activity of RHA. We included full-length recombinant EWS-FLI1 in to the helicase reaction to test the effect of EWS-FLI1. Results: RHA unwinds double stranded RNA in the presence of ATP with Km value of 5.322 nM. We also showed that RHA manifes...
Modern high-throughput structural biology laboratories produce vast amounts of raw experimental d... more Modern high-throughput structural biology laboratories produce vast amounts of raw experimental data. The traditional method of data reduction is very simple-results are summarized in peerreviewed publications, which are hopefully published in high-impact journals. By their nature, publications include only the most important results derived from experiments that may have been performed over the course of many years. The main content of the published paper is a concise compilation of these data, an interpretation of the experimental results, and a comparison of these results with those obtained by other scientists.
Bla g 4 is a male cockroach specific protein and is one of the major allergens produced by Blatte... more Bla g 4 is a male cockroach specific protein and is one of the major allergens produced by Blattella germanica (German cockroach). This protein belongs to the lipocalin family that comprises a set of proteins that characteristically bind small hydrophobic molecules and play a role in a number of processes such as: retinoid and pheromone transport, prostaglandin synthesis and mammalian immune response. Using NMR and Isothermal Titration Calorimetry we demonstrated that Bla g 4 binds tyramine and octopamine in solution. In addition, crystal structure analysis of the complex revealed details of tyramine binding. As tyramine and octopamine play important roles in invertebrates, and are counterparts to vertebrate adrenergic transmitters, we speculate that these molecules are physiological ligands for Bla g 4. The nature of binding these ligands to Bla g 4 sheds light on the possible biological function of the protein. In addition, we performed a large-scale analysis of Bla g 4 and Per a 4 (an allergen from American cockroach) homologs to get insights into the function of these proteins. This analysis together with a structural comparison of Blag 4 and Per a 4 suggests that these proteins may play different roles and most likely bind different ligands.
Journal of Structural and Functional Genomics, 2012
The explosion of the size of the universe of known protein sequences has stimulated two complemen... more The explosion of the size of the universe of known protein sequences has stimulated two complementary approaches to structural mapping of these sequences: theoretical structure prediction and experimental determination by structural genomics (SG). In this work, we assess the accuracy of structure prediction by two automated template-based structure prediction metaservers (genesilico.pl and bioinfo.pl) by measuring the structural similarity of the predicted models to corresponding experimental models determined a posteriori. Of 199 targets chosen from SG programs, the metaservers predicted the structures of about a fourth of them "correctly." (In this case, "correct" was defined as placing more than 70% of the alpha carbon atoms in the model within 2 Å of the experimentally determined positions.) Almost all of the targets that could be modeled to this accuracy were those with an available template in the Protein Data Bank (PDB) with more than 25% sequence identity. The majority of those SG targets with lower sequence identity to structures in the PDB were not predicted by the metaservers with this accuracy. We also compared metaserver results to CASP8 results, finding that the models obtained by participants in the CASP competition were significantly better than those produced by the metaservers.
Acta Crystallographica Section F Structural Biology and Crystallization Communications, 2013
The isochorismate synthase DhbC from Bacillus anthracis is essential for the biosynthesis of the ... more The isochorismate synthase DhbC from Bacillus anthracis is essential for the biosynthesis of the siderophore bacillibactin by this pathogenic bacterium. The structure of the selenomethionine-substituted protein was determined to 2.4 Å resolution using single-wavelength anomalous diffraction. B. anthracis DhbC bears the strongest resemblance to the Escherichia coli isochorismate synthase EntC, which is involved in the biosynthesis of another siderophore, namely enterobactin. Both proteins adopt the characteristic fold of other chorismateutilizing enzymes, which are involved in the biosynthesis of various products, including siderophores, menaquinone and tryptophan. The conservation of the active-site residues, as well as their spatial arrangement, suggests that these enzymes share a common Mg 2+-dependent catalytic mechanism.
Zeitschrift für Kristallographie - New Crystal Structures, 2006
Source of material The compound was isolated as a side product of a reaction involving secondary ... more Source of material The compound was isolated as a side product of a reaction involving secondary propargylic alcohols [1]. It is also available commercially from Sigma Inc. Experimental details All hydrogen atoms were located from the Fourier difference map, but in order to have a favorable NgJN V3I3m ratio, the hydrogen atoms of the benzene ring were placed in calculated positions and refined using a riding-model approximation with C-Η bond lengths of 0.95 Ä. Positional and isotropic displacement parameters were only refined for the Η atom of the aldehyde group.
Acta Crystallographica Section E Structure Reports Online, 2006
Two formula units of disodium 4-nitrophenylphosphate hexahydrate, 2Na+·C6H4NO6P2−·6H2O, are prese... more Two formula units of disodium 4-nitrophenylphosphate hexahydrate, 2Na+·C6H4NO6P2−·6H2O, are present in the asymmetric unit. The 4-nitrophenyl groups of the two dianions are essentially identical in structure, while the phosphate groups differ in conformation.
Members of the Gcn5-related N-acetyltransferase (GNAT) superfamily catalyze the acetylation of a ... more Members of the Gcn5-related N-acetyltransferase (GNAT) superfamily catalyze the acetylation of a wide range of small molecule and protein substrates. Due to their abundance in all kingdoms of life and diversity of their functions, they are implicated in many aspects of eukaryotic and prokaryotic physiology. Although numerous GNATs have been identified thus far, many remain structurally and functionally uncharacterized. The elucidation of their structures and functions is critical for broadening our knowledge of this diverse and important superfamily. In this work, we present the structural and kinetic analyses of two previously uncharacterized bacterial acetyltransferases - SACOL1063 from Staphylococcus aureus strain COL and CD1211 from Clostridium difficile strain 630. Our structures of SACOL1063 show substantial flexibility of a loop that is likely responsible for substrate recognition and binding compared to structures of other homologs. In the CoA complex structure, we found two...
Sperm Lysozyme-Like Protein 1 (SLLP1) is one of the lysozyme-like proteins predominantly expresse... more Sperm Lysozyme-Like Protein 1 (SLLP1) is one of the lysozyme-like proteins predominantly expressed in mammalian testes that lacks bacteriolytic activity, localizes in the sperm acrosome, and exhibits high affinity for an oolemmal receptor, SAS1B. The crystal structure of mouse SLLP1 (mSLLP1) was determined at 2.15Å resolution. mSLLP1 monomer adopts a structural fold similar to that of chicken/mouse lysozymes retaining all four canonical disulfide bonds. mSLLP1 is distinct from c-lysozyme by substituting two essential catalytic residues (E35T/D52N), exhibiting different surface charge distribution, and by forming helical filaments approximately 75Å in diameter with a 25Å central pore comprised of six monomers per helix turn repeating every 33Å. Cross-species alignment of all reported SLLP1 sequences revealed a set of invariant surface regions comprising a characteristic fingerprint uniquely identifying SLLP1 from other clysozyme family members. The fingerprint surface regions reside around the lips of the putative glycan binding groove including three polar residues (Y33/E46/H113). A flexible salt bridge (E46-R61) was observed covering the glycan binding groove. The conservation of these regions may be linked to their involvement in oolemmal protein binding. Interaction between SLLP1 monomer and its oolemmal receptor SAS1B was modeled using protein-protein docking algorithms, utilizing the SLLP1 fingerprint regions along with the SAS1B conserved surface regions. This computational model revealed complementarity between the conserved SLLP1/SAS1B interacting
Journal of structural and functional genomics, 2014
This study describes the structure of the putative ABC-type 2 transporter TM0543 from Thermotoga ... more This study describes the structure of the putative ABC-type 2 transporter TM0543 from Thermotoga maritima MSB8 determined at a resolution of 2.3 Å. In comparative sequence-clustering analysis, TM0543 displays similarity to NatAB-like proteins, which are components of the ABC-type Na(+) efflux pump permease. However, the overall structure fold of the predicted nucleotide-binding domain reveals that it is different from any known structure of ABC-type efflux transporters solved to date. The structure of the putative TM0543 domain also exhibits different dimer architecture and topology of its presumed ATP binding pocket, which may indicate that it does not bind nucleotide at all. Structural analysis of calcium ion binding sites found at the interface between TM0543 dimer subunits suggests that protein may be involved in ion-transporting activity. A detailed analysis of the protein sequence and structure is presented and discussed.
Oncogenic fusion proteins, such as EWS-FLI1, are excellent therapeutic targets as they are only l... more Oncogenic fusion proteins, such as EWS-FLI1, are excellent therapeutic targets as they are only located within the tumor. However, there are currently no agents targeted toward transcription factors, which are often considered to be 'undruggable.' A considerable body of evidence is accruing that refutes this claim based upon the intrinsic disorder of transcription factors. Our previous studies show that RNA Helicase A (RHA) enhances the oncogenesis of EWS-FLI1, a putative intrinsically disordered protein. Interruption of this protein-protein complex by small molecule inhibitors validates this interaction as a unique therapeutic target. Single enantiomer activity from a chiral compound has been recognized as strong evidence for specificity in a small molecule-protein interaction. Our compound, YK-4-279, has a chiral center and can be separated into two enantiomers by chiral HPLC. We show that there is a significant difference in activity between the two enantiomers. (S)-YK-4-...
Background: EWS-FLI1 requires the protein binding of RNA Helicase A (RHA) to enable oncogenic tra... more Background: EWS-FLI1 requires the protein binding of RNA Helicase A (RHA) to enable oncogenic transformation. RHA is a DExH box RNA helicase family member. RHA has critical roles modulating transcription, splicing and translation. RHA also functions as a scaffolding protein in multi-protein complexes including BRCA1, DICER, EGFR, POL2R, TOPO2. Our previous work demonstrated that YK-4-279 inhibited the protein-protein interaction of RHA, with EWS-FLI1 and led to cellular apoptosis. The biochemical mechanism remained cryptic, so we investigated the effect of EWS-FLI1 upon the helicase activity of RHA. Methods: Full-length purified recombinant RHA from insect cells. Infrared-labeled double stranded RNA was used as a substrate to measure helicase activity of RHA. We included full-length recombinant EWS-FLI1 in to the helicase reaction to test the effect of EWS-FLI1. Results: RHA unwinds double stranded RNA in the presence of ATP with Km value of 5.322 nM. We also showed that RHA manifes...
Modern high-throughput structural biology laboratories produce vast amounts of raw experimental d... more Modern high-throughput structural biology laboratories produce vast amounts of raw experimental data. The traditional method of data reduction is very simple-results are summarized in peerreviewed publications, which are hopefully published in high-impact journals. By their nature, publications include only the most important results derived from experiments that may have been performed over the course of many years. The main content of the published paper is a concise compilation of these data, an interpretation of the experimental results, and a comparison of these results with those obtained by other scientists.
Bla g 4 is a male cockroach specific protein and is one of the major allergens produced by Blatte... more Bla g 4 is a male cockroach specific protein and is one of the major allergens produced by Blattella germanica (German cockroach). This protein belongs to the lipocalin family that comprises a set of proteins that characteristically bind small hydrophobic molecules and play a role in a number of processes such as: retinoid and pheromone transport, prostaglandin synthesis and mammalian immune response. Using NMR and Isothermal Titration Calorimetry we demonstrated that Bla g 4 binds tyramine and octopamine in solution. In addition, crystal structure analysis of the complex revealed details of tyramine binding. As tyramine and octopamine play important roles in invertebrates, and are counterparts to vertebrate adrenergic transmitters, we speculate that these molecules are physiological ligands for Bla g 4. The nature of binding these ligands to Bla g 4 sheds light on the possible biological function of the protein. In addition, we performed a large-scale analysis of Bla g 4 and Per a 4 (an allergen from American cockroach) homologs to get insights into the function of these proteins. This analysis together with a structural comparison of Blag 4 and Per a 4 suggests that these proteins may play different roles and most likely bind different ligands.
Journal of Structural and Functional Genomics, 2012
The explosion of the size of the universe of known protein sequences has stimulated two complemen... more The explosion of the size of the universe of known protein sequences has stimulated two complementary approaches to structural mapping of these sequences: theoretical structure prediction and experimental determination by structural genomics (SG). In this work, we assess the accuracy of structure prediction by two automated template-based structure prediction metaservers (genesilico.pl and bioinfo.pl) by measuring the structural similarity of the predicted models to corresponding experimental models determined a posteriori. Of 199 targets chosen from SG programs, the metaservers predicted the structures of about a fourth of them "correctly." (In this case, "correct" was defined as placing more than 70% of the alpha carbon atoms in the model within 2 Å of the experimentally determined positions.) Almost all of the targets that could be modeled to this accuracy were those with an available template in the Protein Data Bank (PDB) with more than 25% sequence identity. The majority of those SG targets with lower sequence identity to structures in the PDB were not predicted by the metaservers with this accuracy. We also compared metaserver results to CASP8 results, finding that the models obtained by participants in the CASP competition were significantly better than those produced by the metaservers.
Acta Crystallographica Section F Structural Biology and Crystallization Communications, 2013
The isochorismate synthase DhbC from Bacillus anthracis is essential for the biosynthesis of the ... more The isochorismate synthase DhbC from Bacillus anthracis is essential for the biosynthesis of the siderophore bacillibactin by this pathogenic bacterium. The structure of the selenomethionine-substituted protein was determined to 2.4 Å resolution using single-wavelength anomalous diffraction. B. anthracis DhbC bears the strongest resemblance to the Escherichia coli isochorismate synthase EntC, which is involved in the biosynthesis of another siderophore, namely enterobactin. Both proteins adopt the characteristic fold of other chorismateutilizing enzymes, which are involved in the biosynthesis of various products, including siderophores, menaquinone and tryptophan. The conservation of the active-site residues, as well as their spatial arrangement, suggests that these enzymes share a common Mg 2+-dependent catalytic mechanism.
Zeitschrift für Kristallographie - New Crystal Structures, 2006
Source of material The compound was isolated as a side product of a reaction involving secondary ... more Source of material The compound was isolated as a side product of a reaction involving secondary propargylic alcohols [1]. It is also available commercially from Sigma Inc. Experimental details All hydrogen atoms were located from the Fourier difference map, but in order to have a favorable NgJN V3I3m ratio, the hydrogen atoms of the benzene ring were placed in calculated positions and refined using a riding-model approximation with C-Η bond lengths of 0.95 Ä. Positional and isotropic displacement parameters were only refined for the Η atom of the aldehyde group.
Acta Crystallographica Section E Structure Reports Online, 2006
Two formula units of disodium 4-nitrophenylphosphate hexahydrate, 2Na+·C6H4NO6P2−·6H2O, are prese... more Two formula units of disodium 4-nitrophenylphosphate hexahydrate, 2Na+·C6H4NO6P2−·6H2O, are present in the asymmetric unit. The 4-nitrophenyl groups of the two dianions are essentially identical in structure, while the phosphate groups differ in conformation.
Uploads
Papers by Wladek Minor