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Solution Structural Studies of GTP:Adenosylcobinamide-Phosphateguanylyl Transferase (CobY) from Methanocaldococcus jannaschii.

Singarapu KK, Otte MM, Tonelli M, Westler WM, Escalante-Semerena JC, Markley JL - PLoS ONE (2015)

Bottom Line: Titration of apo-CobY with GTP resulted in large changes in amide proton chemical shifts that indicated major structural perturbations upon complex formation.However, the CobY:GTP complex as followed by 1H-15N HSQC spectra was found to be unstable over time: GTP hydrolyzed and the protein converted slowly to a species with an NMR spectrum similar to that of apo-CobY.The CobYG153D:GTP complex was also found to be unstable over time.

View Article: PubMed Central - PubMed

Affiliation: National Magnetic Resonance Facility at Madison and Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, United States of America; Center for NMR and Structural Chemistry, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad, Telangana, India.

ABSTRACT
GTP:adenosylcobinamide-phosphate (AdoCbi-P) guanylyl transferase (CobY) is an enzyme that transfers the GMP moiety of GTP to AdoCbi yielding AdoCbi-GDP in the late steps of the assembly of Ado-cobamides in archaea. The failure of repeated attempts to crystallize ligand-free (apo) CobY prompted us to explore its 3D structure by solution NMR spectroscopy. As reported here, the solution structure has a mixed α/β fold consisting of seven β-strands and five α-helices, which is very similar to a Rossmann fold. Titration of apo-CobY with GTP resulted in large changes in amide proton chemical shifts that indicated major structural perturbations upon complex formation. However, the CobY:GTP complex as followed by 1H-15N HSQC spectra was found to be unstable over time: GTP hydrolyzed and the protein converted slowly to a species with an NMR spectrum similar to that of apo-CobY. The variant CobYG153D, whose GTP complex was studied by X-ray crystallography, yielded NMR spectra similar to those of wild-type CobY in both its apo- state and in complex with GTP. The CobYG153D:GTP complex was also found to be unstable over time.

No MeSH data available.


Structure-based Multiple Sequence Alignment of Wild-type apo-CobY with the Five Most Structurally Similar Proteins.The alignment, which was carried out using the PROMALS3D program [32], shows substantial sequence alignment of secondary structural elements derived from the 3D structures: (blue) α-helix; (red) β-strand.
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pone.0141297.g004: Structure-based Multiple Sequence Alignment of Wild-type apo-CobY with the Five Most Structurally Similar Proteins.The alignment, which was carried out using the PROMALS3D program [32], shows substantial sequence alignment of secondary structural elements derived from the 3D structures: (blue) α-helix; (red) β-strand.

Mentions: We used the software programs DALI [26] and ProFunc [27] to search for structural homologues of apo-CobY. The five most similar structures, all determined by X-ray crystallography, contained mononucleotide binding domains with a canonical Rossmann fold (Fig 3). Cytidinyl monophosphate2-keto-3-deoxy-manno-octonic acid synthetase (CMP:Kdo) from Escherichia coli (PDB 1H7F, Z-score 15.8, rmsd 2.6 Å, seq ID 14%) is involved in the synthesis of lipopolysaccharides that are toxic to Gram-negative bacteria [28]. Glucose-1-phosphate cytidylyl transferase from Salmonella typhi (PDB 1WVC, Z-score 15.2, rmsd 3.3 Å, seq ID 18%) catalyzes the transfer of a CMP moiety from CTP to glucose 1-phosphate [29]. Cytidine transferase from Streptococcus pneumonia (PDB 2VSI, Z-score 15.6, rmsd 3.0 Å, seq ID 13%) is involved in the synthesis of cytidine-5'-diphosphate (CDP)-ribitol from ribitol 5-phosphate and CTP [30]. 2-C-methyl-D-erythritol 4-phosphate cytidylyl transferase from Thermotoga maritima (PDB 1VPA, Z-score 15.3, rmsd 3.1 Å, seq ID 18%) catalyzes the formation of 4-diphosphocytidyl-2-C-methyl-D-erythritol from CTP and 2-C-methyl-D-erythritol 4-phosphate. N-acylneuraminate cytidylyl transferase from Neisseria meningitides (PDB 1EYR, Z-score 15.3, rmsd 3.0 Å, seq ID 17%) catalyzes the reaction of CTP and N-acylneuraminate to form CMP-N-acylneuraminate and bisphosphate [31]. A structure-based multiple sequence alignment performed by PROMALS3D [32] revealed several highly-conserved residues (Fig 4): A3, I5, A7, R13, K19, G26, (K/R)27, (D/E)58, T180, D183, and L184. These residues appear to play important roles in nucleotide binding. In particular, residues R13 and K19 directly coordinate the phosphate group of GTP in the X-ray structure of CobYG153D [8].


Solution Structural Studies of GTP:Adenosylcobinamide-Phosphateguanylyl Transferase (CobY) from Methanocaldococcus jannaschii.

Singarapu KK, Otte MM, Tonelli M, Westler WM, Escalante-Semerena JC, Markley JL - PLoS ONE (2015)

Structure-based Multiple Sequence Alignment of Wild-type apo-CobY with the Five Most Structurally Similar Proteins.The alignment, which was carried out using the PROMALS3D program [32], shows substantial sequence alignment of secondary structural elements derived from the 3D structures: (blue) α-helix; (red) β-strand.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4626045&req=5

pone.0141297.g004: Structure-based Multiple Sequence Alignment of Wild-type apo-CobY with the Five Most Structurally Similar Proteins.The alignment, which was carried out using the PROMALS3D program [32], shows substantial sequence alignment of secondary structural elements derived from the 3D structures: (blue) α-helix; (red) β-strand.
Mentions: We used the software programs DALI [26] and ProFunc [27] to search for structural homologues of apo-CobY. The five most similar structures, all determined by X-ray crystallography, contained mononucleotide binding domains with a canonical Rossmann fold (Fig 3). Cytidinyl monophosphate2-keto-3-deoxy-manno-octonic acid synthetase (CMP:Kdo) from Escherichia coli (PDB 1H7F, Z-score 15.8, rmsd 2.6 Å, seq ID 14%) is involved in the synthesis of lipopolysaccharides that are toxic to Gram-negative bacteria [28]. Glucose-1-phosphate cytidylyl transferase from Salmonella typhi (PDB 1WVC, Z-score 15.2, rmsd 3.3 Å, seq ID 18%) catalyzes the transfer of a CMP moiety from CTP to glucose 1-phosphate [29]. Cytidine transferase from Streptococcus pneumonia (PDB 2VSI, Z-score 15.6, rmsd 3.0 Å, seq ID 13%) is involved in the synthesis of cytidine-5'-diphosphate (CDP)-ribitol from ribitol 5-phosphate and CTP [30]. 2-C-methyl-D-erythritol 4-phosphate cytidylyl transferase from Thermotoga maritima (PDB 1VPA, Z-score 15.3, rmsd 3.1 Å, seq ID 18%) catalyzes the formation of 4-diphosphocytidyl-2-C-methyl-D-erythritol from CTP and 2-C-methyl-D-erythritol 4-phosphate. N-acylneuraminate cytidylyl transferase from Neisseria meningitides (PDB 1EYR, Z-score 15.3, rmsd 3.0 Å, seq ID 17%) catalyzes the reaction of CTP and N-acylneuraminate to form CMP-N-acylneuraminate and bisphosphate [31]. A structure-based multiple sequence alignment performed by PROMALS3D [32] revealed several highly-conserved residues (Fig 4): A3, I5, A7, R13, K19, G26, (K/R)27, (D/E)58, T180, D183, and L184. These residues appear to play important roles in nucleotide binding. In particular, residues R13 and K19 directly coordinate the phosphate group of GTP in the X-ray structure of CobYG153D [8].

Bottom Line: Titration of apo-CobY with GTP resulted in large changes in amide proton chemical shifts that indicated major structural perturbations upon complex formation.However, the CobY:GTP complex as followed by 1H-15N HSQC spectra was found to be unstable over time: GTP hydrolyzed and the protein converted slowly to a species with an NMR spectrum similar to that of apo-CobY.The CobYG153D:GTP complex was also found to be unstable over time.

View Article: PubMed Central - PubMed

Affiliation: National Magnetic Resonance Facility at Madison and Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, United States of America; Center for NMR and Structural Chemistry, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad, Telangana, India.

ABSTRACT
GTP:adenosylcobinamide-phosphate (AdoCbi-P) guanylyl transferase (CobY) is an enzyme that transfers the GMP moiety of GTP to AdoCbi yielding AdoCbi-GDP in the late steps of the assembly of Ado-cobamides in archaea. The failure of repeated attempts to crystallize ligand-free (apo) CobY prompted us to explore its 3D structure by solution NMR spectroscopy. As reported here, the solution structure has a mixed α/β fold consisting of seven β-strands and five α-helices, which is very similar to a Rossmann fold. Titration of apo-CobY with GTP resulted in large changes in amide proton chemical shifts that indicated major structural perturbations upon complex formation. However, the CobY:GTP complex as followed by 1H-15N HSQC spectra was found to be unstable over time: GTP hydrolyzed and the protein converted slowly to a species with an NMR spectrum similar to that of apo-CobY. The variant CobYG153D, whose GTP complex was studied by X-ray crystallography, yielded NMR spectra similar to those of wild-type CobY in both its apo- state and in complex with GTP. The CobYG153D:GTP complex was also found to be unstable over time.

No MeSH data available.