Literature summary for 3.6.4.B7 extracted from

Marsh, M.E.; Scott, D.E.; Ehebauer, M.T.; Abell, C.; Blundell, T.L.; Hyvoenen, M.
ATP half-sites in RadA and RAD51 recombinases bind nucleotides (2016), FEBS open bio, 6, 372-385 .

Cloned(Commentary)

Cloned (Comment)	Organism
recombinant expression of the RadAC-terminal domain RadA-ct in Escherichia coli as soluble protein	Pyrococcus horikoshii

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant truncated monomeric mutant RadA-ct bound to ATP, ADP, AMPPNP and GTP, hanging drop vapour diffusion method, mixing protein in 25% v/v glycerol, 15% PEG 1000, and 10 mM MgCl2 with 15% PEG 1000 in 60 mM Na2HPO4 as precipitant, 20°C, crystals are soaked with different nucleotide solutions containing 10 mM nucleotides, X-ray diffraction structure determination and analysis at 1.47-1.88 A resolution. As magnesium is not present in the crystallisation conditions of RadA, the phosphate-bound form also lacks magnesium, but this appears to have little impact on the binding of the phosphate	Pyrococcus horikoshii

Crystallization (Comment)

Organism

purified recombinant truncated monomeric mutant RadA-ct bound to ATP, ADP, AMPPNP and GTP, hanging drop vapour diffusion method, mixing protein in 25% v/v glycerol, 15% PEG 1000, and 10 mM MgCl2 with 15% PEG 1000 in 60 mM Na2HPO4 as precipitant, 20°C, crystals are soaked with different nucleotide solutions containing 10 mM nucleotides, X-ray diffraction structure determination and analysis at 1.47-1.88 A resolution. As magnesium is not present in the crystallisation conditions of RadA, the phosphate-bound form also lacks magnesium, but this appears to have little impact on the binding of the phosphate

Pyrococcus horikoshii

Protein Variants

Protein Variants	Comment	Organism
I169M/Y201A/V202Y/E219S/D220A/K221M	site-directed mutagenesis, construction of mutant, termed HumRadA2, that resembles human RAD51. The mutant shows reduced thermal stability compared to wild-type, while the crystal structure of this mutant shows no structural changes in the ATP-binding site with respect to the wild-type. Fluorescence-based thermal shift and isothermal titration calorimetric analyses of nucleotide binding to recombinant mutant monomeric HumRadA2, overview	Pyrococcus horikoshii
additional information	construction of monomeric form of RAD51 in which BRC repeat 4 from BRCA2 is covalently linked to the N-terminus of the ATPase domain of human RAD51. Fluorescence-based thermal shift and isothermal titration calorimetric analyses of nucleotide binding to recombinant mutant RAD51-BRC4, overview	Homo sapiens
additional information	isolation of the C-terminal ATPase domain by removing the N-terminal domain and the linker that contains the FxxA oligomerisation sequence. To facilitate crystallisation, the unstructured L2 DNA-binding loop is also removed, creating a construct denoted as RadA-ct, analysis of structures of RadA-ct bound to ATP, ADP, AMPPNP and GTP, cyrstal structure of phosphate-bound RadA-ct, overview	Pyrococcus horikoshii

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	required	Homo sapiens
Mg2+	required. A magnesium ion is observed in all nucleotide-bound structures. It is positioned in the same location in ATP and AMPPNP structures, co-ordinated by the beta- and gamma-phosphate groups and the hydroxyl group of Thr145. In the ADP structure, the Mg2+ ion is shifted slightly towards the beta-phosphate group, it is also co-ordinated by the beta-phosphate and the hydroxyl group of Thr145, in addition to four water molecules. The residue Glu174 likely activates a water molecule for hydrolysis of ATP. Glu174 forms an indirect interaction with the Mg2+ ion via a bridging water molecule. As magnesium is not present in the crystallisation conditions of RadA, the phosphate-bound form also lacks magnesium, but this appears to have little impact on the binding of the phosphate	Pyrococcus horikoshii

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.
ATP + H2O	Homo sapiens	-	ADP + phosphate	-	?
ATP + H2O	Pyrococcus horikoshii	-	ADP + phosphate	-	?
ATP + H2O	Pyrococcus horikoshii DSM 12428	-	ADP + phosphate	-	?
ATP + H2O	Pyrococcus horikoshii NBRC 100139	-	ADP + phosphate	-	?
ATP + H2O	Pyrococcus horikoshii JCM 9974	-	ADP + phosphate	-	?
ATP + H2O	Pyrococcus horikoshii ATCC 700860	-	ADP + phosphate	-	?
ATP + H2O	Pyrococcus horikoshii OT-3	-	ADP + phosphate	-	?
GTP + H2O	Homo sapiens	-	GDP + phosphate	-	?
GTP + H2O	Pyrococcus horikoshii	-	GDP + phosphate	-	?
GTP + H2O	Pyrococcus horikoshii DSM 12428	-	GDP + phosphate	-	?
GTP + H2O	Pyrococcus horikoshii NBRC 100139	-	GDP + phosphate	-	?
GTP + H2O	Pyrococcus horikoshii JCM 9974	-	GDP + phosphate	-	?
GTP + H2O	Pyrococcus horikoshii ATCC 700860	-	GDP + phosphate	-	?
GTP + H2O	Pyrococcus horikoshii OT-3	-	GDP + phosphate	-	?

Organism

Organism	UniProt	Comment	Textmining
Homo sapiens	-	-	-
Pyrococcus horikoshii	O58001	-	-
Pyrococcus horikoshii ATCC 700860	O58001	-	-
Pyrococcus horikoshii DSM 12428	O58001	-	-
Pyrococcus horikoshii JCM 9974	O58001	-	-
Pyrococcus horikoshii NBRC 100139	O58001	-	-
Pyrococcus horikoshii OT-3	O58001	-	-

Purification (Commentary)

Purification (Comment)	Organism
soluble recombinant RadAC-terminal domain RadA-ct from Escherichia coli to homogeneity	Pyrococcus horikoshii

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
ATP + H2O	-	Homo sapiens	ADP + phosphate	-	?
ATP + H2O	-	Pyrococcus horikoshii	ADP + phosphate	-	?
ATP + H2O	-	Pyrococcus horikoshii DSM 12428	ADP + phosphate	-	?
ATP + H2O	-	Pyrococcus horikoshii NBRC 100139	ADP + phosphate	-	?
ATP + H2O	-	Pyrococcus horikoshii JCM 9974	ADP + phosphate	-	?
ATP + H2O	-	Pyrococcus horikoshii ATCC 700860	ADP + phosphate	-	?
ATP + H2O	-	Pyrococcus horikoshii OT-3	ADP + phosphate	-	?
GTP + H2O	-	Homo sapiens	GDP + phosphate	-	?
GTP + H2O	-	Pyrococcus horikoshii	GDP + phosphate	-	?
GTP + H2O	-	Pyrococcus horikoshii DSM 12428	GDP + phosphate	-	?
GTP + H2O	-	Pyrococcus horikoshii NBRC 100139	GDP + phosphate	-	?
GTP + H2O	-	Pyrococcus horikoshii JCM 9974	GDP + phosphate	-	?
GTP + H2O	-	Pyrococcus horikoshii ATCC 700860	GDP + phosphate	-	?
GTP + H2O	-	Pyrococcus horikoshii OT-3	GDP + phosphate	-	?
additional information	monomeric RAD51 is able to bind ATP, ADP, AMPPNP, GTP and GDP in an Mg2+-dependent manner. AMP is not able to bind to the protein indicating that both the beta- and gamma-phosphates are essential for this interaction. AMPPNP binds to RAD51, but with reduced affinity in comparison with ATP. This is likely to be caused by the inability of the nitrogen between the band gamma-phosphates to form a hydrogen bond with the backbone amide of Gly141 as observed in the complex with ATP. Comparisons of nucleotide-bound enzyme and enzyme mutant structure, overview. The guanine-containing nucleotides bind to the monomeric form of RadA and RAD51 with a somewhat higher affinity than the adenine-containing equivalents. Selectivity for ATP over GTP appears to be mediated by the binding of the second protomer, which forms specific interactions with ATP but not with GTP	Homo sapiens	?	-	-
additional information	monomeric RadA-ct is able to bind ATP, ADP, AMPPNP, GTP and GDP in an Mg2+-dependent manner. AMP is not able to bind to the protein indicating that both the beta- and gamma-phosphates are essential for this interaction. ADP and GDP bind to RadA with approximately twice the affinity of their triphosphate equivalents, possibly due to the different conformation of the side chain of Phe140 between the di- and triphosphorylated nucleotide complexes, with the additional negative charge of the gamma-phosphate repelling the aromatic ring. AMPPNP binds to mutant RadA-ct, but with reduced affinity in comparison with ATP. This is likely to be caused by the inability of the nitrogen between the band gamma-phosphates to form a hydrogen bond with the backbone amide of Gly141 as observed in the complex with ATP. Comparisons of nucleotide-bound enzyme and enzyme mutant structure, overview. The guanine-containing nucleotides bind to the monomeric form of RadA and RAD51 with a somewhat higher affinity than the adenine-containing equivalents. Selectivity for ATP over GTP appears to be mediated by the binding of the second protomer, which forms specific interactions with ATP but not with GTP	Pyrococcus horikoshii	?	-	-
additional information	monomeric RadA-ct is able to bind ATP, ADP, AMPPNP, GTP and GDP in an Mg2+-dependent manner. AMP is not able to bind to the protein indicating that both the beta- and gamma-phosphates are essential for this interaction. ADP and GDP bind to RadA with approximately twice the affinity of their triphosphate equivalents, possibly due to the different conformation of the side chain of Phe140 between the di- and triphosphorylated nucleotide complexes, with the additional negative charge of the gamma-phosphate repelling the aromatic ring. AMPPNP binds to mutant RadA-ct, but with reduced affinity in comparison with ATP. This is likely to be caused by the inability of the nitrogen between the band gamma-phosphates to form a hydrogen bond with the backbone amide of Gly141 as observed in the complex with ATP. Comparisons of nucleotide-bound enzyme and enzyme mutant structure, overview. The guanine-containing nucleotides bind to the monomeric form of RadA and RAD51 with a somewhat higher affinity than the adenine-containing equivalents. Selectivity for ATP over GTP appears to be mediated by the binding of the second protomer, which forms specific interactions with ATP but not with GTP	Pyrococcus horikoshii DSM 12428	?	-	-
additional information	monomeric RadA-ct is able to bind ATP, ADP, AMPPNP, GTP and GDP in an Mg2+-dependent manner. AMP is not able to bind to the protein indicating that both the beta- and gamma-phosphates are essential for this interaction. ADP and GDP bind to RadA with approximately twice the affinity of their triphosphate equivalents, possibly due to the different conformation of the side chain of Phe140 between the di- and triphosphorylated nucleotide complexes, with the additional negative charge of the gamma-phosphate repelling the aromatic ring. AMPPNP binds to mutant RadA-ct, but with reduced affinity in comparison with ATP. This is likely to be caused by the inability of the nitrogen between the band gamma-phosphates to form a hydrogen bond with the backbone amide of Gly141 as observed in the complex with ATP. Comparisons of nucleotide-bound enzyme and enzyme mutant structure, overview. The guanine-containing nucleotides bind to the monomeric form of RadA and RAD51 with a somewhat higher affinity than the adenine-containing equivalents. Selectivity for ATP over GTP appears to be mediated by the binding of the second protomer, which forms specific interactions with ATP but not with GTP	Pyrococcus horikoshii NBRC 100139	?	-	-
additional information	monomeric RadA-ct is able to bind ATP, ADP, AMPPNP, GTP and GDP in an Mg2+-dependent manner. AMP is not able to bind to the protein indicating that both the beta- and gamma-phosphates are essential for this interaction. ADP and GDP bind to RadA with approximately twice the affinity of their triphosphate equivalents, possibly due to the different conformation of the side chain of Phe140 between the di- and triphosphorylated nucleotide complexes, with the additional negative charge of the gamma-phosphate repelling the aromatic ring. AMPPNP binds to mutant RadA-ct, but with reduced affinity in comparison with ATP. This is likely to be caused by the inability of the nitrogen between the band gamma-phosphates to form a hydrogen bond with the backbone amide of Gly141 as observed in the complex with ATP. Comparisons of nucleotide-bound enzyme and enzyme mutant structure, overview. The guanine-containing nucleotides bind to the monomeric form of RadA and RAD51 with a somewhat higher affinity than the adenine-containing equivalents. Selectivity for ATP over GTP appears to be mediated by the binding of the second protomer, which forms specific interactions with ATP but not with GTP	Pyrococcus horikoshii JCM 9974	?	-	-
additional information	monomeric RadA-ct is able to bind ATP, ADP, AMPPNP, GTP and GDP in an Mg2+-dependent manner. AMP is not able to bind to the protein indicating that both the beta- and gamma-phosphates are essential for this interaction. ADP and GDP bind to RadA with approximately twice the affinity of their triphosphate equivalents, possibly due to the different conformation of the side chain of Phe140 between the di- and triphosphorylated nucleotide complexes, with the additional negative charge of the gamma-phosphate repelling the aromatic ring. AMPPNP binds to mutant RadA-ct, but with reduced affinity in comparison with ATP. This is likely to be caused by the inability of the nitrogen between the band gamma-phosphates to form a hydrogen bond with the backbone amide of Gly141 as observed in the complex with ATP. Comparisons of nucleotide-bound enzyme and enzyme mutant structure, overview. The guanine-containing nucleotides bind to the monomeric form of RadA and RAD51 with a somewhat higher affinity than the adenine-containing equivalents. Selectivity for ATP over GTP appears to be mediated by the binding of the second protomer, which forms specific interactions with ATP but not with GTP	Pyrococcus horikoshii ATCC 700860	?	-	-
additional information	monomeric RadA-ct is able to bind ATP, ADP, AMPPNP, GTP and GDP in an Mg2+-dependent manner. AMP is not able to bind to the protein indicating that both the beta- and gamma-phosphates are essential for this interaction. ADP and GDP bind to RadA with approximately twice the affinity of their triphosphate equivalents, possibly due to the different conformation of the side chain of Phe140 between the di- and triphosphorylated nucleotide complexes, with the additional negative charge of the gamma-phosphate repelling the aromatic ring. AMPPNP binds to mutant RadA-ct, but with reduced affinity in comparison with ATP. This is likely to be caused by the inability of the nitrogen between the band gamma-phosphates to form a hydrogen bond with the backbone amide of Gly141 as observed in the complex with ATP. Comparisons of nucleotide-bound enzyme and enzyme mutant structure, overview. The guanine-containing nucleotides bind to the monomeric form of RadA and RAD51 with a somewhat higher affinity than the adenine-containing equivalents. Selectivity for ATP over GTP appears to be mediated by the binding of the second protomer, which forms specific interactions with ATP but not with GTP	Pyrococcus horikoshii OT-3	?	-	-

Subunits

Subunits	Comment	Organism
More	the C-terminal ATPase domain RadA is monomeric, analysis of binding of ATP and other nucleotides to nonoligomeric RadA	Pyrococcus horikoshii

Synonyms

Synonyms	Comment	Organism
Rad51	-	Homo sapiens
RadA	-	Pyrococcus horikoshii

Temperature Stability [°C]

Temperature Stability Minimum [°C]	Temperature Stability Maximum [°C]	Comment	Organism
81	-	recombinant mutant HumRadA2 (I169M/Y201A/V202Y/E219S/D220A/K221M)	Pyrococcus horikoshii
95	-	full-lengt and truncated RadA-ct prove to be extremely thermostable and fail to denature at a temperature of 95°C	Pyrococcus horikoshii

General Information

General Information	Comment	Organism
additional information	comparisons of nucleotide-bound enzyme and enzyme mutant structure, overview. Nucleotide binding might have an allosteric and/or co-operative effect on the binding to FxxA sequences and plays an additional role in regulating the oligomeric structures of the recombinase	Homo sapiens
additional information	comparisons of nucleotide-bound enzyme and enzyme mutant structure, overview. Nucleotide binding might have an allosteric and/or co-operative effect on the binding to FxxA sequences and plays an additional role in regulating the oligomeric structures of the recombinase	Pyrococcus horikoshii