1lvu: Difference between revisions

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Crystal structure of calf spleen purine nucleoside phosphorylase in a new space group with full trimer in the asymmetric unit

OverviewOverview

The crystal structure of the binary complex of trimeric purine nucleoside phosphorylase (PNP) from calf spleen with the acyclic nucleoside phosphonate inhibitor 2,6-diamino-(S)-9-[2-(phosphonomethoxy)propyl]purine ((S)-PMPDAP) is determined at 2.3A resolution in space group P2(1)2(1)2(1). Crystallization in this space group, which is observed for the first time with a calf spleen PNP crystal structure, is obtained in the presence of calcium atoms. In contrast to the previously described cubic space group P2(1)3, two independent trimers are observed in the asymmetric unit, hence possible differences between monomers forming the biologically active trimer could be detected, if present. Such differences would be expected due to third-of-the-sites binding documented for transition-state events and inhibitors. However, no differences are noted, and binding stoichiometry of three inhibitor molecules per enzyme trimer is observed in the crystal structure, and in the parallel solution studies using isothermal titration calorimetry and spectrofluorimetric titrations. Presence of phosphate was shown to modify binding stoichiometry of hypoxanthine. Therefore, the enzyme was also crystallized in space group P2(1)2(1)2(1) in the presence of (S)-PMPDAP and phosphate, and the resulting structure of the binary PNP/(S)-PMPDAP complex was refined at 2.05A resolution. No qualitative differences between complexes obtained with and without the presence of phosphate were detected, except for the hydrogen bond contact of Arg84 and a phosphonate group, which is observed only in the former complex in three out of six independent monomers. Possible hydrogen bonds observed in the enzyme complexed with (S)-PMPDAP, in particular a putative hydrogen bonding contact N(1)-H cdots, three dots, centered Glu201, indicate that the inhibitor binds in a tautomeric or ionic form in which position N(1) acts as a hydrogen bond donor. This points to a crucial role of this hydrogen bond in defining specificity of trimeric PNPs and is in line with the proposed mechanism of catalysis in which this contact helps to stabilize the negative charge that accumulates on O(6) of the purine base in the transition state. In the present crystal structure the loop between Thr60 and Ala65 was found in a different conformation than that observed in crystal structures of trimeric PNPs up to now. Due to this change a new wide entrance is opened into the active site pocket, which is otherwise buried in the interior of the protein. Hence, our present crystal structure provides no obvious indication for obligatory binding of one of the substrates before binding of a second one; it is rather consistent with random binding of substrates. All these results provide new data for clarifying the mechanism of catalysis and give reasons for the non-Michaelis kinetics of trimeric PNPs.

About this StructureAbout this Structure

1LVU is a Single protein structure of sequence from Bos taurus with and as ligands. Active as Purine-nucleoside phosphorylase, with EC number 2.4.2.1 Full crystallographic information is available from OCA.

ReferenceReference

Crystal structure of calf spleen purine nucleoside phosphorylase with two full trimers in the asymmetric unit: important implications for the mechanism of catalysis., Bzowska A, Koellner G, Wielgus-Kutrowska B, Stroh A, Raszewski G, Holy A, Steiner T, Frank J, J Mol Biol. 2004 Sep 17;342(3):1015-32. PMID:15342253

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-[[Image:1lvu.gif|left|200px]]<br /><applet load="1lvu" size="450" color="white" frame="true" align="right" spinBox="true"
+[[Image:1lvu.gif|left|200px]]<br /><applet load="1lvu" size="350" color="white" frame="true" align="right" spinBox="true"
 caption="1lvu, resolution 2.05&Aring;" />
 '''Crystal structure of calf spleen purine nucleoside phosphorylase in a new space group with full trimer in the asymmetric unit'''<br />
 ==Overview==
-The crystal structure of the binary complex of trimeric purine nucleoside, phosphorylase (PNP) from calf spleen with the acyclic nucleoside, phosphonate inhibitor 2,6-diamino-(S)-9-[2-(phosphonomethoxy)propyl]purine, ((S)-PMPDAP) is determined at 2.3A resolution in space group, P2(1)2(1)2(1). Crystallization in this space group, which is observed for, the first time with a calf spleen PNP crystal structure, is obtained in, the presence of calcium atoms. In contrast to the previously described, cubic space group P2(1)3, two independent trimers are observed in the, asymmetric unit, hence possible differences between monomers forming the, biologically active trimer could be detected, if present. Such differences, would be expected due to third-of-the-sites binding documented for, transition-state events and inhibitors. However, no differences are noted, and binding stoichiometry of three inhibitor molecules per enzyme trimer, is observed in the crystal structure, and in the parallel solution studies, using isothermal titration calorimetry and spectrofluorimetric titrations., Presence of phosphate was shown to modify binding stoichiometry of, hypoxanthine. Therefore, the enzyme was also crystallized in space group, P2(1)2(1)2(1) in the presence of (S)-PMPDAP and phosphate, and the, resulting structure of the binary PNP/(S)-PMPDAP complex was refined at, 2.05A resolution. No qualitative differences between complexes obtained, with and without the presence of phosphate were detected, except for the, hydrogen bond contact of Arg84 and a phosphonate group, which is observed, only in the former complex in three out of six independent monomers., Possible hydrogen bonds observed in the enzyme complexed with (S)-PMPDAP, in particular a putative hydrogen bonding contact N(1)-H cdots, three, dots, centered Glu201, indicate that the inhibitor binds in a tautomeric, or ionic form in which position N(1) acts as a hydrogen bond donor. This, points to a crucial role of this hydrogen bond in defining specificity of, trimeric PNPs and is in line with the proposed mechanism of catalysis in, which this contact helps to stabilize the negative charge that accumulates, on O(6) of the purine base in the transition state. In the present crystal, structure the loop between Thr60 and Ala65 was found in a different, conformation than that observed in crystal structures of trimeric PNPs up, to now. Due to this change a new wide entrance is opened into the active, site pocket, which is otherwise buried in the interior of the protein., Hence, our present crystal structure provides no obvious indication for, obligatory binding of one of the substrates before binding of a second, one; it is rather consistent with random binding of substrates. All these, results provide new data for clarifying the mechanism of catalysis and, give reasons for the non-Michaelis kinetics of trimeric PNPs.
+The crystal structure of the binary complex of trimeric purine nucleoside phosphorylase (PNP) from calf spleen with the acyclic nucleoside phosphonate inhibitor 2,6-diamino-(S)-9-[2-(phosphonomethoxy)propyl]purine ((S)-PMPDAP) is determined at 2.3A resolution in space group P2(1)2(1)2(1). Crystallization in this space group, which is observed for the first time with a calf spleen PNP crystal structure, is obtained in the presence of calcium atoms. In contrast to the previously described cubic space group P2(1)3, two independent trimers are observed in the asymmetric unit, hence possible differences between monomers forming the biologically active trimer could be detected, if present. Such differences would be expected due to third-of-the-sites binding documented for transition-state events and inhibitors. However, no differences are noted, and binding stoichiometry of three inhibitor molecules per enzyme trimer is observed in the crystal structure, and in the parallel solution studies using isothermal titration calorimetry and spectrofluorimetric titrations. Presence of phosphate was shown to modify binding stoichiometry of hypoxanthine. Therefore, the enzyme was also crystallized in space group P2(1)2(1)2(1) in the presence of (S)-PMPDAP and phosphate, and the resulting structure of the binary PNP/(S)-PMPDAP complex was refined at 2.05A resolution. No qualitative differences between complexes obtained with and without the presence of phosphate were detected, except for the hydrogen bond contact of Arg84 and a phosphonate group, which is observed only in the former complex in three out of six independent monomers. Possible hydrogen bonds observed in the enzyme complexed with (S)-PMPDAP, in particular a putative hydrogen bonding contact N(1)-H cdots, three dots, centered Glu201, indicate that the inhibitor binds in a tautomeric or ionic form in which position N(1) acts as a hydrogen bond donor. This points to a crucial role of this hydrogen bond in defining specificity of trimeric PNPs and is in line with the proposed mechanism of catalysis in which this contact helps to stabilize the negative charge that accumulates on O(6) of the purine base in the transition state. In the present crystal structure the loop between Thr60 and Ala65 was found in a different conformation than that observed in crystal structures of trimeric PNPs up to now. Due to this change a new wide entrance is opened into the active site pocket, which is otherwise buried in the interior of the protein. Hence, our present crystal structure provides no obvious indication for obligatory binding of one of the substrates before binding of a second one; it is rather consistent with random binding of substrates. All these results provide new data for clarifying the mechanism of catalysis and give reasons for the non-Michaelis kinetics of trimeric PNPs.
 ==About this Structure==
-LVU is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Bos_taurus Bos taurus] with CA and 9PP as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Purine-nucleoside_phosphorylase Purine-nucleoside phosphorylase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.4.2.1 2.4.2.1] Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1LVU OCA].
+LVU is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Bos_taurus Bos taurus] with <scene name='pdbligand=CA:'>CA</scene> and <scene name='pdbligand=9PP:'>9PP</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Purine-nucleoside_phosphorylase Purine-nucleoside phosphorylase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.4.2.1 2.4.2.1] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1LVU OCA].
 ==Reference==
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 [[Category: purie nucleoside phosphorylase]]
-''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Tue Nov 20 20:55:39 2007''
+''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 13:48:55 2008''