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Crystal structures of IspF from Plasmodium falciparum and Burkholderia cenocepacia: comparisons inform antimicrobial drug target assessment.

O'Rourke PE, Kalinowska-Tłuścik J, Fyfe PK, Dawson A, Hunter WN - BMC Struct. Biol. (2014)

Bottom Line: High-resolution crystal structures of IspF from two important human pathogens have been obtained and compared to orthologues.Ligand binding appears to order a part of the active site involved in substrate recognition.The high degree of structural conservation in and around the IspF active site suggests that any structural model might be suitable to support a program of structure-based drug discovery.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee, DD1 5EH, UK. w.n.hunter@dundee.ac.uk.

ABSTRACT

Background: 2C-methyl-D-erythritol-2,4-cyclodiphosphate synthase (IspF) catalyzes the conversion of 4-diphosphocytidyl-2C-methyl-D-erythritol-2-phosphate to 2C-methyl-D-erythritol-2,4-cyclodiphosphate and cytidine monophosphate in production of isoprenoid-precursors via the methylerythritol phosphate biosynthetic pathway. IspF is found in the protozoan Plasmodium falciparum, a parasite that causes cerebral malaria, as well as in many Gram-negative bacteria such as Burkholderia cenocepacia. IspF represents a potential target for development of broad-spectrum antimicrobial drugs since it is proven or inferred as essential in these pathogens and absent from mammals. Structural studies of IspF from these two important yet distinct pathogens, and comparisons with orthologues have been carried out to generate reagents, to support and inform a structure-based approach to early stage drug discovery.

Results: Efficient recombinant protein production and crystallization protocols were developed, and high-resolution crystal structures of IspF from P. falciparum (Emphasis/Emphasis>IspF) and B. cenocepacia (BcIspF) in complex with cytidine nucleotides determined. Comparisons with orthologues, indicate a high degree of order and conservation in parts of the active site where Zn2+ is bound and where recognition of the cytidine moiety of substrate occurs. However, conformational flexibility is noted in that area of the active site responsible for binding the methylerythritol component of substrate. Unexpectedly, one structure of BcIspF revealed two molecules of cytidine monophosphate in the active site, and another identified citrate coordinating to the catalytic Zn2+. In both cases interactions with ligands appear to help order a flexible loop at one side of the active site. Difficulties were encountered when attempting to derive complex structures with other ligands.

Conclusions: High-resolution crystal structures of IspF from two important human pathogens have been obtained and compared to orthologues. The studies reveal new data on ligand binding, with citrate coordinating to the active site Zn2+ and when present in high concentrations cytidine monophosphate displays two binding modes in the active site. Ligand binding appears to order a part of the active site involved in substrate recognition. The high degree of structural conservation in and around the IspF active site suggests that any structural model might be suitable to support a program of structure-based drug discovery.

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Key residues and interactions to bind CMP 2 in BcIspF. The color scheme is the same as used in Figure 5.
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Figure 6: Key residues and interactions to bind CMP 2 in BcIspF. The color scheme is the same as used in Figure 5.

Mentions: Surprisingly, a second molecule of CMP was observed in the active site when BcIspF was co-crystallized in the presence of 10 mM ligand. This second CMP is likely due to the high concentration used and represents an artifact of crystallization, unlikely to have physiological significance. This new cytidine-binding site is referred to as position 2 and residues that bind this ligand are from the same subunit that binds the active site Zn2+, indeed the CMP 2 phosphate coordinates the metal ion (Figure 5). The binding of CMP 2 within the three active sites of the asymmetric unit is very similar, for example the main chain conformation of the α2-α3 loop and the positions of Leu78 and His36 interacting with van der Waals forces on either side of the pyrimidine and helping to position it is essentially the same. In addition the position of Phe63 that helps to position Leu78 is also conserved. Phe63 is strictly conserved in 96% of IspF sequences, Leu78 in 77%. However, there are differences in the orientation of two side chains that change the detail of the hydrogen bonding interactions with CMP 2. In active site A (Figure 6) two solvent mediated links are noted involving the carbonyl groups of Ser64, Phe70 and Ala73 and cytosine O2 and N3. A direct hydrogen bond is formed between N4 and the Ala73 carbonyl. The Phe70 and Ala73 carbonyl groups position a water molecule that binds cytosine N3 and the Ala73 carbonyl also interacts directly with cytosine N4. The carboxylate of Asp65 binds to the ribose O2’. In the other active sites (not shown), different rotamers of Ser37 place the side chain OG to accept a hydrogen bond from N4 but for Asp65 the rotamer orientations results in functional groups too distant for an interaction with the ribose. Overlay with the EcIspF-product complex shows that CMP 2 binds in the same location as MEcPP [20] and indeed exploits interactions with the same residues, the majority of which are conserved (Figures 3 and 7). Specifically, in common with observations regarding recognition of the pyrimidine moiety of substrates we note that residues that use side chain functional groups to interact with ligands in the MEcPP binding site are well conserved, those that use main chain groups less so. Ser37 is strictly conserved and Asp65 occurs in about 70% of IspF sequences but Ser64, Phe70 and Ala73 are strictly conserved in 4%, 43% and 58% respectively. The latter two residues are not conserved in PfIspF, being replaced by asparagine and lysine respectively (Figure 3).


Crystal structures of IspF from Plasmodium falciparum and Burkholderia cenocepacia: comparisons inform antimicrobial drug target assessment.

O'Rourke PE, Kalinowska-Tłuścik J, Fyfe PK, Dawson A, Hunter WN - BMC Struct. Biol. (2014)

Key residues and interactions to bind CMP 2 in BcIspF. The color scheme is the same as used in Figure 5.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3927217&req=5

Figure 6: Key residues and interactions to bind CMP 2 in BcIspF. The color scheme is the same as used in Figure 5.
Mentions: Surprisingly, a second molecule of CMP was observed in the active site when BcIspF was co-crystallized in the presence of 10 mM ligand. This second CMP is likely due to the high concentration used and represents an artifact of crystallization, unlikely to have physiological significance. This new cytidine-binding site is referred to as position 2 and residues that bind this ligand are from the same subunit that binds the active site Zn2+, indeed the CMP 2 phosphate coordinates the metal ion (Figure 5). The binding of CMP 2 within the three active sites of the asymmetric unit is very similar, for example the main chain conformation of the α2-α3 loop and the positions of Leu78 and His36 interacting with van der Waals forces on either side of the pyrimidine and helping to position it is essentially the same. In addition the position of Phe63 that helps to position Leu78 is also conserved. Phe63 is strictly conserved in 96% of IspF sequences, Leu78 in 77%. However, there are differences in the orientation of two side chains that change the detail of the hydrogen bonding interactions with CMP 2. In active site A (Figure 6) two solvent mediated links are noted involving the carbonyl groups of Ser64, Phe70 and Ala73 and cytosine O2 and N3. A direct hydrogen bond is formed between N4 and the Ala73 carbonyl. The Phe70 and Ala73 carbonyl groups position a water molecule that binds cytosine N3 and the Ala73 carbonyl also interacts directly with cytosine N4. The carboxylate of Asp65 binds to the ribose O2’. In the other active sites (not shown), different rotamers of Ser37 place the side chain OG to accept a hydrogen bond from N4 but for Asp65 the rotamer orientations results in functional groups too distant for an interaction with the ribose. Overlay with the EcIspF-product complex shows that CMP 2 binds in the same location as MEcPP [20] and indeed exploits interactions with the same residues, the majority of which are conserved (Figures 3 and 7). Specifically, in common with observations regarding recognition of the pyrimidine moiety of substrates we note that residues that use side chain functional groups to interact with ligands in the MEcPP binding site are well conserved, those that use main chain groups less so. Ser37 is strictly conserved and Asp65 occurs in about 70% of IspF sequences but Ser64, Phe70 and Ala73 are strictly conserved in 4%, 43% and 58% respectively. The latter two residues are not conserved in PfIspF, being replaced by asparagine and lysine respectively (Figure 3).

Bottom Line: High-resolution crystal structures of IspF from two important human pathogens have been obtained and compared to orthologues.Ligand binding appears to order a part of the active site involved in substrate recognition.The high degree of structural conservation in and around the IspF active site suggests that any structural model might be suitable to support a program of structure-based drug discovery.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee, DD1 5EH, UK. w.n.hunter@dundee.ac.uk.

ABSTRACT

Background: 2C-methyl-D-erythritol-2,4-cyclodiphosphate synthase (IspF) catalyzes the conversion of 4-diphosphocytidyl-2C-methyl-D-erythritol-2-phosphate to 2C-methyl-D-erythritol-2,4-cyclodiphosphate and cytidine monophosphate in production of isoprenoid-precursors via the methylerythritol phosphate biosynthetic pathway. IspF is found in the protozoan Plasmodium falciparum, a parasite that causes cerebral malaria, as well as in many Gram-negative bacteria such as Burkholderia cenocepacia. IspF represents a potential target for development of broad-spectrum antimicrobial drugs since it is proven or inferred as essential in these pathogens and absent from mammals. Structural studies of IspF from these two important yet distinct pathogens, and comparisons with orthologues have been carried out to generate reagents, to support and inform a structure-based approach to early stage drug discovery.

Results: Efficient recombinant protein production and crystallization protocols were developed, and high-resolution crystal structures of IspF from P. falciparum (Emphasis/Emphasis>IspF) and B. cenocepacia (BcIspF) in complex with cytidine nucleotides determined. Comparisons with orthologues, indicate a high degree of order and conservation in parts of the active site where Zn2+ is bound and where recognition of the cytidine moiety of substrate occurs. However, conformational flexibility is noted in that area of the active site responsible for binding the methylerythritol component of substrate. Unexpectedly, one structure of BcIspF revealed two molecules of cytidine monophosphate in the active site, and another identified citrate coordinating to the catalytic Zn2+. In both cases interactions with ligands appear to help order a flexible loop at one side of the active site. Difficulties were encountered when attempting to derive complex structures with other ligands.

Conclusions: High-resolution crystal structures of IspF from two important human pathogens have been obtained and compared to orthologues. The studies reveal new data on ligand binding, with citrate coordinating to the active site Zn2+ and when present in high concentrations cytidine monophosphate displays two binding modes in the active site. Ligand binding appears to order a part of the active site involved in substrate recognition. The high degree of structural conservation in and around the IspF active site suggests that any structural model might be suitable to support a program of structure-based drug discovery.

Show MeSH
Related in: MedlinePlus