Inhibitor-bound complexes of dihydrofolate reductase-thymidylate synthase from<i>Babesia bovis</i>

Begley, Darren W.; Edwards, Thomas E.; Raymond, Amy; Smith, Eric R; Hartley, Robert C.; Abendroth, Jan; Sankaran, Banumathi; Lorimer, Donald D.; Myler, Peter J.; Staker, Bart L.; Stewart, Lance

doi:10.1107/s1744309111029009

Cited by 19 publications

(22 citation statements)

References 54 publications

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“…To elucidate a structural basis for the difference in resistance profiles to DHFR inhibitors of the G137D and A71V mutants in DHFR‐TS1 and DHFR‐TS2, respectively, we modelled the structure of A. thaliana DHFR‐TS1 and DHFR‐TS2 using the crystal structure of protozoan Babesia bovis DHFR‐TS (Begley et al ., ) as it shares ~43% sequence similarity (Figure b). The predicted structures suggest both DHFR‐TS1 and DHFR‐TS2 form canonical bifunctional DHFR‐TS enzymes whereby the DHFR domains (residues 21–198 and 64–242, respectively) form an eight‐stranded β‐sheet encased by four short α‐helical regions, linked to a C‐terminal TS domain (residues 223–519 and 271–565, respectively) by a long Class I type junctional region that has been shown to join adjacent DHFR monomers (O'Neil et al ., ).…”

Section: Resultsmentioning

confidence: 98%

Targeting plant DIHYDROFOLATE REDUCTASE with antifolates and mechanisms for genetic resistance

et al. 2018

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The folate biosynthetic pathway and its key enzyme dihydrofolate reductase (DHFR) is a popular target for drug development due to its essential role in the synthesis of DNA precursors and some amino acids. Despite its importance, little is known about plant DHFRs, which, like the enzymes from the malarial parasite Plasmodium, are bifunctional, possessing DHFR and thymidylate synthase (TS) domains. Here using genetic knockout lines we confirmed that either DHFR-TS1 or DHFR-TS2 (but not DHFR-TS3) was essential for seed development. Screening mutated Arabidopsis thaliana seeds for resistance to antimalarial DHFR-inhibitor drugs pyrimethamine and cycloguanil identified causal lesions in DHFR-TS1 and DHFR-TS2, respectively, near the predicted substrate-binding site. The different drug resistance profiles for the plants, enabled by the G137D mutation in DHFR-TS1 and the A71V mutation in DHFR-TS2, were consistent with biochemical studies using recombinant proteins and could be explained by structural models. These findings provide a great improvement in our understanding of plant DHFR-TS and suggest how plant-specific inhibitors might be developed, as DHFR is not currently targeted by commercial herbicides.

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Section: Resultsmentioning

confidence: 98%

Targeting plant DIHYDROFOLATE REDUCTASE with antifolates and mechanisms for genetic resistance

et al. 2018

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“…The high sequence and structural homology across TS enzymes from human and many parasite species, particularly within active site residues, creates a challenge for obtaining drug selectivity [42], [43]. The B. thailandensis TS protein (BTH_I1680, PDB: 3V8H) has an arginine residue substituted for a canonical active site tryptophan (W83 in E. coli ); arginine is also the side chain found in human TS (Figure 4).…”

Section: Resultsmentioning

confidence: 99%

Combining Functional and Structural Genomics to Sample the Essential Burkholderia Structome

et al. 2013

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BackgroundThe genus Burkholderia includes pathogenic gram-negative bacteria that cause melioidosis, glanders, and pulmonary infections of patients with cancer and cystic fibrosis. Drug resistance has made development of new antimicrobials critical. Many approaches to discovering new antimicrobials, such as structure-based drug design and whole cell phenotypic screens followed by lead refinement, require high-resolution structures of proteins essential to the parasite.Methodology/Principal FindingsWe experimentally identified 406 putative essential genes in B. thailandensis, a low-virulence species phylogenetically similar to B. pseudomallei, the causative agent of melioidosis, using saturation-level transposon mutagenesis and next-generation sequencing (Tn-seq). We selected 315 protein products of these genes based on structure-determination criteria, such as excluding very large and/or integral membrane proteins, and entered them into the Seattle Structural Genomics Center for Infection Disease (SSGCID) structure determination pipeline. To maximize structural coverage of these targets, we applied an “ortholog rescue” strategy for those producing insoluble or difficult to crystallize proteins, resulting in the addition of 387 orthologs (or paralogs) from seven other Burkholderia species into the SSGCID pipeline. This structural genomics approach yielded structures from 31 putative essential targets from B. thailandensis, and 25 orthologs from other Burkholderia species, yielding an overall structural coverage for 49 of the 406 essential gene families, with a total of 88 depositions into the Protein Data Bank. Of these, 25 proteins have properties of a potential antimicrobial drug target i.e., no close human homolog, part of an essential metabolic pathway, and a deep binding pocket. We describe the structures of several potential drug targets in detail.Conclusions/SignificanceThis collection of structures, solubility and experimental essentiality data provides a resource for development of drugs against infections and diseases caused by Burkholderia. All expression clones and proteins created in this study are freely available by request.

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“…The lack of an effect of the C21 peptide on the fluorescence signal of T. gondii TS–DHFR in the presence of dUMP may explain why the inhibitory effect of the peptides was selective for the apo‐enzyme state. The crystal structure of the apo‐TS–DHFR from Babesia bovis , similar to the TS–DHFR from T. gondii in domain organization, showed that the apo form of the enzyme had increased disorder in both the TS and DHFR domains as compared to the dUMP‐bound structure . Peptides from human TS were also dependent on the apo‐enzyme state .…”

Section: Discussionmentioning

confidence: 56%

“…The crystal structure of the apo-TS-DHFR from Babesia bovis, similar to the TS-DHFR from T. gondii in domain organization, showed that the apo form of the enzyme had increased disorder in both the TS and DHFR domains as compared to the dUMP-bound structure. 9 Peptides from human TS were also dependent on the apo-enzyme state. 23 This further validates the role dUMP plays in transitioning the TS enzyme into a stable, active conformation.…”

Section: Discussionmentioning

confidence: 99%

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Selective peptide inhibitors of bifunctional thymidylate synthase‐dihydrofolate reductase from Toxoplasma gondii provide insights into domain–domain communication and allosteric regulation

2013

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The bifunctional enzyme thymidylate synthase-dihydrofolate reductase (TS-DHFR) plays an essential role in DNA synthesis and is unique to several species of pathogenic protozoans, including the parasite Toxoplasma gondii. Infection by T. gondii causes the prevalent disease toxoplasmosis, for which TS-DHFR is a major therapeutic target. Here, we design peptides that target the dimer interface between the TS domains of bifunctional T. gondii TS-DHFR by mimicking bstrands at the interface, revealing a previously unknown allosteric target. The current study shows that these b-strand mimetic peptides bind to the apo-enzyme in a species-selective manner to inhibit both the TS and distal DHFR. Fluorescence spectroscopy was used to monitor conformational switching of the TS domain and demonstrate that these peptides induce a conformational change in the enzyme. Using structure-guided mutagenesis, nonconserved residues in the linker between TS and DHFR were identified that play a key role in domain-domain communication and in peptide inhibition of the DHFR domain. These studies validate allosteric inhibition of apo-TS, specifically at the TS-TS interface, as a potential target for novel, species-specific therapeutics for treating T. gondii parasitic infections and overcoming drug resistance.

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Inhibitor-bound complexes of dihydrofolate reductase-thymidylate synthase fromBabesia bovis

Cited by 19 publications

References 54 publications

Targeting plant DIHYDROFOLATE REDUCTASE with antifolates and mechanisms for genetic resistance

Targeting plant DIHYDROFOLATE REDUCTASE with antifolates and mechanisms for genetic resistance

Combining Functional and Structural Genomics to Sample the Essential Burkholderia Structome

Selective peptide inhibitors of bifunctional thymidylate synthase‐dihydrofolate reductase from Toxoplasma gondii provide insights into domain–domain communication and allosteric regulation

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