Crystallographic analysis reveals a novel second binding site for trimethoprim in active site double mutants of human dihydrofolate reductase

Vivian Cody, Jim Pace, Jennifer Piraino, Sherry Queener

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

In order to produce a more potent replacement for trimethoprim (TMP) used as a therapy for Pneumocystis pneumonia and targets dihydrofolate reductase from Pneumocystis jirovecii (pjDHFR), it is necessary to understand the determinants of potency and selectivity against DHFR from the mammalian host and fungal pathogen cells. To this end, active site residues in human (h) DHFR were replaced with those from pjDHFR. Structural data are reported for two complexes of TMP with the double mutants Gln35Ser/Asn64Phe (Q35S/N64F) and Gln35Lys/Asn64Phe (Q35K/N64F) of hDHFR that unexpectedly show evidence for the binding of two molecules of TMP: one molecule that binds in the normal folate binding site and the second molecule that binds in a novel subpocket site such that the mutated residue Phe64 is involved in van der Waals contacts to the trimethoxyphenyl ring of the second TMP molecule. Kinetic data for the binding of TMP to hDHFR and pjDHFR reveal an 84-fold selectivity of TMP against pjDHFR (K i 49. nM) compared to hDHFR (K i 4093. nM). Two mutants that contain one substitution from pj- and one from the closely related Pneumocystis carinii DHFR (pcDHFR) (Q35K/N64F and Q35S/N64F) show K i values of 593 and 617. nM, respectively; these K i values are well above both the K i for pjDHFR and are similar to pcDHFR (Q35K/N64F and Q35S/N64F) (305. nM). These results suggest that active site residues 35 and 64 play key roles in determining selectivity for pneumocystis DHFR, but that other residues contribute to the unique binding of inhibitors to these enzymes.

Original languageEnglish
Pages (from-to)52-59
Number of pages8
JournalJournal of Structural Biology
Volume176
Issue number1
DOIs
StatePublished - Oct 2011

Fingerprint

Tetrahydrofolate Dehydrogenase
Trimethoprim
Catalytic Domain
Binding Sites
Pneumocystis carinii
Pneumocystis
Pneumocystis Pneumonia
Enzyme Inhibitors
Folic Acid

Keywords

  • Crystal structure
  • Human dihydrofolate reductase
  • Kinetics
  • Mutagenesis
  • Pneumocystis jirovecii

ASJC Scopus subject areas

  • Structural Biology

Cite this

Crystallographic analysis reveals a novel second binding site for trimethoprim in active site double mutants of human dihydrofolate reductase. / Cody, Vivian; Pace, Jim; Piraino, Jennifer; Queener, Sherry.

In: Journal of Structural Biology, Vol. 176, No. 1, 10.2011, p. 52-59.

Research output: Contribution to journalArticle

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