Table 5 Binding affinity data of NADPH and TMP to EcDHFR, DfrA1 and DfrA5 by microscale thermophoresis (MST).

From: Structure-guided functional studies of plasmid-encoded dihydrofolate reductases reveal a common mechanism of trimethoprim resistance in Gram-negative pathogens

Complex

Titrant

KD (nM)

Fold change

NADPH KD ± Trimethoprim

 

EcDHFR apo

NADPH

771 ± 145

̶

EcDHFR:TMP

NADPH

8.1 ± 4.0

95.2 ± 50.3

DfrA1 apo

NADPH

1650 ± 230

̶

DfrA1:TMP

NADPH

430 ± 95

3.8 ± 1.0

DfrA5 apo

NADPH

7470 ± 1670

̶

DfrA5:TMP

NADPH

1540 ± 380

4.9 ± 1.6

Trimethoprim KD ± NADPH

EcDHFR apo

TMP

23.6 ± 5.1

-

EcDHFR:NADPH

TMP

0.011 ± 0.007

2145.5 ± 1441

DfrA1 apo

TMP

27,792 ± 4877

̶

DfrA1:NADPH

TMP

1308 ± 451

21.2 ± 8.2

DfrA5 apo

TMP

11,929 ± 4448

̶

DfrA5:NADPH

TMP

113 ± 56

105.6 ± 65.5

NADPH KD ± UCP1228

DfrA1 apo

NADPH

1044 ± 106

̶

DfrA1:UCP1228

NADPH

289 ± 51

3.6 ± 0.7

DfrA5 apo

NADPH

6837 ± 1608

̶

DfrA5:UCP1228

NADPH

459 ± 157

14.9 ± 6.2

  1. Binding response of the fluorescently labeled dihydrofolate reductases, pre and post incubation step to the increasing concentration of the non-fluorescent ligand. The Data were fitted to a single-site binding model accounting for ligand depletion. Binding constants, KD ± KD confidence (±68% confidence) and the fold change in KD value ± relative error on the fold change were determined from two independent measurements using MO Affinity Analysis v2.1.3 software (NanoTemper Technologies GmbH). Each binding experiment was prepared independently, in parallel using the same preparation of proteins.
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