Figure 3.

Walker A motif is essential to ATP binding and ATP-dependent dimerization. (A) ATP interacts with the residues in the Walker A motif and signature motif of RecF. All of the interacting residues are represented by sticks (magenta). (B) Extensive interactions between TTERecF and ATP. The plots were generated using LIGPLOT³³. RecF residues and ATP are shown in pink and blue, respectively. H bonds are indicated by dashed lines (green). (C) ITC curves of TTERecF and the mutants of Walker A motif titrated into ATP. The ITC experiments involved 20 injections of 2 µL 1 mM ATP into 300 µL 70 µM RecF native or the mutants. (D) Size-exclusion chromatography analysis of TTERecF, TTERecF-ATP, ATP, G35A-ATP, K36A-ATP, S37A-ATP and N38A-ATP. Size exclusion chromatography was performed using a HiLord 16/60 Superdex 200 column (GE Health Life Sciences) at 0.5 ml/min in 50 mM Tris-HCl pH 7.0 and 300 mM NaCl. Protein elution was monitored by measuring the absorbance at 280 nm.