Mapping the subunit interface of ribonucleotide reductase (RNR) using photo cross-linking

Abstract

Escherichia coli ribonucleotide reductase (RNR) catalyzes the conversion of nucleoside 5'-diphosphates to deoxynucleoside 5'-diphosphates and is a 1:1 complex of two homodimeric subunits: alpha2 and beta2. As a first step towards mapping the subunit interface, beta2 (V365C) was labeled with [(14)C]-benzophenone (BP) iodoacetamide. The resulting [(14)C]-BP-beta2 (V365C) was complexed with alpha2 and irradiated at 365nm for 30min at 4 degrees C. The cross-linked mixture was purified by anion exchange chromatography and digested with trypsin. The peptides were purified by reverse phase chromatography, identified by scintillation counting and analyzed by Edman sequencing. Three [(14)C]-labeled peptides were identified: two contained a peptide in beta to which the BP was attached. The third contained the same beta peptide and a peptide in alpha found in its alphaD helix. These results provide direct support for the proposed docking model of alpha2beta2.

Fig 1

Purification of the photo cross-linked mixture by anion exchange chromatography (POROS 10 HQ) in 50 mM Tris pH 7.6 with the indicated NaCl gradient. The regions I–VI are shown by vertical lines (from left to right). (Inset) SDS-PAGE gel of region I–VI.

Fig 2

Purification of the peptides generated by trypsin digestion of the photo cross-linked mixture with reverse phase (Jupitor®) HPLC in 0.1% TFA/H₂O with the indicated gradient (---) of 0.1% TFA/CH₃CN. The radioactivity of each fraction (1 mL, -■-) is overlaid on the absorbance (—).

Fig 3

The crystal structure of α2 (green) in complex with C-terminal tail (cyan) of β2. The orientation and distances of V365 with respect to S₄₀₀LMMQERAST₄₀₉ (αD, light blue) are shown.