Structural basis on the dityrosyl-diiron radical cluster and the functional differences of human ribonucleotide reductase small subunits hp53R2 and hRRM2

Abstract

Ribonucleotide reductase (RNR) is an enzyme for the de novo conversion of ribonucleotides to deoxyribonucleotides. The two human RNR small subunits hRRM2 and hp53R2 share 83% sequence homology but show distinct expression patterns and function. Structural analyses of the oxidized form of hRRM2 and hp53R2 indicate that both proteins contain a conserved Gln127-hp53R2/Gln165-hRRM2 close to the dinuclear iron center and the essential tyrosine residue Tyr124-hp53R2/Tyr162-hRRM2 forms hydrogen bonds with the tyrosine and iron ligands, implying a critical role for the glutamine residue in assembling the dityrosyl-diiron radical cofactor. The present work also showed that Tyr221 in hRRM2, which is replaced by Phe183 in hp53R2, forms a hydrogen bond with Tyr162 to extend the hydrogen bond network from Gln165-hRRM2. Mutagenesis and spectroscopic experiments suggested that the tyrosine-to-phenylalanine switch at Phe183-hp53R2/Tyr221-hRRM2 could lead to differences in radical generation or enzymatic activity for hp53R2 and hRRM2. This study correlates the distinct catalytic mechanisms of the small subunits hp53R2 and hRRM2 with a hydrogen-bonding network and provides novel directions for designing and developing subunit-specific therapeutic agents for human RNR enzymes.

Figure 1

A) The dityrosyl-diiron center of hRRM2 (PDB ID 2UW2) is shown with the protein secondary structures at the background. Two iron atoms are colored red. The iron ligands and Q165 (highlighted as ball-and-stick) are shown. B) The hydrogen bond network of Q165 is depicted. Hydrogen bonds are colored yellow and labeled with bond lengths. Distances between Q165 to Y162 and Fe2 are marked in green. The secondary structures with the residues attached are shown. Five residues Y176, H172, E169, Q165, and Y162 are at five continuous helical turns on C-helix. C) Comparison of the residues surrounding Y124/162 of hp53R2/hRRM2. In hp53R2, Y124 and Q127 are colored yellow; F183 in magenta. The other residues are colored cyan. Residues in hRRM2 are colored by atom types (carbon – white, oxygen – red, hydrogen – cyan, and nitrogen – blue). Y124/Y162, Q127/Q165, F183/Y221 are highlighted in stick rendering. Residues were labeled as hp53R2/hRRM2. D) Hydrogen bond networks around Y124-hp53R2 and Y162-hRRM2.

Figure 2

Circular dichroism spectra of human RRM2 and mutants (A), p53R2 and the mutants (B), comparison of hRRM2 and hp53R2 spectrum (C), recorded at 37°C in 50 mM Tris buffer (pH 7.4) using a 0.01 cm path length cell. Each spectrum shown represents the average of five.

Figure 3

A) EPR spectra of hp53R2 and hRRM2 wild types and the mutants Q127V, Q127K, Q127E, Q127N, and Q165V, Q165K, Q165E, Q165N. B) EPR spectra of wide types and mutants F183Y, Y124W, Y124W/F183Y in hp53R2 and Y221F, Y162W, Y162W/Y221F in hRRM2. EPR spectra of human RRM2 and p53R2 proteins recorded at 20K in a Bruker 300E spectrometer with microwave power 0.1 mW, microwave frequency 9.375 GHz, and modulation amplitude 2.0 G.

Figure 3

A) EPR spectra of hp53R2 and hRRM2 wild types and the mutants Q127V, Q127K, Q127E, Q127N, and Q165V, Q165K, Q165E, Q165N. B) EPR spectra of wide types and mutants F183Y, Y124W, Y124W/F183Y in hp53R2 and Y221F, Y162W, Y162W/Y221F in hRRM2. EPR spectra of human RRM2 and p53R2 proteins recorded at 20K in a Bruker 300E spectrometer with microwave power 0.1 mW, microwave frequency 9.375 GHz, and modulation amplitude 2.0 G.