The structural basis for peptidomimetic inhibition of eukaryotic ribonucleotide reductase: a conformationally flexible pharmacophore

Abstract

Eukaryotic ribonucleotide reductase (RR) catalyzes nucleoside diphosphate conversion to deoxynucleoside diphosphate. Crucial for rapidly dividing cells, RR is a target for cancer therapy. RR activity requires formation of a complex between subunits R1 and R2 in which the R2 C-terminal peptide binds to R1. Here we report crystal structures of heterocomplexes containing mammalian R2 C-terminal heptapeptide, P7 (Ac-1FTLDADF7) and its peptidomimetic P6 (1Fmoc(Me)PhgLDChaDF7) bound to Saccharomyces cerevisiae R1 (ScR1). P7 and P6, both of which inhibit ScRR, each bind at two contiguous sites containing residues that are highly conserved among eukaryotes. Such binding is quite distinct from that reported for prokaryotes. The Fmoc group in P6 peptide makes several hydrophobic interactions that contribute to its enhanced potency in binding to ScR1. Combining all of our results, we observe three distinct conformations for peptide binding to ScR1. These structures provide pharmacophores for designing highly potent nonpeptide class I RR inhibitors.

Figure. 1

ScR1-P7 complex. (a) Stereo view of the 2Fo-Fc difference Fourier electron density map contoured at 1 σ, displayed in blue. The peptide is color coded as carbon yellow, nitrogen blue and oxygen red. (b-c) The electrostatic potential surface showing the R2 binding site on R1. Left: Overall electrostatic surface of the protein. Right: A zoom view of the electrostatic surface of the P7 binding site that includes pockets A and B. Red indicates negative surface charges, blue indicates positive surface charges, and gray represents uncharged surfaces. Peptide residues (carbon atoms, yellow; oxygens, red; and nitrogens, blue). (d) Stereo pictures of the interactions between ScR1 and P7. Hydrogen bonds are shown in blue dash lines and salt-bridges in red. (e) ScR2 peptide superimposed on P7. The peptide is drawn from right to left. P7 is drawn in yellow and ScR2 peptide in cyan. Only P7 is labeled in red. Nearby helices are drawn from the P7-R1 complex (green).

Figure. 2

ScR1-P6 complex. (a) Stereo view of the 2Fo-Fc difference Fourier map contoured at 0.7 σ, displayed in blue. The peptide is color coded as carbon orange, nitrogen blue and oxygen red. (b) Stereo view showing the interactions between R1 and P6. (c) P6 superimposed with P7. Carbon atoms for P7 (cyan) and P6 (orange) and nearby helices are drawn from the P7-R1 complex (blue). The alternative conformation of the P6 Fmoc group is shown in green in all the figures. Only P6 is labeled in red.

Figure. 3

(a) Structural comparison of P7, P6 and ScR4 peptide binding to ScR1. Carbon atoms for P7 (yellow), P6 (magenta) and ScR4 (cyan). Surface is drawn from the ScR1-P6 complex. (b) Stereo view of P7 superposed with S. typhimurium R2. S. typhimurium is drawn in orange and its R2pep in cyan, while ScR1 in green and P7 is in yellow. Labels for S. typhimurium R1 are shown in parenthesis.

Scheme 1