Mutational and Structural Analyses of the Ribonucleotide Reductase Inhibitor Sml1 Define Its Rnr1 Interaction Domain Whose Inactivation Allows Suppression of mec1 andrad53 Lethality

Abstract

In budding yeast, MEC1 and RAD53 are essential for cell growth. Previously we reported that mec1or rad53 lethality is suppressed by removal of Sml1, a protein that binds to the large subunit of ribonucleotide reductase (Rnr1) and inhibits RNR activity. To understand further the relationship between this suppression and the Sml1-Rnr1 interaction, we randomly mutagenized the SML1 open reading frame. Seven mutations were identified that did not affect protein expression levels but relieved mec1 and rad53inviability. Interestingly, all seven mutations abolish the Sml1 interaction with Rnr1, suggesting that this interaction causes the lethality observed in mec1 and rad53strains. The mutant residues all cluster within the 33 C-terminal amino acids of the 104-amino-acid-long Sml1 protein. Four of these residues reside within an alpha-helical structure that was revealed by nuclear magnetic resonance studies. Moreover, deletions encompassing the N-terminal half of Sml1 do not interfere with its RNR inhibitory activity. Finally, the seven sml1 mutations also disrupt the interaction with yeast Rnr3 and human R1, suggesting a conserved binding mechanism between Sml1 and the large subunit of RNR from different species.

ACKNOWLEDGMENTS

We are grateful to Marisa Wagner for critically reading the manuscript.

This work was supported by National Institutes of Health grant GM50237 (R.R.), by the Alexander and Margaret Stewart Trust Pilot Project in Cancer Research (X.Z. and R.R.), by the Swedish Natural Sciences Research Council (S.W. and L.T.), and by The Royal Swedish Academy of Sciences (V.D.).