Mutational and structural analyses of the ribonucleotide reductase inhibitor Sml1 define its Rnr1 interaction domain whose inactivation allows suppression of mec1 and rad53 lethality.
In budding yeast, MEC1 and RAD53 are essential for cell growth. Previously we reported that mec1 or 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 rad53 inviability. Interestingly, all seven mutations abolish the Sml1 interaction with Rnr1, suggesting that this interaction causes the lethality observed in mec1 and rad53 strains. 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.
Zhao
X
X
Department of Genetics & Development, Columbia University, College of Physicians & Surgeons, New York, New York 10032, USA.
Georgieva
B
B
Chabes
A
A
Domkin
V
V
Ippel
J H
JH
Schleucher
J
J
Wijmenga
S
S
Thelander
L
L
Rothstein
R
R
D018797:Cell Cycle Proteins; D064447:Checkpoint Kinase 2; D015825:Chromosomes, Fungal; D004252:DNA Mutational Analysis; D004791:Enzyme Inhibitors; D005656:Fungal Proteins / Q000235:genetics*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D020125:Mutation, Missense; D011485:Protein Binding; D011494:Protein Kinases / Q000235:genetics*; D017346:Protein Serine-Threonine Kinases; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012264:Ribonucleotide Reductases / Q000037:antagonists & inhibitors*; D029701:Saccharomyces cerevisiae Proteins; D012996:Solutions; D013045:Species Specificity; D013489:Suppression, Genetic; D000071164:Trefoil Factor-2; D020798:Two-Hybrid System Techniques
D018797:Cell Cycle Proteins; D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D047908:Intracellular Signaling Peptides and Proteins; C115052:SML1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D012996:Solutions; C000604007:TFF2 protein, human; D000071164:Trefoil Factor-2; D012264:Ribonucleotide Reductases; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; C088937:MEC1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae