Subunit and small-molecule interaction of ribonucleotide reductases via surface plasmon resonance biosensor analyses.
Ribonucleotide reductase (RNR) synthesizes deoxyribonucleotides for DNA replication and repair and is controlled by sophisticated allosteric regulation involving differential affinity of nucleotides for regulatory sites. We have developed a robust and sensitive method for coupling biotinylated RNRs to surface plasmon resonance streptavidin biosensor chips via a 30.5 A linker. In comprehensive studies on three RNRs effector nucleotides strengthened holoenzyme interactions, whereas substrate had no effect on subunit interactions. The RNRs differed in their response to the negative allosteric effector dATP that binds to an ATP-cone domain. A tight RNR complex was formed in Escherichia coli class Ia RNR with a functional ATP cone. No strengthening of subunit interactions was observed in the class Ib RNR from the human pathogen Bacillus anthracis that lacks the ATP cone. A moderate strengthening was seen in the atypical Aeromonas hydrophila phage 1 class Ia RNR that has a split catalytic subunit and a non-functional ATP cone with remnant dATP-mediated regulatory features. We also successfully immobilized a functional catalytic NrdA subunit of the E.coli enzyme, facilitating study of nucleotide interactions. Our surface plasmon resonance methodology has the potential to provide biological insight into nucleotide-mediated regulation of any RNR, and can be used for high-throughput screening of potential RNR inhibitors.
Department of Molecular Biology and Functional Genomics, Stockholm University, SE-10691 Stockholm, Sweden.
Mikael
M
Crona
Ernst
E
Furrer
Eduard
E
Torrents
David R
DR
Edgell
Britt-Marie
BM
Sjöberg
D000255:Adenosine Triphosphate / Q000378:metabolism; D000494:Allosteric Regulation; D001408:Bacillus anthracis / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D015374:Biosensing Techniques; D001710:Biotin / Q000737:chemistry / Q000378:metabolism; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D011485:Protein Binding; D012262:Ribonucleoside Diphosphate Reductase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012264:Ribonucleotide Reductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019809:Streptavidin / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance
D001426:Bacterial Proteins; D004800:Enzymes, Immobilized; D029968:Escherichia coli Proteins; D006153:Guanosine Diphosphate; D001710:Biotin; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D019809:Streptavidin; D012264:Ribonucleotide Reductases; C449642:NrdA protein, E coli; D012262:Ribonucleoside Diphosphate Reductase