Nucleotide-dependent formation of catalytically competent dimers from engineered monomeric ribonucleotide reductase protein R1.

Each catalytic turnover by aerobic ribonucleotide reductase requires the assembly of the two proteins, R1 (alpha(2)) and R2 (beta(2)), to produce deoxyribonucleotides for DNA synthesis. The R2 protein forms a tight dimer, whereas the strength of the R1 dimer differs between organisms, being monomeric in mouse R1 and dimeric in Escherichia coli. We have used the known E. coli R1 structure as a framework for design of eight different mutations that affect the helices and proximal loops that comprise the dimer interaction area. Mutations in loop residues did not affect dimerization, whereas mutations in the helices had very drastic effects on the interaction resulting in monomeric proteins with very low or no activity. The monomeric N238A protein formed an interesting exception, because it unexpectedly was able to reduce ribonucleotides with a comparatively high capacity. Gel filtration studies revealed that N238A was able to dimerize when bound by both substrate and effector, a result in accordance with the monomeric R1 protein from mouse. The effects of the N238A mutation, fit well with the notion that E. coli protein R1 has a comparatively small dimer interaction surface in relation to its size, and the results illustrate the stabilization effects of substrates and effectors in the dimerization process. The identification of key residues in the dimerization process and the fact that there is little sequence identity between the interaction areas of the mammalian and the prokaryotic enzymes may be of importance in drug design, similar to the strategy used in treatment of HSV infection.

Enhancement by effectors and substrate nucleotides of R1-R2 interactions in Escherichia coli class Ia ribonucleotide reductase.

Ribonucleotide reductases are a family of essential enzymes that catalyze the reduction of ribonucleotides to their corresponding deoxyribonucleotides and provide cells with precursors for DNA synthesis. The different classes of ribonucleotide reductase are distinguished based on quaternary structures and enzyme activation mechanisms, but the components harboring the active site region in each class are evolutionarily related. With a few exceptions, ribonucleotide reductases are allosterically regulated by nucleoside triphosphates (ATP and dNTPs). We have used the surface plasmon resonance technique to study how allosteric effects govern the strength of quaternary interactions in the class Ia ribonucleotide reductase from Escherichia coli, which like all class I enzymes has a tetrameric alpha(2) beta(2) structure. The component alpha(2)called R1 harbors the active site and two types of binding sites for allosteric effector nucleotides, whereas the beta(2) component called R2 harbors the tyrosyl radical necessary for catalysis. Our results show that only the known allosteric effector nucleotides, but not non-interacting nucleotides, promote a specific interaction between R1 and R2. Interestingly, the presence of substrate together with allosteric effector nucleotide strengthens the complex 2-3 times with a similar free energy change as the mutual allosteric effects of substrate and effector nucleotide binding to protein R1 in solution experiments. The dual allosteric effects of dATP as positive allosteric effector at low concentrations and as negative allosteric effector at high concentrations coincided with an almost 100-fold stronger R1-R2 interaction. Based on the experimental setup, we propose that the inhibition of enzyme activity in the E. coli class Ia enzyme occurs in a tight 1:1 complex of R1 and R2. Most intriguingly, we also discovered that thioredoxin, one of the physiological reductants of ribonucleotide reductases, enhances the R1-R2 interaction 4-fold.

Mutant R1 proteins from Escherichia coli class Ia ribonucleotide reductase with altered responses to dATP inhibition.

Aerobic ribonucleotide reductase from Escherichia coli regulates its level of activity by binding of effectors to an allosteric site in R1, located to the proposed interaction area of the two proteins that comprise the class I enzyme. Activity is increased by ATP binding and decreased by dATP binding. To study the mechanism governing this regulation, we have constructed three R1 proteins with mutations at His-59 in the activity site and one R1 protein with a mutation at His-88 close to the activity site and compared their allosteric behavior to that of the wild type R1 protein. All mutant proteins retained about 70% of wild type enzymatic activity. We found that if residue His-59 was replaced with alanine or asparagine, the enzyme lost its normal response to the inhibitory effect of dATP, whereas the enzyme with a glutamine still managed to elicit a normal response. We saw a similar result if residue His-88, which is proposed to hydrogen-bond to His-59, was replaced with alanine. Nucleotide binding experiments ruled out the possibility that the effect is due to an inability of the mutant proteins to bind effector since little difference in binding constants was observed for wild type and mutant proteins. Instead, the interaction between proteins R1 and R2 was perturbed in the mutant proteins. We propose that His-59 is important in the allosteric effect triggered by dATP binding, that the conserved hydrogen bond between His-59 and His-88 is important for the communication of the allosteric effect, and that this effect is exerted on the R1/R2 interaction.