Crystallization and crystallographic investigations of ribonucleotide reductase protein R1 from Escherichia coli

Abstract

Crystals of Escherichia coli ribonucleotide reductase protein Rl have been grown in complex with a synthetic peptide corresponding to the carboxyl end of protein R2. Good quality crystals could only be obtained after improvement of the purification protocol and are of the space group R32 with hexagonal cell axes a = b = 226 8, and c = 341 A. They contain 3 subunits per asymmetric unit and diffract to 2.5 8, resolution in synchrotron radiation. A multiple isomorphous replacement map at 5.5 A, improved by solvent flattening, shows that the dimeric molecules are elongated, about 110 A long. The dimer is thin in the middle around the molecular two-fold axis. The subunit is shaped like a bowl, probably with the active site in its center.Crystallization; Ribonucleotide reductase; Protein purification; Peptide complex; Crystal packing; Heavy atom derivative I~RDDUCTIONRibonucleotide reductase (RNR) is an essential enzyme for DNA synthesis. It catalyses the reduction of all four ribonucleotides to their corresponding deoxyribonucleotides [l]. In Escherichiu coli and higher organisms RNR consists of two homodimeric unequal proteins Rl and R2 (for reviews see [24]). A catalytically active enzyme is formed by a one to one complex of the two proteins in the presence of Mg" and ATP. The carboxyl end of R2 contributes to a large extent to the binding between the two proteins, and peptides of the carboxyl end of R2 have been shown to inhibit formation of the active holoenzyme [5][6][7][8].Each subunit of the protein Rl has two different effector binding sites for allosteric regulation. One site regulates the overall activity with ATP and dATP as effecters while the other site regulates substrate specificity with deoxyribonucleoside triphosphates as effecters. Rl also contains redox active cysteines which become oxidized during catalytis. To regenerate active enzyme, this disulfide bridge has to be reduced. This can be done by at least two different systems, the glutaredoxin and thioredoxin systems 19).The Rl protein is about twice as large as the R2 protein in most species. In E. co/i, Rl has a molecular weight of 2 x 83 kDa where each peptide consists of 761 amino acid residues [lo]. The smaller R2 protein (2 x 43.4 kDa) contains a tyrosyl free radical close to a binuclear iron center [I 11. *Corresponding author.For several years we have worked on the structure dete~ination of ribonucleotide reductase from E. co&. The main difficulty of this work has been to obtain useful crystals for crystallographic studies. The R 1 protein has been crystallized in at least ten different crystal forms, and R2 in three orthorhombic crystal forms [12]. Most of the crystal forms were not suitable for highresolution crystallography. Crystals of the holoenzyme have been obtained, but they have large cell dimensions and are X-ray sensitive and thus not useful for structure determination. By extensive exploration of different crystallization conditions in combination with improved enzyme preparation we were able to obtain crystals of good quality...

Orientation of the Tyrosyl Radical inSalmonella typhimuriumClass Ib Ribonucleotide Reductase Determined by High Field EPR of R2F Single Crystals

“…Otherwise the g x -value would shift from 2.0091 (hydrophobic environment, E. coli (38,39)) to values of 2.0076 for the case of a more polar environment (52-58) (see Table 1). This finding clearly indicates that also in the R1E⅐R2F complex the radical Tyr-105 ⅐ is disconnected from a hydrogen bond network along a pathway of amino acid residues connecting the diiron site in R2 with the substrate binding site in R1 (61,64). …”

“…In the widely accepted model of the catalytic mechanism of the class Ia RNR, the binding of the substrate in subunit R1 and docking of both subunits R1 and R2 initiates the radical transfer process, mostly described as coupled proton-electron transfer, from the tyrosyl radical Tyr-122 ⅐ in protein subunit R2 to the transient cysteine radical Cys-439 (E. coli numbering) in protein subunit R1 (1,5,7,25,26,61). Details of this proposed radical transfer reaction are still not revealed.…”

Two Conserved Tyrosine Residues in Protein R1 Participate in an Intermolecular Electron Transfer in Ribonucleotide Reductase

“…Crystallization Conditions and Data Collection-The Y730F and Y731F proteins were crystallized in the space group R32 with hexagonal cell axes a ϭ b ϭ 226 Å and c ϭ 341 Å as in the wild type (20). The crystals were obtained by hanging drops of 10 l (5 l of protein mixture ϩ 5 l of reservoir solution).…”

“…The UV-visible absorbance spectra were collected on a Perkin-Elmer Lambda 2 spectrophotometer. For crystallization, proteins were purified as described previously (20).…”

A New Mechanism-based Radical Intermediate in a Mutant R1 Protein Affecting the Catalytically Essential Glu441 inEscherichia coli Ribonucleotide Reductase

“…Crystallization and Data Collection of Glu 441 Mutant Proteins-All mutants were crystallized in the space group R32 (42). The crystals were obtained by hanging drops of 10 l (5 l of protein mixture and 5 l of reservoir solution).…”