3.3-Å resolution cryo-EM structure of human ribonucleotide reductase with substrate and allosteric regulators bound.

Ribonucleotide reductases (RNRs) convert ribonucleotides into deoxyribonucleotides, a reaction essential for DNA replication and repair. Human RNR requires two subunits for activity, the α subunit contains the active site, and the ß subunit houses the radical cofactor. Here, we present a 3.3-Å resolution structure by cryo-electron microscopy (EM) of a dATP-inhibited state of human RNR. This structure, which was determined in the presence of substrate CDP and allosteric regulators ATP and dATP, has three α2 units arranged in an α6 ring. At near-atomic resolution, these data provide insight into the molecular basis for CDP recognition by allosteric specificity effectors dATP/ATP. Additionally, we present lower-resolution EM structures of human α6 in the presence of both the anticancer drug clofarabine triphosphate and ß2. Together, these structures support a model for RNR inhibition in which ß2 is excluded from binding in a radical transfer competent position when α exists as a stable hexamer.

Genetic incorporation of histidine derivatives using an engineered pyrrolysyl-tRNA synthetase.

A polyspecific amber suppressor aminoacyl-tRNA synthetase/tRNA pair was evolved that genetically encodes a series of histidine analogues in both Escherichia coli and mammalian cells. In combination with tRNACUA(Pyl), a pyrrolysyl-tRNA synthetase mutant was able to site-specifically incorporate 3-methyl-histidine, 3-pyridyl-alanine, 2-furyl-alanine, and 3-(2-thienyl)-alanine into proteins in response to an amber codon. Substitution of His66 in the blue fluorescent protein (BFP) with these histidine analogues created mutant proteins with distinct spectral properties. This work further expands the structural and chemical diversity of unnatural amino acids (UAAs) that can be genetically encoded in prokaryotic and eukaryotic organisms and affords new probes of protein structure and function.

Generation of a stable, aminotyrosyl radical-induced α2ß2 complex of Escherichia coli class Ia ribonucleotide reductase.

Ribonucleotide reductase (RNR) catalyzes the conversion of nucleoside diphosphates to deoxynucleoside diphosphates (dNDPs). The Escherichia coli class Ia RNR uses a mechanism of radical propagation by which a cysteine in the active site of the RNR large (α2) subunit is transiently oxidized by a stable tyrosyl radical (Y•) in the RNR small (ß2) subunit over a 35-Å pathway of redox-active amino acids: Y122• ↔ [W48?] ↔ Y356 in ß2 to Y731 ↔ Y730 ↔ C439 in α2. When 3-aminotyrosine (NH2Y) is incorporated in place of Y730, a long-lived NH2Y730• is generated in α2 in the presence of wild-type (wt)-ß2, substrate, and effector. This radical intermediate is chemically and kinetically competent to generate dNDPs. Herein, evidence is presented that NH2Y730• induces formation of a kinetically stable α2ß2 complex. Under conditions that generate NH2Y730•, binding between Y730NH2Y-α2 and wt-ß2 is 25-fold tighter (Kd = 7 nM) than for wt-α2

Clofarabine targets the large subunit (α) of human ribonucleotide reductase in live cells by assembly into persistent hexamers.

Clofarabine (ClF) is a drug used in the treatment of leukemia. One of its primary targets is human ribonucleotide reductase (hRNR), a dual-subunit, (α(2))(m)(ß(2))(n), regulatory enzyme indispensable in de novo dNTP synthesis. We report that, in live mammalian cells, ClF targets hRNR by converting its α-subunit into kinetically stable hexamers. We established mammalian expression platforms that enabled isolation of functional α and characterization of its altered oligomeric associations in response to ClF treatment. Size exclusion chromatography and electron microscopy documented persistence of in-cell-assembled-α(6). Our data validate hRNR as an important target of ClF, provide evidence that in vivo α's quaternary structure can be perturbed by a nonnatural ligand, and suggest small-molecule-promoted, persistent hexamerization as a strategy to modulate hRNR activity. These studies lay foundations for documentation of RNR oligomeric state within a cell.

Iterative stable alignment and clustering of 2D transmission electron microscope images.

Identification of homogeneous subsets of images in a macromolecular electron microscopy (EM) image data set is a critical step in single-particle analysis. The task is handled by iterative algorithms, whose performance is compromised by the compounded limitations of image alignment and K-means clustering. Here we describe an approach, iterative stable alignment and clustering (ISAC) that, relying on a new clustering method and on the concepts of stability and reproducibility, can extract validated, homogeneous subsets of images. ISAC requires only a small number of simple parameters and, with minimal human intervention, can eliminate bias from two-dimensional image clustering and maximize the quality of group averages that can be used for ab initio three-dimensional structural determination and analysis of macromolecular conformational variability. Repeated testing of the stability and reproducibility of a solution within ISAC eliminates heterogeneous or incorrect classes and introduces critical validation to the process of EM image clustering.

A soluble α-synuclein construct forms a dynamic tetramer.

A heterologously expressed form of the human Parkinson disease-associated protein α-synuclein with a 10-residue N-terminal extension is shown to form a stable tetramer in the absence of lipid bilayers or micelles. Sequential NMR assignments, intramonomer nuclear Overhauser effects, and circular dichroism spectra are consistent with transient formation of α-helices in the first 100 N-terminal residues of the 140-residue α-synuclein sequence. Total phosphorus analysis indicates that phospholipids are not associated with the tetramer as isolated, and chemical cross-linking experiments confirm that the tetramer is the highest-order oligomer present at NMR sample concentrations. Image reconstruction from electron micrographs indicates that a symmetric oligomer is present, with three- or fourfold symmetry. Thermal unfolding experiments indicate that a hydrophobic core is present in the tetramer. A dynamic model for the tetramer structure is proposed, based on expected close association of the amphipathic central helices observed in the previously described micelle-associated "hairpin" structure of α-synuclein.

Structure and nucleosome interaction of the yeast NuA4 and Piccolo-NuA4 histone acetyltransferase complexes.

We have used EM and biochemistry to characterize the structure of NuA4, an essential yeast histone acetyltransferase (HAT) complex conserved throughout eukaryotes, and we have determined the interaction of NuA4 with the nucleosome core particle (NCP). The ATM-related Tra1 subunit, which is shared with the SAGA coactivator complex, forms a large domain joined to a second region that accommodates the catalytic subcomplex Piccolo and other NuA4 subunits. EM analysis of a NuA4-NCP complex shows the NCP bound at the periphery of NuA4. EM characterization of Piccolo and Piccolo-NCP provided further information about subunit organization and confirmed that histone acetylation requires minimal contact with the NCP. A small conserved region at the N terminus of Piccolo subunit enhancer of Polycomb-like 1 (Epl1) is essential for NCP interaction, whereas the subunit yeast homolog of mammalian Ing1 2 (Yng2) apparently positions Piccolo for efficient acetylation of histone H4 or histone H2A tails. Taken together, these results provide an understanding of the NuA4 subunit organization and the NuA4-NCP interactions.

A promiscuous aminoacyl-tRNA synthetase that incorporates cysteine, methionine, and alanine homologs into proteins.

A mutant Escherichia coli leucyl-tRNA synthetase has been evolved for the selective incorporation of the methionine homolog 1 into proteins in yeast. This single aminoacyl-tRNA synthetase is capable of charging an amber suppressor EctRNA(CUA)(Leu) with at least eight different amino acids including methionine and cysteine homologs, as well as straight chain aliphatic amino acids. In addition we show that incorporation yields for these amino acids can be increased substantially by mutations in the editing CP1 domain of the E. coli leucyl-tRNA synthetase.

Acidophilic adaptations in the structure of Acetobacter aceti N5-carboxyaminoimidazole ribonucleotide mutase (PurE).

The crystal structure of Acetobacter aceti PurE was determined to a resolution of 1.55 A and is compared with the known structures of the class I PurEs from a mesophile, Escherichia coli, and a thermophile, Thermotoga maritima. Analyses of the general factors that increase protein stability are examined as potential explanations for the acid stability of A. aceti PurE. Increased inter-subunit hydrogen bonding and an increased number of arginine-containing salt bridges appear to account for the bulk of the increased acid stability. A chain of histidines linking two active sites is discussed in the context of the proton transfers catalyzed by the enzyme.