Essential for DNA biosynthesis and repair, ribonucleotide reductases (RNRs) convert ribonucleotides to deoxyribonucleotides via radical-based chemistry. Although long known that allosteric regulation of RNR activity is vital for cell health, the molecular basis of this regulation has been enigmatic, largely due to a lack of structural information about how the catalytic subunit (α 2 ) and the radical-generation subunit (β 2 ) interact. Here we present the first structure of a complex between α 2 and β 2 subunits for the prototypic RNR from Escherichia coli. Using four techniques (small-angle X-ray scattering, X-ray crystallography, electron microscopy, and analytical ultracentrifugation), we describe an unprecedented α 4 β 4 ring-like structure in the presence of the negative activity effector dATP and provide structural support for an active α 2 β 2 configuration. We demonstrate that, under physiological conditions, E. coli RNR exists as a mixture of transient α 2 β 2 and α 4 β 4 species whose distributions are modulated by allosteric effectors. We further show that this interconversion between α 2 β 2 and α 4 β 4 entails dramatic subunit rearrangements, providing a stunning molecular explanation for the allosteric regulation of RNR activity in E. coli.allostery | protein-protein interactions | conformational equilibria | nucleotide metabolism I mportant targets of anticancer and antiviral drugs, ribonucleotide reductases (RNRs) are classified by the metallocofactor used to generate a thiyl radical (1) that initiates reduction of ribonucleotides to deoxyribonucleotides (2, 3). Class Ia RNRs are found in all eukaryotes and many aerobic bacteria, with the Escherichia coli enzyme serving as the prototype. These RNRs reduce ribonucleoside 5′-diphosphates and are composed of two homodimeric subunits: α 2 and β 2 (Fig. 1A). The α 2 subunit contains the active site, where ribonucleotide reduction occurs, and two types of allosteric effector binding sites (4, 5). One effector site tunes the specificity for all four ribonucleotide substrates in response to intracellular levels of deoxyribonucleoside 5′-triphosphates (dATP, dTTP, dGTP) and ATP (6, 7) such that balanced pools of deoxyribonucleotides are maintained (8). The second effector site controls the rate of reduction, binding ATP to turn the enzyme on or dATP to turn it off (4, 6). This activity site is housed in an N-terminal cone domain (5, 9) and provides a means for negative feedback regulation to safeguard against cytotoxic elevation of deoxyribonucleotide levels (2, 3, 10). The β 2 subunit harbors the essential diferric-tyrosyl radical (Y122• in E. coli) cofactor (11) that initiates radical chemistry.Active RNR has long been proposed to be a transient α 2 β 2 complex (Fig. 1B) with enhanced subunit affinity upon binding of substrates and effectors (12-15). For each turnover, α 2 , β 2 , substrate and effector must interact, triggering long-range proton-coupled electron transfer (PCET) reducing Y122• in β 2 and oxidizing C439 to a thiyl radical in the active s...

Mammalian phenylalanine hydroxylase (PheH) is an allosteric enzyme that catalyzes the first step in the catabolism of the amino acid phenylalanine. Following allosteric activation by high phenylalanine levels, the enzyme catalyzes the pterin-dependent conversion of phenylalanine to tyrosine. Inability to control elevated phenylalanine levels in the blood leads to increased risk of mental disabilities commonly associated with the inherited metabolic disorder, phenylketonuria. Although extensively studied, structural changes associated with allosteric activation in mammalian PheH have been elusive. Here, we examine the complex allosteric mechanisms of rat PheH using X-ray crystallography, isothermal titration calorimetry (ITC), and small-angle X-ray scattering (SAXS). We describe crystal structures of the pre-activated state of the PheH tetramer depicting the regulatory domains docked against the catalytic domains and preventing substrate binding. Using SAXS, we further describe the domain movements involved in allosteric activation of PheH in solution and present the first demonstration of chromatography-coupled SAXS with Evolving Factor Analysis (EFA), a powerful method for separating scattering components in a model-independent way. Together, these results support a model for allostery in PheH in which phenylalanine stabilizes the dimerization of the regulatory domains and exposes the active site for substrate binding and other structural changes needed for activity.

Summary With recent advances in data analysis algorithms, X-ray detectors, and synchrotron sources, small-angle X-ray scattering (SAXS) has become much more accessible to the structural biology community than ever before. Although limited to ~10 Å resolution, SAXS can provide a wealth of structural information on biomolecules in solution and is compatible with a wide range of experimental conditions. SAXS is thus an attractive alternative when crystallography is not possible. Moreover, advanced usage of SAXS can provide unique insight into biomolecular behavior that can only be observed in solution, such as large conformational changes and transient protein-protein interactions. Unlike crystal diffraction data, however, solution scattering data are subtle in appearance, highly sensitive to sample quality and experimental errors, and easily misinterpreted. In addition, synchrotron beamlines that are dedicated to SAXS are often unfamiliar to the non-specialist. Here, we present a series of procedures that can be used for SAXS data collection and basic cross-checks designed to detect and avoid aggregation, concentration effects, radiation damage, buffer mismatch, and other common problems. The protein, human serum albumin (HSA), serves as a convenient and easily replicated example of just how subtle these problems can sometimes be, but also of how proper technique can yield pristine data even in problematic cases. Because typical data collection times at a synchrotron are only one to several days, we recommend that the sample purity, homogeneity, and solubility be extensively optimized prior to the experiment.

Protein dynamics are integral to biological function, yet few techniques are sensitive to collective atomic motions. A long-standing goal of X-ray crystallography has been to combine structural information from Bragg diffraction with dynamic information contained in the diffuse scattering background. However, the origin of macromolecular diffuse scattering has been poorly understood, limiting its applicability. We present a finely sampled diffuse scattering map from triclinic lysozyme with unprecedented accuracy and detail, clearly resolving both the inter-and intramolecular correlations. These correlations are studied theoretically using both all-atom molecular dynamics and simple vibrational models. Although lattice dynamics reproduce most of the diffuse pattern, protein internal dynamics, which include hinge-bending motions, are needed to explain the short-ranged correlations revealed by Patterson analysis. These insights lay the groundwork for animating crystal structures with biochemically relevant motions.