P. Andrew Karplus

Linking Crystallographic Model and Data Quality

Finessing Crystal Analysis Protein crystallography has revolutionized our understanding of a whole variety of biological processes (see the Perspective by Evans). In crystallography, the measure of agreement between the data and the calculated model is not on the same scale as the measure of data quality, making it challenging to choose an optimal high resolution limit beyond which the data should be discarded. Now, Karplus and Diederichs (p. 1030) introduce a statistical model that assesses agreement of model and data accuracy on the same scale. Determining the structures of biological macromolecules by x-ray crystallography requires solving the phase problem. The two techniques that dominate phase evaluation (multi- and single-wavelength anomalous diffraction) rely on element-specific scattering from incorporated heavy atoms. Liu et al. (p. 1033) present procedures for routine structure determination of native proteins with no heavy atom incorporation. The technique, which relies on combining data from multiple crystals, was used to determine the structures of four native proteins, including a 1200-residue complex. A statistical method places model and data quality on the same scale and indicates how far one can model. In macromolecular x-ray crystallography, refinement R values measure the agreement between observed and calculated data. Analogously, Rmerge values reporting on the agreement between multiple measurements of a given reflection are used to assess data quality. Here, we show that despite their widespread use, Rmerge values are poorly suited for determining the high-resolution limit and that current standard protocols discard much useful data. We introduce a statistic that estimates the correlation of an observed data set with the underlying (not measurable) true signal; this quantity, CC*, provides a single statistically valid guide for deciding which data are useful. CC* also can be used to assess model and data quality on the same scale, and this reveals when data quality is limiting model improvement.

Structure of the ERM Protein Moesin Reveals the FERM Domain Fold Masked by an Extended Actin Binding Tail Domain

The ezrin-radixin-moesin (ERM) protein family link actin filaments of cell surface structures to the plasma membrane, using a C-terminal F-actin binding segment and an N-terminal FERM domain, a common membrane binding module. ERM proteins are regulated by an intramolecular association of the FERM and C-terminal tail domains that masks their binding sites. The crystal structure of a dormant moesin FERM/tail complex reveals that the FERM domain has three compact lobes including an integrated PTB/PH/ EVH1 fold, with the C-terminal segment bound as an extended peptide masking a large surface of the FERM domain. This extended binding mode suggests a novel mechanism for how different signals could produce varying levels of activation. Sequence conservation suggests a similar regulation of the tumor suppressor merlin.

Old yellow enzyme at 2 A resolution: overall structure, ligand binding, and comparison with related flavoproteins.

BACKGROUND Old yellow enzyme (OYE) was the first flavoenzyme purified, but its function is still unknown. Nevertheless, the NADPH oxidase activity, the flavin mononucleotide environment and the ligand-binding properties of OYE have been extensively studied by biochemical and spectroscopic approaches. Full interpretation of these data requires structural information. RESULTS The crystal structures of oxidized and reduced OYE at 2 A resolution reveal an alpha/beta-barrel topology clearly related to trimethylamine dehydrogenase. Complexes of OYE with p-hydroxybenzaldehyde, beta-estradiol, and an NADPH analog show all three binding at a common site, stacked on the flavin. The putative NADPH binding mode is novel as it involves primary recognition of the nicotinamide mononucleotide portion. CONCLUSIONS This work shows that the striking spectral changes seen upon phenol binding are due to close physical association of the flavin and phenolate. It also identifies the structural class of OYE and suggests that if NADPH is its true substrate, then OYE has adopted NADPH dependence during evolution.

Cysteine-based redox switches in enzymes.

The enzymes involved in metabolism and signaling are regulated by posttranslational modifications that influence their catalytic activity, rates of turnover, and targeting to subcellular locations. Most prominent among these has been phosphorylation/dephosphorylation, but now a distinct class of modification coming to the fore is a set of versatile redox modifications of key cysteine residues. Here we review the chemical, structural, and regulatory aspects of such redox regulation of enzymes and discuss examples of how these regulatory modifications often work in concert with phosphorylation/dephosphorylation events, making redox dependence an integral part of many cell signaling processes. Included are the emerging roles played by peroxiredoxins, a family of cysteine-based peroxidases that now appear to be major players in both antioxidant defense and cell signaling.

Structure-based Insights into the Catalytic Power and Conformational Dexterity of Peroxiredoxins

Peroxiredoxins (Prxs), some of natures dominant peroxidases, use a conserved Cys residue to reduce peroxides. They are highly expressed in organisms from all kingdoms, and in eukaryotes they participate in hydrogen peroxide signaling. Seventy-two Prx structures have been determined that cover much of the diversity of the family. We review here the current knowledge and show that Prxs can be effectively classified by a structural/evolutionary organization into six subfamilies followed by specification of a 1-Cys or 2-Cys mechanism, and for 2-Cys Prxs, the structural location of the resolving Cys. We visualize the varied catalytic structural transitions and highlight how they differ depending on the location of the resolving Cys. We also review new insights into the question of how Prxs are such effective catalysts: the enzyme activates not only the conserved Cys thiolate but also the peroxide substrate. Moreover, the hydrogen-bonding network created by the four residues conserved in all Prx active sites stabilizes the transition state of the peroxidatic S(N)2 displacement reaction. Strict conservation of the peroxidatic active site along with the variation in structural transitions provides a fascinating picture of how the diverse Prxs function to break down peroxide substrates rapidly.

Structural comparisons among the short-chain helical cytokines

BACKGROUND Cytokines and growth factors are soluble proteins that regulate the development and activities of many cell types. One group of these proteins have structures based on a four-helix bundle, though this similarity is not apparent from amino acid sequence comparisons. An understanding of how diverse sequences can adopt the same fold would be useful for recognizing and aligning distant homologs and for applying structural information gained from one protein to other sequences. RESULTS We have approached this problem by comparing the five known structures which adopt a granulocyte-macrophage colony-stimulating factor (GM-CSF)-like, or short-chain fold: interleukin (IL)-4, GM-CSF, IL-2, IL-5, and macrophage colony-stimulating factor. The comparison reveals a common structural framework of five segments including 31 inner-core and 30 largely exposed residues. Buried polar interactions found in each protein illustrate how complementary substitutions maintain protein stability and may help specify unique core packing. A profile based on the known structures is not sufficient to guarantee accurate amino acid sequence alignments with other family members. Comparisons of the conserved short-chain framework with growth hormone define the optimal structural alignment. CONCLUSIONS Our results are useful for extrapolating functional results among the short-chain cytokines and growth hormone, and provide a foundation for similar characterization of other subfamilies. These results also show that the placement of polar residues at different buried positions in each protein complicates sequence comparisons, and they document a challenging test case for methods aimed at recognizing and aligning distant homologs.

Peroxiredoxins: guardians against oxidative stress and modulators of peroxide signaling

Peroxiredoxins (Prxs) are a ubiquitous family of cysteine-dependent peroxidase enzymes that play dominant roles in regulating peroxide levels within cells. These enzymes, often present at high levels and capable of rapidly clearing peroxides, display a remarkable array of variations in their oligomeric states and susceptibility to regulation by hyperoxidative inactivation and other post-translational modifications. Key conserved residues within the active site promote catalysis by stabilizing the transition state required for transferring the terminal oxygen of hydroperoxides to the active site (peroxidatic) cysteine residue. Extensive investigations continue to expand our understanding of the scope of their importance as well as the structures and forces at play within these critical defense and regulatory enzymes.

Hydrophobicity regained: Hydrophobicity regained

A widespread practice is to use free energies of transfer between organic solvents and water (ΔGtransfer°) to define hydrophobicity scales for the amino acid side chains. A comparison of four ΔGtransfer° scales reveals that the values for hydrogen‐bonding side chains are highly dependent on the non‐aqueous environment. This property of polar side chains violates the assumptions underlying the paradigm of equating ΔGtransfer° with hydrophobicity or even with a generic solvation energy that is directly relevant to protein stability and ligand binding energetics. This simple regaining of the original concept of hydrophobicity reveals a flaw in approaches that use ΔGtransfer° values to derive generic estimates of the energetics of the burial of polar groups, and allows the introduction of a “pure” hydrophobicity scale for the amino acid residues.

Substrate specificity and redox potential of AhpC, a bacterial peroxiredoxin

Typical 2-Cys peroxiredoxins (Prxs) are ubiquitous peroxidases that are involved in peroxide scavenging and/or the regulation of peroxide signaling in eukaryotes. Despite their prevalence, very few Prxs have been reliably characterized in terms of their substrate specificity profile and redox potential even though these values are important for gaining insight into physiological function. Here, we present such studies focusing on Salmonella typhimurium alkyl hydroperoxide reductase C component (StAhpC), an enzyme that has proven to be an excellent prototype of this largest and most widespread class of Prxs that includes mammalian Prx I–Prx IV. The catalytic efficiencies of StAhpC (kcat/Km) are >107 M−1·s−1 for inorganic and primary hydroperoxide substrates and ≈100-fold less for tertiary hydroperoxides, with the difference being exclusively caused by changes in Km. The oxidative inactivation of AhpC through reaction with a second molecule of peroxide shows parallel substrate specificity. The midpoint reduction potential of StAhpC is determined to be −178 ± 0.4 mV, a value much higher than most other thiol-based redox proteins. The relevance of these results for our understanding of Prx and the physiological role of StAhpC is discussed.

Structural Evidence that Peroxiredoxin Catalytic Power Is Based on Transition-State Stabilization

Peroxiredoxins (Prxs) are important peroxidases associated with both antioxidant protection and redox signaling. They use a conserved Cys residue to reduce peroxide substrates. The Prxs have a remarkably high catalytic efficiency that makes them a dominant player in cell-wide peroxide reduction, but the origins of their high activity have been mysterious. We present here a novel structure of human PrxV at 1.45 A resolution that has a dithiothreitol bound in the active site with its diol moiety mimicking the two oxygens of a peroxide substrate. This suggests diols and similar di-oxygen compounds as a novel class of competitive inhibitors for the Prxs. Common features of this and other structures containing peroxide, peroxide-mimicking ligands, or peroxide-mimicking water molecules reveal hydrogen bonding and steric factors that promote its high reactivity by creating an oxygen track along which the peroxide oxygens move as the reaction proceeds. Key insights include how the active-site microenvironment activates both the peroxidatic cysteine side chain and the peroxide substrate and how it is exquisitely well suited to stabilize the transition state of the in-line S(N)2 substitution reaction that is peroxidation.