The Unusual Cosubstrate Specificity of NQO2: Conservation Throughout the Amniotes and Implications for Cellular Function.

Human Quinone Reductase 2 (NQO2) is a pharmacological target and has appeared in numerous screening efforts as an off-target interactor with kinase-targeted drugs. However the cellular functions of NQO2 are not known. To gain insight into the potential cellular functions of NQO2, we have carried out a detailed evolutionary analysis. One of the most striking characteristics of NQO2 is that it uses conventional dihydronicotinamide cosubstrates, NADH and NADPH, extremely inefficiently, raising questions about an enzymatic function in cells. To characterize the ability of NQO2 to serve as an enzyme, the NQO2 gene was disrupted in HCT116 cells. These NQO2 knockouts along with the parental cells were used to demonstrate that cellular NQO2 is unable to catalyze the activation of the DNA cross-linking reagent, CB1954, without the addition of exogenous dihydronicotinamide riboside (NRH). To find whether the unusual cosubstrate specificity of NQO2 has been conserved in the amniotes, recombinant NQO2 from a reptile, Alligator mississippiensis, and a bird, Anas platyrhynchos, were cloned, purified, and their catalytic activity characterized. Like the mammalian enzymes, the reptile and bird NQO2 were efficient catalysts with the small and synthetic cosubstrate N-benzyl-1,4-dihydronicotinamide but were inefficient in their use of NADH and NADPH. Therefore, the unusual cosubstrate preference of NQO2 appears to be conserved throughout the amniotes; however, we found that NQO2 is not well-conserved in the amphibians. A phylogenetic analysis indicates that NQO1 and NQO2 diverged at the time, approximately 450 MYA, when tetrapods were beginning to evolve.

Variants with increased negative electrostatic potential in the Cx50 gap junction pore increased unitary channel conductance and magnesium modulation.

Gap junction (GJ) channels are oligomers of connexins forming channels linking neighboring cells. GJs formed by different connexins show distinct unitary channel conductance (γj), transjunctional voltage-dependent gating (Vj-gating) properties, and modulation by intracellular magnesium ([Mg2+]i). The underlying molecular determinants are not fully clear. Previous experimental evidence indicates that residues in the amino terminal (NT) and initial segment of the first extracellular (E1) domain influence the γj, Vj-gating, and/or [Mg2+]i modulation in several GJs. Increasing negatively charged residues in Cx50 (connexin50) E1 (G46D or G46E) increased γj, while increasing positively charged residue (G46K) reduced the γj Sequence alignment of Cx50 and Cx37 in the NT and E1 domains revealed that in Cx50 G8 and V53, positions are negatively charged residues in Cx37 (E8 and E53, respectively). To evaluate these residues together, we generated a triple variant in Cx50, G8E, G46E, and V53E simultaneously to study its γj, Vj-gating properties, and modulation by [Mg2+]i Our data indicate that the triple variant and individual variants G8E, G46E, and V53E significantly increased Cx50 GJ γj without a significant change in the Vj gating. In addition, elevated [Mg2+]i reduced γj in Cx50 and all the variant GJs. These results and our homology structural models suggest that these NT/E1 residues are likely to be pore-lining and the variants increased the negative electrostatic potentials along the GJ pore to facilitate the γj of this cation-preferring GJ channel. Our results indicate that electrostatic properties of the Cx50 GJ pore are important for the γj and the [Mg2+]i modulation.

ADP-Ribosylation, a Multifaceted Posttranslational Modification Involved in the Control of Cell Physiology in Health and Disease.

Posttranslational modifications (PTMs) regulate protein functions and interactions. ADP-ribosylation is a PTM, in which ADP-ribosyltransferases use nicotinamide adenine dinucleotide (NAD+) to modify target proteins with ADP-ribose. This modification can occur as mono- or poly-ADP-ribosylation. The latter involves the synthesis of long ADP-ribose chains that have specific properties due to the nature of the polymer. ADP-Ribosylation is reversed by hydrolases that cleave the glycosidic bonds either between ADP-ribose units or between the protein proximal ADP-ribose and a given amino acid side chain. Here we discuss the properties of the different enzymes associated with ADP-ribosylation and the consequences of this PTM on substrates. Furthermore, the different domains that interpret either mono- or poly-ADP-ribosylation and the implications for cellular processes are described.

An alternate mode of oligomerization for E. coli SecA.

SecA is the ATPase of preprotein translocase. SecA is a dimer in solution and changes in its oligomeric state may function in preprotein translocation. The SecA-N68 construct, in which the C-terminal helical domains of SecA are deleted, was used to investigate the mechanism of SecA oligomerization. SecA-N68 is in equilibrium between monomers, dimers, and tetramers. Subunit interactions in the SecA-N68 tetramer are mediated entirely by unstructured regions at its N- and C-termini: when the termini are deleted to yield SecA-N68∆NC, the construct is completely monomeric. This monomeric construct yielded crystals diffracting to 2.6 Å that were used to solve the structure of SecA-N68, including the "preprotein crosslinking domain" (PPXD) that was missing from previous E. coli SecA structures. The SecA-N68 structure was combined with small angle X-ray scattering (SAXS) data to construct a model of the SecA-N68 tetramer that is consistent with the essential roles of the extreme N- and C-termini in oligomerization. This mode of oligomerization, which depends on binding of the extreme N-terminus to the DEAD motor domains, NBD1 and NBD2, was used to model a novel parallel and flexible SecA solution dimer that agrees well with SAXS data.

Characterization of a polypeptide-binding site in the DEAD Motor of the SecA ATPase.

We coupled peptides from a CNBr digest of signal-sequenceless maltose-binding protein (MBP) to a surface plasmon resonance chip. SecA-N95, SecA-N68, and SecA-DM (which consists of only the DEAD Motor domains NBD1 and NBD2) bound to the immobilized peptides; ADP weakened the binding. SecA-DM, which lacks the 'preprotein cross-linking domain' (PPXD), displayed the most extensive binding, while an MBP-PPXD chimera showed no binding, demonstrating that the PPXD does not contribute to the binding. We characterized the sequence specificity using oriented peptide libraries; these results enabled synthesis of a 20-residue peptide that was used to recapitulate the results obtained with MBP-derived peptides. This study shows that there is a promiscuous and nucleotide-modulated peptide-binding site in the DEAD Motor domains of SecA.

Genetic selection for mistranslation rescues a defective co-chaperone in yeast.

Despite the general requirement for translation fidelity, mistranslation can be an adaptive response. We selected spontaneous second site mutations that suppress the stress sensitivity caused by a Saccharomyces cerevisiae tti2 allele with a Leu to Pro mutation at residue 187, identifying a single nucleotide mutation at the same position (C70U) in four tRNAProUGG genes. Linkage analysis and suppression by SUF9G3:U70 expressed from a centromeric plasmid confirmed the causative nature of the suppressor mutation. Since the mutation incorporates the G3:U70 identity element for alanyl-tRNA synthetase into tRNAPro, we hypothesized that suppression results from mistranslation of Pro187 in Tti2L187P as Ala. A strain expressing Tti2L187A was not stress sensitive. In vitro, tRNAProUGG (C70U) was mis-aminoacylated with alanine by alanyl-tRNA synthetase, but was not a substrate for prolyl-tRNA synthetase. Mass spectrometry from protein expressed in vivo and a novel GFP reporter for mistranslation confirmed substitution of alanine for proline at a rate of ∼6%. Mistranslating cells expressing SUF9G3:U70 induce a partial heat shock response but grow nearly identically to wild-type. Introducing the same G3:U70 mutation in SUF2 (tRNAProAGG) suppressed a second tti2 allele (tti2L50P). We have thus identified a strategy that allows mistranslation to suppress deleterious missense Pro mutations in Tti2.

Enhanced ubiquitination and proteasomal degradation of catalytically deficient human choline acetyltransferase mutants.

Choline acetyltransferase (ChAT) is essential for cholinergic neuron function as it mediates synthesis of the neurotransmitter acetylcholine. ChAT mutations have been linked to the neuromuscular disorder congenital myasthenic syndrome (CMS). One CMS-related ChAT mutation, V18M, reduces enzyme activity and cellular protein levels, and is positioned within a highly conserved proline-rich motif with the sequence 14 PKLPVPP20 . We demonstrate that N-terminal truncation that includes this proline-rich motif, as well as mutation of prolines-17/19 together to alanine (P17A/P19A), dramatically reduces ChAT steady-state protein levels and cellular activity when expressed in cholinergic SN56 neural cells. The in vitro activity of bacterially expressed recombinant P17A/P19A-ChAT is also reduced, although this is not caused by changes in protein secondary structure or thermal stability. Treatment of SN56 cells with the proteasome inhibitor MG132 increases cellular P17A/P19A-ChAT steady-state protein levels, and by immunoprecipitation we found that ChAT is ubiquitinated and that polyubiquitination of P17A/P19A-ChAT is increased compared to wild-type (WT) ChAT. Using a novel fluorescent-biorthogonal pulse-chase protocol in SN56 cells, we determined that the protein half-life of P17A/P19A-ChAT (2.2 h) is substantially reduced compared to WT-ChAT (19.7 h). Lastly, we show that two CMS-related ChAT mutants (V18M and A513T) have enhanced ubiquitination, and that treatment with MG132 can partially restore both the steady-state protein levels as well as cellular activity of some CMS-mutant ChAT. These results identify a novel mechanism for regulation of ChAT through the ubiquitin-proteasome system that is influenced by the conserved N-terminal proline-rich motif of ChAT and may be implicated in CMS pathology. Choline acetyltransferase (ChAT) synthesizes acetylcholine in cholinergic neurons. In this study we find that steady-state protein levels of human 69-kDa ChAT are regulated by the ubiquitin-proteasome system. Mutation of a highly conserved N-terminal proline-rich motif in human 69-kDa ChAT reduces both cellular ChAT protein levels, through enhanced ubiquitination and proteasomal degradation, and enzyme activity. Ubiquitination of catalytically deficient congenital myasthenic syndrome (CMS)-mutant ChAT is increased in cells, and importantly proteasome inhibition partially restores steady-state protein levels as well as cellular activity of some CMS-mutant ChAT proteins.

Binding of DNA-Intercalating Agents to Oxidized and Reduced Quinone Reductase 2.

Quinone reductase 2 (NQO2) is an enzyme that might have intracellular signaling functions. NQO2 can exist in either an oxidized state or a reduced state, and binding of compounds to one or both of these states inhibits enzymatic activity and could also affect intracellular signaling. A wide range of planar aromatic compounds bind NQO2, and we have identified three DNA-intercalating agents [ethidium bromide, acridine orange (AO), and doxorubicin] as novel nanomolar inhibitors of NQO2. Ethidium and AO, which carry a positive charge in their aromatic ring systems, bound reduced NQO2 with an affinity 50-fold higher than that of oxidized NQO2, while doxorubicin bound only oxidized NQO2. Crystallographic analyses of oxidized NQO2 in complex with the inhibitors indicated that the inhibitors were situated deep in the active site. The aromatic faces were sandwiched between the isoalloxazine ring of FAD and the phenyl ring of F178, with their edges making direct contact with residues lining the active site. In reduced NQO2, ethidium and AO occupied a more peripheral position in the active site, allowing several water molecules to interact with the polar end of the negatively charged isoalloxazine ring. We also showed that AO inhibited NQO2 at a nontoxic concentration in cells while ethidium was less effective at inhibiting NQO2 in cells. Together, this study shows that reduced NQO2 has structural and electrostatic properties that yield a preference for binding of planar, aromatic, and positively charged molecules that can also function as DNA-intercalating agents.

The First Extracellular Domain Plays an Important Role in Unitary Channel Conductance of Cx50 Gap Junction Channels.

Gap junction (GJ) channels provide direct passage for ions and small molecules to be exchanged between neighbouring cells and are crucial for many physiological processes. GJ channels can be gated by transjunctional voltage (known as Vj-gating) and display a wide range of unitary channel conductance (γj), yet the domains responsible for Vj-gating and γj are not fully clear. The first extracellular domain (E1) of several connexins has been shown to line part of their GJ channel pore and play important roles in Vj-gating properties and/or ion permeation selectivity. To test roles of the E1 of Cx50 GJ channels, we generated a chimera, Cx50Cx36E1, where the E1 domain of Cx50 was replaced with that of Cx36, a connexin showing quite distinct Vj-gating and γj from those of Cx50. Detailed characterizations of the chimera and three point mutants in E1 revealed that, although the E1 domain is important in determining γj, the E1 domain of Cx36 is able to effectively function within the context of the Cx50 channel with minor changes in Vj-gating properties, indicating that sequence differences between the E1 domains in Cx36 and Cx50 cannot account for their drastic differences in Vj-gating and γj. Our homology models of the chimera and the E1 mutants revealed that electrostatic properties of the pore-lining residues and their contribution to the electric field in the pore are important factors for the rate of ion permeation of Cx50 and possibly other GJ channels.

Active transporters as enzymes: an energetic framework applied to major facilitator superfamily and ABC importer systems.

Active membrane transporters are dynamic molecular machines that catalyse transport across a membrane by coupling solute movement to a source of energy such as ATP or a secondary ion gradient. A central question for many active transporters concerns the mechanism by which transport is coupled to a source of energy. The transport process and associated energetic coupling involve conformational changes in the transporter. For efficient transport, the conformational changes must be tightly regulated and they must link energy use to movement of the substrate across the membrane. The present review discusses active transport using the well-established energetic framework for enzyme-mediated catalysis. In particular, membrane transport systems can be viewed as ensembles consisting of low-energy and high-energy conformations. The transport process involves binding interactions that selectively stabilize the higher energy conformations, and in this way promote conformational changes in the system that are coupled to decreases in free energy and substrate translocation. The major facilitator superfamily of secondary active transporters is used to illustrate these ideas, which are then be expanded to primary active transport mediated by ABC (ATP-binding cassette) import systems, with a focus on the well-studied maltose transporter.

Structural Analysis of the 14-3-3ζ/Chibby Interaction Involved in Wnt/ß-Catenin Signaling.

The partially disordered Chibby (Cby) is a conserved nuclear protein that antagonizes the Wnt/ß-catenin signaling pathway. By competing with the Tcf/Lef family proteins for binding to ß-catenin, Cby abrogates the ß-catenin-mediated transcription of Wnt signaling genes. Additionally, upon phosphorylation on S20 by the kinase Akt, Cby forms a complex with 14-3-3 to facilitate the nuclear export of ß-catenin, which represents another crucial mechanism for the regulation of Wnt signaling. To obtain a mechanistic understanding of the 14-3-3/Cby interaction, we have extensively characterized the complex using X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and isothermal titration calorimetry (ITC). The crystal structure of the human 14-3-3ζ/Cby protein-peptide complex reveals a canonical binding mode; however the residue at the +2 position from the phosphorylated serine is shown to be uniquely oriented relative to other solved structures of 14-3-3 complexes. Our ITC results illustrate that although the phosphorylation of S20 is essential for Cby to recognize 14-3-3, residues flanking the phosphorylation site also contribute to the binding affinity. However, as is commonly observed in other 14-3-3/phosphopeptide crystal structures, residues of Cby flanking the 14-3-3 binding motif lack observable electron density. To obtain a more detailed binding interface, we have completed the backbone NMR resonance assignment of 14-3-3ζ. NMR titration experiments reveal that residues outside of the 14-3-3 conserved binding cleft, namely a flexible loop consisting of residues 203-210, are also involved in binding Cby. By using a combined X-ray and NMR approach, we have dissected the molecular basis of the 14-3-3/Cby interaction.

The HIP2~ubiquitin conjugate forms a non-compact monomeric thioester during di-ubiquitin synthesis.

Polyubiquitination is a post-translational event used to control the degradation of damaged or unwanted proteins by modifying the target protein with a chain of ubiquitin molecules. One potential mechanism for the assembly of polyubiquitin chains involves the dimerization of an E2 conjugating enzyme allowing conjugated ubiquitin molecules to be put into close proximity to assist reactivity. HIP2 (UBE2K) and Ubc1 (yeast homolog of UBE2K) are unique E2 conjugating enzymes that each contain a C-terminal UBA domain attached to their catalytic domains, and they have basal E3-independent polyubiquitination activity. Although the isolated enzymes are monomeric, polyubiquitin formation activity assays show that both can act as ubiquitin donors or ubiquitin acceptors when in the activated thioester conjugate suggesting dimerization of the E2-ubiquitin conjugates. Stable disulfide complexes, analytical ultracentrifugation and small angle x-ray scattering were used to show that the HIP2-Ub and Ubc1-Ub thioester complexes remain predominantly monomeric in solution. Models of the HIP2-Ub complex derived from SAXS data show the complex is not compact but instead forms an open or backbent conformation similar to UbcH5b~Ub or Ubc13~Ub where the UBA domain and covalently attached ubiquitin reside on opposite ends of the catalytic domain. Activity assays showed that full length HIP2 exhibited a five-fold increase in the formation rate of di-ubiquitin compared to a HIP2 lacking the UBA domain. This difference was not observed for Ubc1 and may be attributed to the closer proximity of the UBA domain in HIP2 to the catalytic core than for Ubc1.

Pin1: Intimate involvement with the regulatory protein kinase networks in the global phosphorylation landscape.

BACKGROUND: Protein phosphorylation is a universal regulatory mechanism that involves an extensive network of protein kinases. The discovery of the phosphorylation-dependent peptidyl-prolyl isomerase Pin1 added an additional layer of complexity to these regulatory networks. SCOPE OF REVIEW: We have evaluated interactions between Pin1 and the regulatory kinome and proline-dependent phosphoproteome taking into consideration findings from targeted studies as well as data that has emerged from systematic phosphoproteomic workflows and from curated protein interaction databases. MAJOR CONCLUSIONS: The relationship between Pin1 and the regulatory protein kinase networks is not restricted simply to the recognition of proteins that are substrates for proline-directed kinases. In this respect, Pin1 itself is phosphorylated in cells by protein kinases that modulate its functional properties. Furthermore, the phosphorylation-dependent targets of Pin1 include a number of protein kinases as well as other enzymes such as phosphatases and regulatory subunits of kinases that modulate the actions of protein kinases. GENERAL SIGNIFICANCE: As a result of its interactions with numerous protein kinases and their substrates, as well as itself being a target for phosphorylation, Pin1 has an intricate relationship with the regulatory protein kinase and phosphoproteomic networks that orchestrate complex cellular processes and respond to environmental cues. This article is part of a Special Issue entitled Proline-directed Foldases: Cell Signaling Catalysts and Drug Targets.

Peroxide-mediated oxidation and inhibition of the peptidyl-prolyl isomerase Pin1.

Pin1 is a phosphorylation-dependent peptidyl-prolyl isomerase that plays a critical role in mediating protein conformational changes involved in signaling processes related to cell cycle control. Pin1 has also been implicated as being neuroprotective in aging-related neurodegenerative disorders including Alzheimer's disease where Pin1 activity is diminished. Notably, recent proteomic analysis of brain samples from patients with mild cognitive impairment revealed that Pin1 is oxidized and also displays reduced activity. Since the Pin1 active site contains a functionally critical cysteine residue (Cys113) with a low predicted pK(a), we hypothesized that Cys113 is sensitive to oxidation. Consistent with this hypothesis, we observed that treatment of Pin1 with hydrogen peroxide results in a 32Da mass increase, likely resulting from the oxidation of Cys113 to sulfinic acid (Cys-SO(2)H). This modification results in loss of peptidyl-prolyl isomerase activity. Notably, Pin1 with Cys113 substituted by aspartic acid retains activity and is no longer sensitive to oxidation. Structural studies by X-ray crystallography revealed increased electron density surrounding Cys113 following hydrogen peroxide treatment. At lower concentrations of hydrogen peroxide, oxidative inhibition of Pin1 can be partially reversed by treatment with dithiothreitol, suggesting that oxidation could be a reversible modification with a regulatory role. We conclude that the loss of Pin1 activity upon oxidation results from oxidative modification of the Cys113 sulfhydryl to sulfenic (Cys-SOH) or sulfinic acid (Cys-SO(2)H). Given the involvement of Pin1 in pathological processes related to neurodegenerative diseases and to cancer, these findings could have implications for the prevention or treatment of disease.

Quinone reductase 2 is an adventitious target of protein kinase CK2 inhibitors TBBz (TBI) and DMAT.

Quinone reductase 2 (NQO2) exhibits off-target interactions with two protein kinase CK2 inhibitors, 4,5,6,7-1H-tetrabromobenzimidazole (TBBz) and 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT). TBBz and DMAT induce apoptosis in cells expressing an inhibitor-resistant CK2, suggesting that the interaction with NQO2 may mediate some of their pharmacological effects. In this study, we have fully characterized the binding of TBBz and DMAT to NQO2. Fluorescence titrations showed that TBBz and DMAT bind oxidized NQO2 in the low nanomolar range; in the case of TBBz, the affinity for NQO2 was 40-fold greater than its affinity for CK2. A related CK2 inhibitor, 4,5,6,7-tetrabromobenzotriazole (TBB), which failed to cause apoptosis in cells expressing inhibitor-resistant CK2, binds NQO2 with an affinity 1000-fold lower than those of TBBz and DMAT. Kinetic analysis indicated that DMAT inhibits NQO2 by binding with similar affinities to the oxidized and reduced forms. Crystal structure analysis showed that DMAT binds reduced NQO2 in a manner different from that in the oxidized state. In oxidized NQO2, TBBz and DMAT are deeply buried in the active site and make direct hydrogen and halogen bonds to the enzyme. In reduced NQO2, DMAT occupies a more peripheral region and hydrogen and halogen bonds with the enzyme are mediated through three water molecules. Therefore, although TBB, TBBz, and DMAT are all potent inhibitors of CK2, they exhibit different activity profiles toward NQO2. We conclude that the active site of NQO2 is fundamentally different from the ATP binding site of CK2 and the inhibition of NQO2 by CK2 inhibitors is adventitious.

Crystal and solution structure analysis of FhuD2 from Staphylococcus aureus in multiple unliganded conformations and bound to ferrioxamine-B.

Iron acquisition is a central process for virtually all organisms. In Staphylococcus aureus, FhuD2 is a lipoprotein that is a high-affinity receptor for iron-bound hydroxamate siderophores. In this study, FhuD2 was crystallized bound to ferrioxamine-B (FXB), and also in its ligand-free state; the latter structures are the first for hydroxamate-binding receptors within this protein family. The structure of the FhuD2-FXB conformation shows that residues W197 and R199 from the C-terminal domain donate hydrogen bonds to the hydroxamate oxygens, and a ring of aromatic residues cradles the aliphatic arms connecting the hydroxamate moieties of the siderophore. The available ligand-bound structures of FhuD from Escherichia coli and YfiY from Bacillus cereus show that, despite a high degree of structural conservation, three protein families have evolved with critical siderophore binding residues on either the C-terminal domain (S. aureus), the N-terminal domain (E. coli), or both (B. cereus). Unliganded FhuD2 was crystallized in five conformations related by rigid body movements of the N- and C-terminal domains. Small-angle X-ray scattering (SAXS) indicates that the solution conformation of unliganded FhuD2 is more compact than the conformations observed in crystals. The ligand-induced conformational changes for FhuD2 in solution are relatively modest and depend on the identity of the siderophore. The crystallographic and SAXS results are used to discuss roles for the liganded and unliganded forms of FhuD2 in the siderophore transport mechanism.

The PS1 hairpin of Mcm3 is essential for viability and for DNA unwinding in vitro.

The pre-sensor 1 (PS1) hairpin is found in ring-shaped helicases of the AAA+ family (ATPases associated with a variety of cellular activities) of proteins and is implicated in DNA translocation during DNA unwinding of archaeal mini-chromosome maintenance (MCM) and superfamily 3 viral replicative helicases. To determine whether the PS1 hairpin is required for the function of the eukaryotic replicative helicase, Mcm2-7 (also comprised of AAA+ proteins), we mutated the conserved lysine residue in the putative PS1 hairpin motif in each of the Saccharomyces cerevisiae Mcm2-7 subunits to alanine. Interestingly, only the PS1 hairpin of Mcm3 was essential for viability. While mutation of the PS1 hairpin in the remaining MCM subunits resulted in minimal phenotypes, with the exception of Mcm7 which showed slow growth under all conditions examined, the viable alleles were synthetic lethal with each other. Reconstituted Mcm2-7 containing Mcm3 with the PS1 mutation (Mcm3(K499A)) had severely decreased helicase activity. The lack of helicase activity provides a probable explanation for the inviability of the mcm3(K499A) strain. The ATPase activity of Mcm2-7(3K499A) was similar to the wild type complex, but its interaction with single-stranded DNA in an electrophoretic mobility shift assay and its associations in cells were subtly altered. Together, these findings indicate that the PS1 hairpins in the Mcm2-7 subunits have important and distinct functions, most evident by the essential nature of the Mcm3 PS1 hairpin in DNA unwinding.

Chloroquine binding reveals flavin redox switch function of quinone reductase 2.

Quinone reductase 2 (NQO2) is an FAD-linked enzyme and the only known human target of two antimalarial drugs, primaquine (PQ) and chloroquine (CQ). The structural differences between oxidized and reduced NQO2 and the structural basis for inhibition by PQ and CQ were investigated by x-ray crystallography. Structures of oxidized NQO2 in complex with PQ and CQ were solved at 1.4 Å resolution. CQ binds preferentially to reduced NQO2, and upon reduction of NQO2-CQ crystals, the space group changed from P2(1)2(1)2(1) to P2(1), with 1-Å decreases in all three unit cell dimensions. The change in crystal packing originated in the negative charge and 4-5º bend in the reduced isoalloxazine ring of FAD, which resulted in a new mode of CQ binding and closure of a flexible loop (Phe(126)-Leu(136)) over the active site. This first structure of a reduced quinone reductase shows that reduction of the FAD cofactor and binding of a specific inhibitor lead to global changes in NQO2 structure and is consistent with a functional role for NQO2 as a flavin redox switch.

Non-catalytic participation of the Pin1 peptidyl-prolyl isomerase domain in target binding.

Pin1 is a phosphorylation-dependent peptidyl-prolyl isomerase (PPIase) that has the potential to add an additional level of regulation within protein kinase mediated signaling pathways. Furthermore, there is a mounting body of evidence implicating Pin1 in the emergence of pathological phenotypes in neurodegeneration and cancer through the isomerization of a wide variety of substrates at peptidyl-prolyl bonds where the residue preceding proline is a phosphorylated serine or threonine residue (i.e., pS/T-P motifs). A key step in this regulatory process is the interaction of Pin-1 with its substrates. This is a complex process since Pin1 is composed of two domains, the catalytic PPIase domain, and a type IV WW domain, both of which recognize pS/T-P motifs. The observation that the WW domain exhibits considerably higher binding affinity for pS/T-P motifs has led to predictions that the two domains may have distinct roles in mediating the actions of Pin1 on its substrates. To evaluate the participation of its individual domains in target binding, we performed GST pulldowns to monitor interactions between various forms of Pin1 and mitotic phospho-proteins that revealed two classes of Pin-1 interacting proteins, differing in their requirement for residues within the PPIase domain. From these observations, we consider models for Pin1-substrate interactions and the potential functions of the different classes of Pin1 interacting proteins. We also compare sequences that are recognized by Pin1 within its individual interaction partners to investigate the underlying basis for its different types of interactions.

Identification of basepairs within Tn5 termini that are critical sfor H-NS binding to the transpososome and regulation of Tn5 transposition.

BACKGROUND: The H-NS protein is a global regulator of gene expression in bacteria and can also bind transposition complexes (transpososomes). In Tn5 transposition H-NS promotes transpososome assembly in vitro and disruption of the hns gene causes a modest decrease in Tn5 transposition (three- to five-fold). This is consistent with H-NS acting as a positive regulator of Tn5 transposition. Molecular determinants for H-NS binding to the Tn5 transpososome have not been determined, nor has the strength of the interaction been established. There is also uncertainty as to whether H-NS regulates Tn5 transposition in vivo through an interaction with the transposition machinery as disruption of the hns gene has pleiotropic effects on Escherichia coli, the organism used in this study. RESULTS: In the current work we have further examined determinants for H-NS binding to the Tn5 transpososome through both mutational studies on Tn5 termini (or 'transposon ends') and protein-protein cross-linking analysis. We identify mutations in two different segments of the transposon ends that abrogate H-NS binding and characterize the affinity of H-NS for wild type transposon ends in the context of the transpososome. We also show that H-NS forms cross-links with the Tn5 transposase protein specifically in the transpososome, an observation consistent with the two proteins occupying overlapping binding sites in the transposon ends. Finally, we make use of the end mutations to test the idea that H-NS exerts its impact on Tn5 transposition in vivo by binding directly to the transpososome. Consistent with this possibility, we show that two different end mutations reduce the sensitivity of the Tn5 system to H-NS regulation. CONCLUSIONS: H-NS typically regulates cellular functions through its potent transcriptional repressor function. Work presented here provides support for an alternative mechanism of H-NS-based regulation, and adds to our understanding of how bacterial transposition can be regulated.