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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.

Despite the innovations in chemotherapeutic treatment of pancreatic cancer, the generally poor outcome of this disease begs the search for novel targets. RAD51 is a critical component of homologous recombination DNA repair, binding with BRCA2 and forming polymeric filaments essential for its function. RAD51 is often elevated in cancer cells compared to normal cells, particularly in pancreatic cancer: RAD51 is a negative prognostic biomarker and promotes tumor cell proliferation in that disease (Cancer Cell Int 19: 356, 2019; doi: 10.1186/s12935-019-1077-6). Therefore, RAD51 is an attractive target for anticancer treatment in that its elevated level and activity in tumor cells provide a potential level of selectivity for such an agent, increasing the therapeutic index. We demonstrated previously that novel inhibitors of RAD51, IBR2 [2-(Benzylsulfonyl)-1-(1H-indol-3-yl)-1,2-dihydroisoquinoline] and IBR120 (an isoindolinyl derivative of IBR2) (Eur J Med Chem. 96:196-208, 2015; doi: 10.1016/j.ejmech.2015.04.021), not only inhibited proliferation of a range of tumor cell lines at micromolar concentrations but also acted in combination with commonly used cytotoxic chemotherapy and molecularly targeted drugs to synergistically inhibit proliferation (J Pharmacol Expt Ther, 364: 46-54, 2018; doi.org/10.1124/jpet.117.241661). To improve ADME properties, enhance activity, and increase potential to selectively target RAD51, advanced computational tools and rational approaches were employed to predict possible modification to the IBR chemical backbone. This effort resulted in discovery of the compound JKYN-1. As a model system in which to test the activity of JKYN-1 in vitro, human pancreatic cancer cell lines PANC-1, Capan-1 and Capan-2 were used. All lines have mutant K-Ras and p53, and Capan-1 has mutant BRCA2 (https://www.cancer.gov/research/key-initiatives/ras; https://web.expasy.org/cellosaurus). JKYN-1 or its water-soluble methylsulfonate salt (JKYN-1-mesylate) inhibit proliferation of PANC-1 cells approximately 5 times more effectively than IBR120 and inhibit Capan-1 and Capan-2 cells even better. Against PANC-1, JKYN-1-mesylate inhibits proliferation synergistically in combination with afatinib (inhibitor of epidermal growth factor receptor) or with IBR120, and is additive with gemcitabine. Biochemical characterization of the interaction between RAD51 and these inhibitors demonstrated the following: (1) IBR2 and IBR120 promote disassembly of RAD51 multimers in an ex vivo native polyacrylamide gel electrophoresis assay using clonal constructs of RAD51, and (2) JKYN-1 binds to RAD51 with higher affinity than IBR120 and B02, a commercially available RAD51 inhibitor, in a surface plasmon resonance spectroscopy assay. Therefore, JKYN-1 is a potential novel small molecule therapeutic agent for treatment of pancreatic cancer, both as a single agent and in combination with standard chemotherapy drugs. Citation Format: Peter Ferguson, Mark D. Vincent, Yousef Najajreh, Brian Shilton, Stephen Ritter, Rima Al-awar, Richard Marcellus, Mohammed Mohammed, Methvin Isaac, James Koropatnnick. Antiproliferative activity of inhibitors of RAD51, singly and in combination with chemotherapy drugs, against pancreatic cancer cell lines [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-039.

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.

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.

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.

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.

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.

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.

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.

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.

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.

The ATPase activity of the maltose transporter (MalFGK(2)) is dependent on interactions with the maltose-binding protein (MBP). To determine whether direct interactions between the translocated sugar and MalFGK(2) are important for the regulation of ATP hydrolysis, we used an MBP mutant (sMBP) that is able to bind either maltose or sucrose. We observed that maltose- and sucrose-bound sMBP stimulate equal levels of MalFGK(2) ATPase activity. Therefore, the ATPase activity of MalFGK(2) is coupled to translocation of maltose solely by interactions between MalFGK(2) and MBP. For both maltose and sucrose, the ability of sMBP to stimulate the MalFGK(2) ATPase was greatly reduced compared with wild-type MBP, indicating that the mutations in sMBP have interfered with important interactions between MBP and MalFGK(2). High resolution crystal structure analysis of sMBP shows that in the closed conformation with bound sucrose, three of four mutations are buried, and the fourth causes only a minor change in the accessible surface. In contrast, in the open form of sMBP, all of the mutations are accessible, and the main chain of Tyr(62)-Gly(69) is destabilized and occupies an alternative conformation due to the W62Y mutation. On this basis, the compromised ability of sMBP to stimulate ATP hydrolysis by MalFGK(2) is most likely due to a disruption of interactions between MalFGK(2) and the open, rather than the closed, conformation of sMBP. Modeling the open sMBP structure bound to MalFGK(2) in the transition state for ATP hydrolysis points to an important site of interaction and suggests a mechanism for coupling ATP hydrolysis to substrate translocation that is independent of the exact structure of the substrate.

ADP-ribosylation controls many processes, including transcription, DNA repair, and bacterial toxicity. ADP-ribosyltransferases and poly-ADP-ribose polymerases (PARPs) catalyze mono- and poly-ADP-ribosylation, respectively, and depend on a highly conserved glutamate residue in the active center for catalysis. However, there is an apparent absence of this glutamate for the recently described PARP6-PARP16, raising questions about how these enzymes function. We find that PARP10, in contrast to PARP1, lacks the catalytic glutamate and has transferase rather than polymerase activity. Despite this fundamental difference, PARP10 also modifies acidic residues. Consequently, we propose an alternative catalytic mechanism for PARP10 compared to PARP1 in which the acidic target residue of the substrate functionally substitutes for the catalytic glutamate by using substrate-assisted catalysis to transfer ADP-ribose. This mechanism explains why the novel PARPs are unable to function as polymerases. This discovery will help to illuminate the different biological functions of mono- versus poly-ADP-ribosylation in cells.

This review is focused on the interaction between maltose binding protein (MBP) and the maltose transporter complex, MalFGK(2), which is a member of the ATP Binding Cassette (ABC) superfamily. The interaction between MBP and MalFGK(2) has a critical role in maltose transport, but a coherent description of the interaction is complicated because both MBP and MalFGK(2) can adopt multiple conformations. Drawing on genetic, structural, and biochemical data, the different conformations of MBP and MalFGK(2) are described and incorporated into a model for their interaction. The most important feature of this model is that ligand-bound MBP initiates the process of ATP-dependent maltose transport by stabilizing a high-energy conformation of MalFGK(2). In this model of the MBP-MalFGK(2) interaction, stabilization of a high-energy conformation of MalFGK(2) allows ATP to drive conformational changes in the system - in particular the opening of bound MBP - that leads to formation of a transition state for ATP hydrolysis. Such a role for ligand-bound MBP explains how MBP-independent MalFGK(2) mutants work, and represents a general mechanism for binding-protein dependent ABC import systems. In ABC export systems, which do not use a binding protein, the substrate itself is expected to play a role similar to ligand-bound MBP in the maltose transport system. The mechanistic model for the maltose transporter suggests that ABC-type import systems evolved to make use of a peripheral binding protein so that the transport process is essentially irreversible.

© 2015 Elsevier B.V. All rights reserved. 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.

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.

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 pKa, we hypothesized that Cys113 is sensitive to oxidation. Consistent with this hypothesis, we observed that treatment of Pin1 with hydrogen peroxide results in a 32 Da mass increase, likely resulting from the oxidation of Cys113 to sulfinic acid (Cys-SO2H). 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-SO2H). 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.

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.

The partially disordered Chibby (Cby) is a conserved nuclear protein that antagonizes the Wnt/β-catenin signaling pathway. By competing with the Tcf/Lef family proteins for binding to β-catenin, Cby abrogates the β-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 β-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.

© 2015 Published by NRC Research Press. Pin1 is a peptidyl-prolyl isomerase (PPIase) that plays a central role in eukaryotic cell cycle regulation, making this protein an interesting target for cancer therapy. Pin1 exhibits high specificity for substrates where proline is preceded by phosphoserine or phosphothreonine. The protein comprises an N-terminal WW (tryptophan-tryptophan) domain and a C-terminal PPIase domain. The cyclic peptide [CRYPEVEIC] (square brackets are used to denote the cyclic structure) represents a lead compound for a new class of nonphosphorylated Pin1 inhibitors. Unfortunately, it has not been possible thus far to characterize the Pin1-[CRYPEVEIC] complex by X-ray crystallography. Thus, the exact binding mode remains unknown. The current work employs hydrogen/deuterium exchange mass spectrometry for gaining insights into the Pin1-[CRYPEVEIC] interactions. The WW domain shows extensive conformational dynamics, both in the presence and in the absence of ligand. In contrast, profound changes in deuteration kinetics are observed in the PPIase domain after the addition of [CRYPEVEIC]. The secondary structure elements 2,3, and 4 exhibit markedly reduced deuteration, consistent with their postulated involvement in ligand binding. Unexpectedly, [CRYPEVEIC] destabilizes the range of residues 61-86, a segment that comprises basic side chains that normally interact with the substrate phosphate. This destabilization is likely caused by steric clashes with Y3 or E5 of the inhibitor. Ligand-induced destabilization has previously been reported for a few other proteins, but effects of this type are not very common. Our findings suggest that future crystallization trials on Pin1 variants deleted for residues in the 61-86 range might provide a path towards high-resolution X-ray structures of Pin1 bound to cyclic peptide inhibitors.

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.

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.

Transposition reactions take place in the context of higher-order protein-DNA complexes called transpososomes. In the Tn10 transpososome, IHF binding to an "outside end" creates a bend in the DNA that allows the transposase protein to contact the end at two different sites, the terminal and subterminal binding sites. Presumably this helps to stabilize the transposase-end interaction. However, the DNA loop that is formed must be unfolded at a later stage in order for the transposon to integrate into other DNA molecules. It has been proposed that transpososome unfolding also plays a role in transposon excision. To investigate this possibility further, we have isolated and characterized transposase mutants with altered transpososome unfolding properties. Two such mutants were identified, R182A and R184A. Both mutants fail to carry out hairpin formation, an intermediate step in transposon excision, specifically with outside end-containing substrates. These results support the idea that transpososome unfolding and excision are linked. Also, based on the importance of residues R182 and R184 in transpososome unfolding, we propose a new model for the Tn10 transpososome, wherein both DNA ends of the transpososome make subterminal contacts with transposase.

Staphylococcus aureus can utilize ferric hydroxamates as a source of iron under iron-restricted growth conditions. Proteins involved in this transport process are: FhuCBG, which encodes a traffic ATPase; FhuD2, a post-translationally modified lipoprotein that acts as a high affinity receptor at the cytoplasmic membrane for the efficient capture of ferric hydroxamates; and FhuD1, a protein with similarity to FhuD2. Gene duplication likely gave rise to fhuD1 and fhuD2. While the genomic locations of fhuCBG and fhuD2 in S. aureus strains are conserved, both the presence and the location of fhuD1 are variable. The apparent redundancy of FhuD1 led us to examine the role of this protein. We demonstrate that FhuD1 is expressed only under conditions of iron limitation through the regulatory activity of Fur. FhuD1 fractions with the cell membrane and binds hydroxamate siderophores but with lower affinity than FhuD2. Using small angle x-ray scattering, the solution structure of FhuD1 resembles that of FhuD2, and only a small conformational change is associated with ferrichrome binding. FhuD1, therefore, appears to be a receptor for ferric hydroxamates, like FhuD2. Our data to date suggest, however, that FhuD1 is redundant to FhuD2 and plays a minor role in hydroxamate transport. However, given the very real possibility that we have not yet identified the proper conditions where FhuD1 does provide an advantage over FhuD2, we anticipate that FhuD1 serves an enhanced role in the transport of untested hydroxamate siderophores and that it may play a prominent role during the growth of S. aureus in its natural environments.

The fhuD2 gene encodes a lipoprotein that has previously been shown to be important for the utilization of iron(III)-hydroxamates by Staphylococcus aureus. We have studied the function of the FhuD2 protein in greater detail, and demonstrate here that the protein binds several iron(III)-hydroxamates. Mutagenesis of FhuD2 identified several residues that were important for the ability of the protein to function in iron(III)-hydroxamate transport. Several residues, notably Tyr-191, Trp-197, and Glu-202, were found to be critical for ligand binding. Moreover, mutation of two highly conserved glutamate residues, Glu-97 and Glu-231, had no affect on ligand binding, but did impair iron(III)-hydroxamate transport. Interestingly, the transport defect was not equivalent for all iron(III)-hydroxamates. We modeled FhuD2 against the high resolution structures of Escherichia coli FhuD and BtuF, two structurally related proteins, and showed that the three proteins share a similar overall structure. FhuD2 Glu-97 and Glu-231 were positioned on the surface of the N and C domains, respectively. Characterization of E97A, E231A, or E97A/E231A mutants suggests that these residues, along with the ligand itself, play a cumulative role in recognition by the ABC transporter FhuBGC2. In addition, small angle x-ray scattering was used to demonstrate that, in solution, FhuD2 does not undergo a detectable change in conformation upon binding iron(III)-hydroxamates. Therefore, the mechanism of binding and transport of ligands for binding proteins within this family is significantly different from that of other well studied binding protein families, such as that represented by maltose-binding protein.

Visual arrestin is converted from a ‘basal’ state to an ‘activated’ state by interaction with the phosphorylated C-terminus of photoactivated rhodopsin (R*), but the conformational changes in arrestin that lead to activation are unknown. Small-angle X-ray scattering (SAXS) was used to investigate the solution structure of arrestin and characterize changes attendant upon activation. Wild-type arrestin forms dimers with a dissociation constant of 60 µm. Small conformational changes, consistent with local movements of loops or the mobile N- or C-termini of arrestin, were observed in the presence of a phosphopeptide corresponding to the C-terminus of rhodopsin, and with an R175Q mutant. Because both the phosphopeptide and the R175Q mutation promote binding to unphosphorylated R*, we conclude that arrestin is activated by subtle conformational changes. Most of the arrestin will be in a dimeric state in vivo. Using the arrestin structure as a guide [Hirsch, J.A., Schubert, C., Gurevich, V.V. & Sigler, P.B. (1999) Cell 97, 257–269], we have identified a model for the arrestin dimer that is consistent with our SAXS data. In this model, dimerization is mediated by the C-terminal domain of arrestin, leaving the N-terminal domains free for interaction with phosphorylated R*.

The b subunit of E. coli F(0)F(1)-ATPase links the peripheral F(1) subunits to the membrane-integral F(0) portion and functions as a "stator", preventing rotation of F(1). The b subunit is present as a dimer in ATP synthase, and residues 62-122 are required to mediate dimerization. To understand how the b subunit dimer is formed, we have studied the structure of the isolated dimerization domain, b(62-122). Analytical ultracentrifugation and solution small-angle X-ray scattering (SAXS) indicate that the b(62-122) dimer is extremely elongated, with a frictional ratio of 1.60, a maximal dimension of 95 A, and a radius of gyration of 27 A, values that are consistent with an alpha-helical coiled-coil structure. The crystal structure of b(62-122) has been solved and refined to 1.55 A. The protein crystallized as an isolated, monomeric alpha helix with a length of 90 A. Combining the crystal structure of monomeric b(62-122) with SAXS data from the dimer in solution, we have constructed a model for the b(62-122) dimer in which the two helices form a coiled coil with a right-handed superhelical twist. Analysis of b sequences from E. coli and other prokaryotes indicates conservation of an undecad repeat, which is characteristic of a right-handed coiled coil and consistent with our structural model. Mutation of residue Arg-83, which interrupts the undecad pattern, to alanine markedly stabilized the dimer, as expected for the proposed two-stranded, right-handed coiled-coil structure.

Preprotein translocase is a general and essential system for bacterial protein export, the minimal components of which are SecA and SecYEG. SecA is a peripheral ATPase that associates with nucleotide, preprotein, and the membrane integral SecYEG to form a translocation-competent complex. SecA can be separated into two domains: an N-terminal 68 kDa ATPase domain (N68) that binds preprotein and catalyzes ATP hydrolysis, and a 34 kDa C-terminal domain that regulates the ATPase activity of N68 and mediates dimerization. We have carried out gel filtration chromatography, analytical ultracentrifugation, and small-angle X-ray scattering (SAXS) to demonstrate that isolated N68 self-associates to form a tetramer in solution, indicating that removal of the C-terminal domain facilitates the formation of a higher-order SecA structure. The associative process is best modelled as a monomer-tetramer equilibrium, with a KD value of 63 mM 3 (where KDa [monomer] 4 /[tetramer]) so that at moderate concentrations (10 mM and above), the tetramer is the major species in solution. Hydrodynamic properties of the N68 monomer indicate that it is almost globular in shape, but the N68 tetramer has a more ellipsoidal structure. Analysis of SAXS data indicates that the N68 tetramer is a flattened, bi-lobed structure with dimensions of approximately 13.5 nm 9.0 nm 6.5 nm, that appears to contain a central pore. # 2002 Academic Press

Poxviruses encode a number of secreted virulence factors that function to mitigate or modulate the host immune response. M-T1 is a secreted 43-kDa glycoprotein produced by the myxoma virus, a poxvirus pathogen of rabbits, that binds CC-chemokines with high affinity, blocks binding to their cognate G-protein coupled receptors, and thereby inhibits chemokine-induced leukocyte chemotaxis. The present study indicates that M-T1, but not the related vaccinia virus 35-kDa CC-chemokine-binding protein, can localize to cell surfaces through an interaction with glycosaminoglycan molecules. In addition to biochemically characterizing the nature of this interaction, we demonstrate that M-T1 can also simultaneously interact with CC-chemokines while bound to heparin, suggesting that the binding sites on M-T1 for chemokines and heparin are distinct. Furthermore, using recombinant baculovirus-expressed M-T1 truncation and internal deletion mutants, we localize the heparin-binding region of M-T1 to the C terminus of the protein, a region that contains a high abundance of basic residues and includes two clusters of basic amino acid residues that resemble Cardin and Weintraub heparin-binding consensus sequences. The ability of M-T1 to simultaneously interact with chemokines and glycosaminoglycans may enable M-T1 to tether to endothelial surfaces or extracellular matrix and capture host chemokines that are expressed close to sites of virus infection.

The b subunit of ATP synthase is a major component of the second stalk connecting the F1and F0 sectors of the enzyme and is essential for normal assembly and function. The156-residue b subunit of the Escherichia coli ATP synthase has been investigated extensivelythrough mutagenesis, deletion analysis, and biophysical characterization. The two copies ofb exist as a highly extended, helical dimer extending from the membrane to near the top ofF1, where they interact with the δ subunit. The sequence has been divided into four domains:the N-terminal membrane-spanning domain, the tether domain, the dimerization domain, andthe C-terminal δ-binding domain. The dimerization domain, contained within residues 60–122,has many properties of a coiled-coil, while the δ-binding domain is more globular. Sites ofcrosslinking between b and the a, α, β, and δ subunits of ATP synthase have been identified,and the functional significance of these interactions is under investigation. The b dimer mayserve as an elastic element during rotational catalysis in the enzyme, but also directly influencesthe catalytic sites, suggesting a more active role in coupling.

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.

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.

A mutant of the periplasmic maltose-binding protein (MBP) with altered transport properties was studied. A change of residue 230 from tryptophan to arginine results in dominant-negative MBP: expression of this protein against a wild-type background causes inhibition of maltose transport. As part of an investigation of the mechanism of such inhibition, we have solved crystal structures of both unliganded and liganded mutant protein. In the closed, liganded conformation, the side-chain of R230 projects into a region of the surface of MBP that has been identified as important for transport while in the open form, the same side-chain takes on a different, and less ordered, conformation. The crystallographic work is supplemented with a small-angle X-ray scattering study that provides evidence that the solution conformation of unliganded mutant is similar to that of wild-type MBP. It is concluded that dominant-negative inhibition of maltose transport must result from the formation of a non-productive complex between liganded-bound mutant MBP and wild-type MalFGK2. A general kinetic framework for transport by either wild-type MalFGK2 or MBP-independent MalFGK2 is used to understand the effects of dominant-negative MBP molecules on both of these systems.

Small-angle X-ray scattering experiments were carried out for the maltose-, glucose/galactose- and ribose-binding proteins of Gram negative bacteria. All were shown to be monomers that decrease in radius of gyration on ligand binding. The results obtained for the maltose-binding protein agree well with crystal structures of the closed, ligand-bound, and open, ligand-free protein, suggesting that these are indeed the primary forms in solution. The closed form is stabilized by protein-sugar interactions, while the open conformation is stabilized by close contacts between the two domains. Since it is the proper special relationship of the domains in the closed form that is most important for interaction with chemotaxis and transport partners, the stabilization of the open form would help keep ligand-free molecules from interfering in function. The scattering results also provide evidence that a large conformational change takes place in association with ligand binding to the glucose/galactose- and ribose-binding proteins, and that the two changes are similar. Modeling suggests that the open forms resemble those found in the related leucine and leucine/isoleucine/valine-binding proteins, but are different from those observed for the maltose-binding protein and the related purine repressor.

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.

Quinone reductase 2 (NQO2) is a broadly expressed enzyme implicated in responses to a number of compounds, including protein kinase inhibitors, resveratrol, and antimalarial drugs. NQO2 includes a flavin adenine dinucleotide (FAD) cofactor, but X-ray crystallographic analysis of human NQO2 expressed in Escherichia coli showed that electron density for the isoalloxazine ring of FAD was weak and there was no electron density for the adenine mononucleotide moiety. Reversed-phase high-performance liquid chromatography (HPLC) of the NQO2 preparation indicated that FAD was not present and only 38% of the protomers contained flavin mononucleotide (FMN), explaining the weak electron density for FAD in the crystallographic analysis. A method for purifying NQO2 and reconstituting with FAD such that the final content approaches 100% occupancy with FAD is presented here. The enzyme prepared in this manner has a high specific activity, and there is strong electron density for the FAD cofactor in the crystal structure. Analysis of NQO2 crystal structures present in the Protein Data Bank indicates that many may have sub-stoichiometric cofactor content and/or contain FMN rather than FAD. This method of purification and reconstitution will help to optimize structural and functional studies of NQO2 and possibly other flavoproteins.

Phage panning led to the discovery of a disulfide-cyclized peptide CRYPEVEIC that inhibits Pin1 activity with a K(I) of 0.5 μM. NMR chemical shift perturbation experiments show that cyclic CRYPEVEIC binds to the active site of Pin1. Pin1 residues K63 and R68, which bind the phosphate of substrate peptides, do not show a significant chemical shift change in response to binding of cyclic CRYPEVEIC, consistent with absence of phosphate on the peptide. Cyclic CRYPEVEIC adopts a stable conformation with the side chains of the Y, P, V, and I residues packed together on one side of the ring. Cyclic CRYPEVEIC in solution exists as a mixture of two species, with a 1:4 cis/trans ratio for the Y-P bond. This mixture is stabilized to a single conformation when bound to Pin1. The constrained structure of cyclic CRYPEVEIC apparently facilitates high affinity binding without the presence of a phosphate group.

The site specificity of in vitro glycation of horse liver alcohol dehydrogenase (ADH) was examined and the results interpreted in terms of structural features of the enzyme molecule. In a phosphate buffer solution, glycation occurred at Lys231 (the main site of glycation in vivo), at Lys228 (which is not glycated in vivo), and at several unidentified positions. Buffer anions or NAD+ did not affect glycation of Lys231; this supported our hypothesis that the base catalyst which removes a proton from carbon 2 of a Lys231-attached aldimine is part of the ADH molecule [Shilton, B. H. & Walton, D. J. (1991) J. Biol. Chem. 266, 5587–5592]. Use of a molecular modelling programme indicated that this catalyst was likely to be the imidazole group of His348, exerting its effect through the hydroxyl of Thr347. Glycation of Lys228 occurred only in the presence of phosphate; in this case molecular modelling showed that the base catalyst could be a phosphate ion, bound to ADH at a positive region of the coenzyme binding site. NAD+ inhibited glycation of Lys228 by binding to the enzyme and restricting access to glucose.

Synthetic triterpenoids are anti-tumor agents that affect numerous cellular functions including apoptosis and growth inhibition. Here, we used mass spectrometric and protein array approaches and uncovered that triterpenoids associate with proteins of the actin cytoskeleton, including actin-related protein 3 (Arp3). Arp3, a subunit of the Arp2/3 complex, is involved in branched actin polymerization and the formation of lamellipodia. 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO)-Im and CDDO-Me were observed to 1) inhibit the localization of Arp3 and actin at the leading edge of cells, 2) abrogate cell polarity, and 3) inhibit Arp2/3-dependent branched actin polymerization. We confirmed our drug effects with siRNA targeting of Arp3 and observed a decrease in Rat2 cell migration. Taken together, our data suggest that synthetic triterpenoids target Arp3 and branched actin polymerization to inhibit cell migration.

Phosphorylation enhances IGFBP-1 binding to IGF-I, thereby limiting the bioavailability of IGF-I that may be important in fetal growth. Our goal in this study was to determine whether changes in site-specific IGFBP-1 phosphorylation were unique to fetal growth restriction. To establish a link, we compared IGFBP-1 phosphorylation (sites and degree) in amniotic fluid from FGR (N = 10) and controls (N = 12). The concentration of serine phosphorylated IGFBP-1 showed a negative correlation with birth weight in FGR (P = 0.049). LC-MS/MS analysis revealed all four previously identified phosphorylation sites (Ser98, Ser101, Ser119, and Ser169) to be common to FGR and control groups. Relative phosphopeptide intensities (LC-MS) between FGR and controls demonstrated 4-fold higher intensity for Ser101 (P = 0.026), 7-fold for Ser98/Ser101 (P = 0.02), and 23-fold for Ser169 (P = 0.002) in the FGR group. Preliminary BIAcore data revealed 4-fold higher association and 1.7-fold lower dissociation constants for IGFBP-1/IGF-I in FGR. A structural model of IGFBP-1 bound to IGF-I indicates that all the phosphorylation sites are on relatively mobile regions of the IGFBP-1 sequence. Residues Ser98, Ser101, and Ser169 are close to structured regions that are involved in IGF-I binding and, therefore, could potentially make direct contact with IGF-I. On the other hand, residue Ser119 is in the middle of the unstructured linker that connects the N- and C-terminal domains of IGFBP-1. The model is consistent with the assumption that residues Ser98, Ser101, and Ser169 could directly interact with IGF-I, and therefore phosphorylation at these sites could change IGF-I interactions. We suggest that site-specific increase in IGFBP-1 phosphorylation limits IGF-I bioavailability, which directly contributes to the development of FGR. This study delineates the potential role of higher phosphorylation of IGFBP-1 in FGR and provides the basis to substantiate these findings with larger sample size.

ATP hydrolysis by the maltose transporter (MalFGK(2)) is regulated by maltose binding protein (MBP). Binding of maltose to MBP brings about a conformational change from open to closed that leads to a strong stimulation of the MalFGK(2) ATPase. In this study, we address the long-standing but enigmatic observation that unliganded MBP is also able to stimulate MalFGK(2). Although the mechanism of this stimulation is not understood, it is sometimes attributed to a small amount of closed (but unliganded) MBP that may exist in solution. To gain insight into how MBP regulates the MalFGK(2) ATPase, we have investigated whether the open or the closed conformation of MBP is responsible for MalFGK(2) stimulation in the absence of maltose. The effect of MBP concentration on the stimulation of MalFGK(2) was assessed: for unliganded MBP, the apparent K(M) for stimulation of MalFGK(2) was below 1 microM, while for maltose-bound MBP, the K(M) was approximately 15 microM. We show that engineered MBP molecules in which the open-closed equilibrium has been shifted toward the closed conformation have a decreased ability to stimulate MalFGK(2). These results indicate that stimulation of the MalFGK(2) ATPase by unliganded MBP does not proceed through a closed conformation and instead must operate through a different mechanism than stimulation by liganded MBP. One possible explanation is that the open conformation is able to activate the MalFGK(2) ATPase directly.

Sites of in vivo glycation of human and horse liver alcohol dehydrogenase were identified by cleavage of the borotritide-treated enzyme with trypsin, followed by gas-phase sequencing of the resulting tritium-labeled glycated peptides. A blank sequencing result, i.e. failure to detect an amino acid phenylthiohydantoin after completion of an Edman degradation cycle, was ascribed to an N-(1-deoxyhexitolyl)lysyl residue, which represented a glycation site on the original enzyme subunit. In human liver alcohol dehydrogenase the sites affected were the epsilon-amino groups of lysines 10, 39, 231, 248, and 325, which were glycated to the relative extents of 10, 5, 75, 5, and 5%, respectively. The site specificity of in vivo glycation of the horse enzyme is similar; 70-75% of it had occurred at lysine 231. A computer image of the crystal structure of horse liver alcohol dehydrogenase was examined. As a result, it was proposed that the high rate of glycation at lysine 231 is due to acid-base catalysis of the Amadori rearrangement by the imidazole group of histidine 348. This hypothesis was supported by showing that imidazole groups were close to sites of glycation in several other proteins.

The catalytic domain of the peptidyl-prolyl cis/ trans isomerase Pin1 is a member of the FKBP superfold family. Within its active site are two highly conserved histidine residues, H59 and H157. Despite their sequence conservation in parvulin PPIase domains, the role of these histidine residues remains unclear. Our previous work (Behrsin et al. (2007) J. Mol. Biol. 365, 1143- 1162.) was consistent with a model where one or both histidines had critical roles in a hydrogen bonding network in the active site. Here, we test this model by looking at the effect of mutations to H59 and H157 on Pin1 function, activity, and protein stability. Using a yeast complementation assay, we show that both H59 and H157 can be mutated to non-hydrogen bonding residues and still support viability. Surprisingly, a nonfunctional H59L mutation can be rescued by a mutation of H157, to leucine. This double mutation (H59L/H157L) also had about 5-fold greater isomerase activity than the H59L mutation with a phosphorylated substrate. Structural analyses suggest that rescue of function and activity results from partial rescue of protein stability. Our findings indicate that H59 and H157 are not required for hydrogen bonding within the active site, and in contrast to the active site C113, they do not participate directly in catalysis. Instead, we suggest these histidines play a key role in domain structure or stability.

The preprotein cross-linking domain and C-terminal domains of Escherichia coli SecA were removed to create a minimal DEAD motor, SecA-DM. SecA-DM hydrolyzes ATP and has the same affinity for ADP as full-length SecA. The crystal structure of SecA-DM in complex with ADP was solved and shows the DEAD motor in a closed conformation. Comparison with the structure of the E. coli DEAD motor in an open conformation (Protein Data Bank ID 2FSI) indicates main-chain conformational changes in two critical sequences corresponding to Motif III and Motif V of the DEAD helicase family. The structures that the Motif III and Motif V sequences adopt in the DEAD motor open conformation are incompatible with the closed conformation. Therefore, when the DEAD motor makes the transition from open to closed, Motif III and Motif V are forced to change their conformations, which likely functions to regulate passage through the transition state for ATP hydrolysis. The transition state for ATP hydrolysis for the SecA DEAD motor was modeled based on the conformation of the Vasa helicase in complex with adenylyl imidodiphosphate and RNA (Protein Data Bank ID 2DB3). A mechanism for chemical-mechanical coupling emerges, where passage through the transition state for ATP hydrolysis is hindered by the conformational changes required in Motif III and Motif V, and may be promoted by binding interactions with the preprotein substrate and/or other translocase domains and subunits.

It remains an open question as to whether experiments involving collision-induced dissociation (CID) can provide a viable approach for monitoring spatially resolved deuteration levels in electrosprayed polypeptide ions. A number of laboratories reported the successful application of CID following solution-phase H/D exchange (HDX), whereas others found that H/D scrambling precluded site-specific measurements. The aim of the current work is to help clarify the general feasibility of HDX-CID methods, using a 22-residue zinc-bound protein domain (Zn-ZBD) as model system. Metal binding in Zn-ZBD should confer structural rigidity, and the presence of several basic residues should sequester mobile charge carriers in the gas phase. Both of these factors were expected to suppress the extent of scrambling. HDX was carried out by employing rapid on-line mixing, thereby mimicking conditions typically encountered in kinetic pulse-labeling studies. Quadrupole time-of-flight MS/MS of pulse-labeled Zn-ZBD provides high sequence coverage. However, the measured fragment deuteration levels do not correlate with the known H-bonding pattern of Zn-ZBD, suggesting the occurrence of extensive scrambling. Instead of showing a uniform distribution, the fragment ions reveal a distinct nonrandom pattern of deuteration levels. In the absence of prior information, these data could erroneously be ascribed to the presence of protected sites. However, the observed patterns clearly originate from other factors; possibly they are caused by modulations of the amide CID efficiency by kinetic isotope effects. It is concluded that scrambling does not represent the only conceptual problem in HDX-CID studies and that control experiments on uniformly labeled samples are essential for ruling out interpretation artifacts.