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1.  Determination of the Substrate Binding Mode to the Active Site Iron of (S)-2-Hydroxypropylphosphonic Acid Epoxidase Using 17O-Enriched Substrates and Substrate Analogues† 
Biochemistry  2007;46(44):12628-12638.
(S)-2-hydroxypropylphosphonic acid epoxidase (HppE) is an O2-dependent, nonheme Fe(II)-containing oxidase that converts (S)-2-hydroxypropylphosphonic acid ((S)-HPP) to the regio-and enantiomerically specific epoxide, fosfomycin. Use of (R)-2-hydroxypropylphosphonic acid ((R)-HPP) yields the 2-keto-adduct rather than the epoxide. Here we report the chemical synthesis of a range of HPP analogs designed to probe the basis for this specificity. In past studies, NO has been used as an O2 surrogate to provide an EPR probe of the Fe(II) environment. These studies suggest that O2 binds to the iron, and substrates bind in a single orientation that strongly perturbs the iron environment. Recently, the X-ray crystal structure showed direct binding of the substrate to the iron, but both monodentate (via the phosphonate) and chelated (via the hydroxyl and phosphonate) orientations were observed. In the current study, hyperfine broadening of the homogeneous S = 3/2 EPR spectrum of the HppE-NO-HPP complex was observed when either the hydroxyl or the phosphonate group of HPP was enriched with 17O (I = 5/2). These results indicate that both functional groups of HPP bind to Fe(II) ion at the same time as NO, suggesting that the chelated substrate binding mode dominates in solution. (R)- and (S)-analog compounds that maintained the core structure of HPP but added bulky terminal groups were turned over to give products analogous to those from (R)- and (S)-HPP, respectively. In contrast, substrate analogs lacking either the phosphonate or hydroxyl group were not turned over. Elongation of the carbon chain between the hydroxyl and phosphonate allowed binding to the iron in a variety of orientations to give keto and diol products at positions determined by the hydroxyl substituent, but no stable epoxide was formed. These studies show the importance of the Fe(II)-substrate chelate structure to active antibiotic formation. This fixed orientation may align the substrate next to the iron-bound activated oxygen species thought to mediate hydrogen atom abstraction from the nearest substrate carbon.
PMCID: PMC2780580  PMID: 17927218
2.  A distal phenylalanine clamp in a hydrophobic channel controls the substrate specificity in the quorum-quenching metallo-γ-lactonase (AiiA) from Bacillus thuringiensis† 
Biochemistry  2013;52(9):1603-1610.
AiiA is a metal-dependent N-acyl homoserine lactone hydrolase that displays broad substrate specificity, but shows preference for substrates with long N-acyl substitutions. Previously, crystal structures of AiiA in complex with the ring-opened product N-hexanoyl-l-homoserine revealed binding interactions near the metal center, but did not identify a binding pocket for the N-acyl chains of longer substrates. Here we report the crystal structure of an AiiA mutant, F107W, determined in the presence and absence of N-decanoyl-l-homoserine. F107 is located in a hydrophobic cavity adjacent to the previously identified ligand binding pocket, and F107W results in the formation of an unexpected interaction with the ring-opened product. Notably, the structure reveals a previously unidentified hydrophobic binding pocket for the substrate’s N-acyl chain. Two aromatic residues, F64 and F68 form a hydrophobic clamp, centered around the seventh carbon in the product-bound structure’s decanoyl chain, making an interaction that would also be available for longer substrates, but not for shorter substrates. Steady-state kinetics using substrates of various lengths with AiiA bearing mutations at the hydrophobic clamp, including insertion of a redox sensitive cysteine pair, confirms the importance of this hydrophobic feature for substrate preference. Identifying the specificity determinants of AiiA will aid the development of more selective quorum-quenching enzymes as tools and as potential therapeutics.
PMCID: PMC3603367  PMID: 23387521
AiiA; lactonase; dizinc hydrolase; substrate specificity; quorum quenching; N-acyl homoserine lactone
3.  Design of High-Activity Mutants of Human Butyrylcholinesterase against (−)-Cocaine: Structural and Energetic Factors Affecting the Catalytic Efficiency† 
Biochemistry  2010;49(42):9113-9119.
The present study was aimed to explore the correlation between the protein structure and catalytic efficiency of butyrylcholinesterase (BChE) mutants against (−)-cocaine by modeling the rate-determining transition state (TS1), i.e. the transition state for the first step of chemical reaction process, of (−)-cocaine hydrolysis catalyzed by various mutants of human BChE in comparison with the wild-type. Molecular modeling of the TS1 structures revealed that mutations on certain non-active site residues can indirectly affect the catalytic efficiency of the enzyme against (−)-cocaine through enhancing or weakening the overall hydrogen bonding between the carbonyl oxygen of (−)-cocaine benzoyl ester and the oxyanion hole of the enzyme. Computational insights and predictions were supported by the catalytic activity data obtained from wet experimental tests on the mutants of human BChE, including five new mutants reported for the first time. The BChE mutants with at least ~1000-fold improved catalytic efficiency against (−)-cocaine compared to the wild-type BChE are all associated with the TS1 structures having stronger overall hydrogen bonding between the carbonyl oxygen of (−)-cocaine benzoyl ester and the oxyanion hole of the enzyme. The combined computational and experimental data demonstrate a reasonable correlation relationship between the hydrogen bonding distances in the TS1 structure and the catalytic efficiency of the enzyme against (−)cocaine.
PMCID: PMC2963158  PMID: 20886866
4.  Heme Iron Nitrosyl Complex of MauG Reveals an Efficient Redox Equilibrium Between Hemes with Only One Heme Exclusively Binding Exogenous Ligands 
Biochemistry  2009;48(49):11603-11605.
MauG is a diheme enzyme that oxidizes two protein-bound tryptophan residues to generate a catalytic tryptophan tryptophylquinone cofactor within methylamine dehydrogenase. Upon the two-electron oxidation of bis-ferric MauG, the two c-type hemes exist as a spin-uncoupled bis-Fe(IV) species with only one binding oxygen, which is chemically equivalent to a single ferryl heme plus a π porphyrin cation radical (Li, X. et al. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 8597–8600). The EPR spectrum of the nitrosyl complex of fully reduced MauG shows a single six-coordinate Fe(II)-NO species, which is characteristic of a histidine-ligated Fe(II)-NO moiety in the heme environment. Exposure of partially reduced MauG to NO reveals a redox equilibrium with facile electron transfer between hemes, but with only one binding nitric oxide. Thus, the second heme is able to stabilize all three redox states of iron (Fe(II), Fe(III) and Fe(IV)) in a six-coordinate protein-bound heme without binding exogenous ligands. This is unprecedented behavior for a protein-bound heme for which each of these redox states is relevant to the overall catalytic mechanism. The results also illustrate the electronic communication between the two iron centers which function as a diheme unit rather than independent heme cofactors.
PMCID: PMC2801551  PMID: 19911786
5.  Purification and Characterization of the Epoxidase Catalyzing the Formation of Fosfomycin from Pseudomonas syringae 
Biochemistry  2008;47(33):8726-8735.
The final step in the biosynthesis of fosfomycin in Streptomyces wedmorensis is catalyzed by (S)-2-hydroxypropylphosphonic acid (HPP) epoxidase (Sw-HppE). A homologous enzyme from Pseudomonas syringae has recently been isolated whose encoding gene (orf3) shares relatively low sequence homology to the corresponding Sw-HppE gene. This purified P. syringae protein was determined to catalyze the epoxidation of (S)-HPP to fosfomycin and the oxidation of (R)-HPP to 2-oxopropylphosphonic acid under the same conditions as Sw-HppE. Therefore, this protein is indeed a true HPP epoxidase and is termed Ps-HppE. Like Sw-HppE, Ps-HppE was determined to be post-translationally modified by the hydroxylation of a putative active site tyrosine (Tyr95). Analysis of the Fe(II)-center by EPR spectroscopy using NO as a spin probe and molecular oxygen surrogate reveals that Ps-HppE’s metal center is similar, but not identical, to that of Sw-HppE. The identity of the rate determining step for the (S)-HPP and (R)-HPP reactions was determined by measuring primary deuterium kinetic effects, and the outcome of these results were correlated with density functional theory calculations. Interestingly, the reaction using the non-physiological substrate (R)-HPP was 1.9 times faster than that with (S)-HPP for both Ps-HppE and Sw-HppE. This is likely due to the difference in bond dissociation energy of the abstracted hydrogen atom for each respective reaction. Thus, despite low amino acid sequence identity, Ps-HppE is a close mimic of Sw-HppE, representing a second example of a non-heme iron-dependent enzyme capable of catalyzing dehydrogenation of a secondary alcohol to form a new C-O bond.
PMCID: PMC2780581  PMID: 18656958
6.  Overexpression and functional characterization of the extracellular domain of the human α1 glycine receptor 
Biochemistry  2008;47(37):9803-9810.
A novel truncated form (residues 1–214, with a randomized C-terminal tail) of the ligand-binding extracellular domain (ECD) of the human α1 glycine receptor (GlyR), with amino acids from the corresponding sequence of an acetylcholine binding protein (AChBP) substituted for two relatively hydrophobic membrane-proximal loops, was overexpressed using a baculovirus expression system. The mutant GlyR ECD, named GlyBP, was present in both soluble and membrane-associated fractions after cell lysis, though only the latter appeared to be in a native-like conformation capable of binding strychnine, a GlyR specific antagonist. The membrane-associated GlyBP was solubilized and detergent/lipid/protein micelles were affinity purified. After detergent removal, GlyBP may be isolated in either aqueous or vesicular form. Binding assays and spectroscopic studies using circular dichroism and FRET are consistent with both forms adopting equivalent native-like conformations. Thus GlyBP may be isolated as a soluble or membrane-associated assembly that serves as a structural and functional homolog of the ECD of GlyR.
PMCID: PMC2705929  PMID: 18710260
7.  The role of protein dynamics in thymidylate synthase catalysis 
Biochemistry  2006;45(24):7415-7428.
The enzyme thymidylate synthase (TS) catalyzes the reductive methylation of 2′-deoxyuridine 5′-monophosphate (dUMP) to 2′-deoxythymidine 5′-monophosphate. Using kinetic and x-ray crystallography experiments, we have examined the role of the highly conserved Tyr-261 in the catalytic mechanism of TS. While Tyr-261 is distant from the site of methyl transfer, mutants at this position show a marked decrease in enzymatic activity. Given that Tyr-261 forms a hydrogen bond with the dUMP 3′-O, we hypothesized that this interaction would be important for substrate binding, orientation, and specificity. Our results, surprisingly, show that Tyr-261 contributes little to these features of the mechanism of TS. However, the residue is part of the structural core of closed ternary complexes of TS, and conservation of the size and shape of the Tyr side chain is essential for maintaining wild-type values of kcat/Km. Moderate increases in Kms for both substrate and the cofactor upon mutation of Tyr-261 arise mainly from destabilization of the active conformation of a loop containing a dUMP-binding arginine. Besides binding dUMP, this loop has a key role in stabilizing the closed conformation of the enzyme and in shielding the active site from bulk solvent during catalysis. Changes to atomic vibrations in crystals of a ternary complex of E. coli Tyr261Trp are associated with a greater than 2000-fold drop in kcat/Km. These results underline the important contribution of dynamics to catalysis in TS.
PMCID: PMC2556892  PMID: 16768437
8.  A Retro-Evolution Study of CDP-6-deoxy-D-glycero-L-threo-4-hexulose-3-dehydrase (E1) from Yersinia pseudotuberculosis: Implications for C-3 Deoxygenation in the Biosynthesis of 3,6-Dideoxyhexoses† 
Biochemistry  2007;46(12):3759-3767.
CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase (E1), which catalyzes C-3 deoxygenation of CDP-4-keto-6-deoxyglucose in the biosynthesis of 3,6-dideoxyhexoses, shares a modest sequence identity with other B6-dependent enzymes, albeit with two important distinctions. It is a rare example of a B6-dependent enzyme that harbors a [2Fe-2S] cluster, and a highly conserved lysine that serves as an anchor for PLP in most B6-dependent enzymes is replaced by histidine at position 220 in E1. Since alteration of His220 to a lysine residue may produce a putative progenitor of E1, the H220K mutant was constructed and tested for the ability to process the predicted substrate, CDP-4-amino-4,6-dideoxyglucose, using PLP as the coenzyme. Our data showed that H220K-E1 has no dehydrase activity, but can act as a PLP-dependent transaminase. However, the reaction is not catalytic since PLP cannot be regenerated during turnover. Reported herein are the results of this investigation and the implications for the role of His220 in the catalytic function and mechanism of E1.
PMCID: PMC2515278  PMID: 17323931
9.  Characterization of TDP-4-Keto-6-Deoxy-D-Glucose-3,4-Ketoisomerase (Tyl1a) from the D-Mycaminose Biosynthetic Pathway of Streptomyces fradiae: in vitro Activity and Substrate Specificity Studies 
Biochemistry  2007;46(2):577-590.
Deoxysugars are critical structural elements for the bioactivity of many natural products. Ongoing work toward elucidating a variety of deoxysugar biosynthetic pathways has paved the way for manipulation of these pathways to generate structurally diverse glycosylated natural products. In the course of this work, the biosynthesis of D-mycaminose in the tylosin pathway of Streptomyces fradiae was investigated. Attempts to reconstitute the entire mycaminose biosynthetic machinery in a heterologous host led to the discovery of a previously overlooked gene, tyl1a, encoding an enzyme thought to convert TDP-4-keto-6-deoxy-D-glucose to TDP-3-keto-6-deoxy-D-glucose, a 3,4-ketoisomerization reaction in the pathway. Tyl1a has now been overexpressed, purified, assayed, and its activity verified by product analysis. Incubation of Tyl1a and the C-3 aminotransferase TylB, the next enzyme in the pathway, produced TDP-3-amino-3,6-dideoxy-D-glucose, confirming that these two enzymes act sequentially. Steady state kinetic parameters of the Tyl1a-catalyzed reaction were determined, and the ability of Tyl1a and TylB to process a C-2 deoxygenated substrate and a CDP-linked substrate was also demonstrated. Enzymes catalyzing 3,4-ketoisomerization of hexoses represent a new class of enzymes involved in unusual sugar biosynthesis. The fact that Tyl1a shows relaxed substrate specificity holds potential for future deoxysugar biosynthetic engineering endeavors.
PMCID: PMC2515277  PMID: 17209568
10.  Characterization and Mechanistic Studies of Type II Isopentenyl Diphosphate:Dimethylallyl Diphosphate Isomerase from Staphylococcus aureus 
Biochemistry  2007;46(28):8401-8413.
The recently identified type II isopentenyl diphosphate (IPP):dimethylallyl diphosphate (DMAPP) isomerase (IDI-2) is a flavoenzyme that requires FMN and NAD(P)H for activity. IDI-2 is an essential enzyme for the biosynthesis of isoprenoids in several pathogenic bacteria including Staphylococcus aureus, Streptococcus pneumoniae, and Enterococcus faecalis, and thus is considered as a potential new drug target to battle bacterial infections. One notable feature of the IDI-2 reaction is that there is no net change in redox state between the substrate (IPP) and product (DMAPP), indicating that the FMN cofactor must start and finish each catalytic cycle in the same redox state. Here, we report the characterization and initial mechanistic studies of the S. aureus IDI-2. The steady-state kinetic analyses under aerobic and anaerobic conditions show that FMN must be reduced to be catalytically active and the overall IDI-2 reaction is O2 sensitive. Interestingly, our results demonstrate that NADPH is needed only in catalytic amounts to activate the enzyme for multiple turnovers of IPP to DMAPP. The hydride transfer from NAD(P)H to reduce FMN is determined to be pro-S stereospecific. Photoreduction and oxidation-reduction potential studies reveal that the S. aureus IDI-2 can stabilize significant amounts of the neutral FMN semiquinone. In addition, reconstitution of apo-IDI-2 with 5-deazaFMN resulted in a dead enzyme, whereas reconstitution with 1-deazaFMN led to the full recovery of enzyme activity. Taken together, these studies of S. aureus IDI-2 support a catalytic mechanism in which the reduced flavin coenzyme mediates a single electron transfer to and from the IPP substrate during catalysis.
PMCID: PMC2515275  PMID: 17585782
11.  Biosynthesis of Fosfomycin, Re-examination and Re-confirmation of A Unique Fe(II) and NAD(P)H-dependent Epoxidation Reaction 
Biochemistry  2006;45(38):11473-11481.
(S)-2-hydroxypropylphosphonic acid epoxidase (HppE) catalyzes the epoxide ring closure of (S)-HPP to form fosfomycin, a clinically useful antibiotic. Early investigation showed that its activity can be reconstituted with Fe(II), FMN, NADH, and O2, and identified HppE as a new type of mononuclear non-heme iron-dependent oxygenase involving high valent iron-oxo species in the catalysis. However, a recent study showed that the Zn(II)-reconstituted HppE is active, and HppE exhibits modest affinity for FMN. Thus, a new mechanism is proposed in which the active site bound Fe2+ or Zn2+ serves as a Lewis acid to activate the 2-OH group of (S)-HPP, and the epoxide ring is formed by the attack of the 2-OH group at C-1 coupled with the transfer of the C-1 hydrogen as a hydride ion to the bound FMN. To distinguish between these mechanistic discrepancies, we re-examined the bioautography assay, the basis for the alternative mechanism, and showed that Zn(II) cannot replace Fe(II) in the HppE reaction, and NADH is indispensable. Moreover, we demonstrated that the proposed role for FMN as a hydride acceptor is inconsistent with the finding that FMN cannot bind to HppE in the presence of substrate. In addition, using a newly developed HPLC assay we showed that several non-flavin electron mediators could replace FMN in the HppE-catalyzed epoxidation. Taken together, these results argue against the newly proposed “nucleophilic displacement-hydride transfer” mechanism, but are fully consistent with the previously proposed iron-redox mechanism for HppE catalysis, which is unique within the mononuclear non-heme iron enzyme superfamily.
PMCID: PMC2515266  PMID: 16981707
12.  Cooperative Interaction of Human XPA Stabilizes and Enhances Specific Binding of XPA to DNA Damage† 
Biochemistry  2005;44(19):7361-7368.
Human xeroderma pigmentosum group A (XPA) is an essential protein for nucleotide excision repair (NER). We have previously reported that XPA forms a homodimer in the absence of DNA. However, what oligomeric forms of XPA are involved in DNA damage recognition and how the interaction occurs in terms of biochemical understanding remain unclear. Using the homogeneous XPA protein purified from baculovirus-infected sf21 insect cells and the methods of gel mobility shift assays, gel filtration chromatography, and UV-cross-linking, we demonstrated that both monomeric and dimeric XPA bound to the DNA adduct of N-acetyl-2-aminofluorene (AAF), while showing little affinity for nondamaged DNA. The binding occurred in a sequential and protein concentration-dependent manner. At relatively low-protein concentrations, XPA formed a complex with DNA adduct as a monomer, while at the higher concentrations, an XPA dimer was involved in the specific binding. Results from fluorescence spectroscopic and competitive binding analyses indicated that the specific binding of XPA to the adduct was significantly facilitated and stabilized by the presence of the second XPA in a positive cooperative manner. This cooperative binding exhibited a Hill coefficient of 1.9 and the step binding constants of K1 = 1.4 × 106 M-1 and K2) = 1.8 × 107 M-1. When interaction of XPA and RPA with DNA was studied, even though binding of RPA-XPA complex to adducted DNA was observed, the presence of RPA had little effect on the overall binding efficiency. Our results suggest that the dominant form for XPA to efficiently bind to DNA damage is the XPA dimer. We hypothesized that the concentration-dependent formation of different types of XPA-damaged DNA complex may play a role in cellular regulation of XPA activity.
PMCID: PMC1475732  PMID: 15882075
13.  Molecular Mechanism of Photoactivation and Structural Location of the Cyanobacterial Orange Carotenoid Protein 
Biochemistry  2013;53(1):13-19.
The Orange Carotenoid Protein (OCP) plays a similar photoprotective role in cyanobacterial photosynthesis to that of non-photochemical quenching in higher plants. Under high-light conditions, OCP binds to the phycobilisome (PBS) and reduces energy transfer to the photosystems. The protective cycle starts from a light-induced activation of OCP. Detailed information on the molecular mechanism of this process as well as the subsequent recruitment of active OCP to the phycobilisome is not known. We report here our investigation on OCP photoactivation from the cyanobacterium Synechocystis sp. PCC 6803 by using a combination of native mass spectrometry (MS) and protein cross-linking. We demonstrate that Native MS is able to capture OCP with its intact pigment and further reveal that OCP undergoes a dimer-tomonomer transition upon light illumination. The reversion of activated form of OCP to inactive, dark form was also observed by using native MS. Furthermore, in vitro reconstitution of OCP and PBS allowed to perform protein chemical cross-linking experiments. LC-MS/MS analysis identified cross-linking species between OCP and the PBS core components. Our result indicates that the N-terminal domain of OCP is closely involved in the association with a site formed by two allophycocyanin trimers in the basal cylinders of the phycobilisome core. This report helps to understand the activation mechanism of OCP and the structural binding site of OCP during the cyanobacterial non-photochemical quenching process.
PMCID: PMC3963514  PMID: 24359496
14.  Selectivity of Vibrio cholerae H-NOX for Gaseous Ligands Follows “Sliding Scale Rule” Hypothesis 
Biochemistry  2013;52(52):9432-9446.
Vc H-NOX (or VCA0720) is an H-NOX (heme-nitric oxide and oxygen binding) protein from facultative aerobic bacterium Vibrio cholerae. It shares significant sequence homology with soluble guanylyl cyclase (sGC), a NO sensor protein commonly found in animals. Similar to sGC, Vc H-NOX binds strongly to NO and CO with affinities of 0.27 nM and 0.77 μM, respectively, but weakly to O2. When positioned in “sliding scale” plot {Tsai, A.-L. et. al. (2012) Biochemistry, 51, pp172-86}, the line connecting logKD(NO) and logKD(CO) of Vc H-NOX is almost superimposable with that of Ns H-NOX. Therefore, the measured affinities and kinetic parameters of gaseous ligands to Vc H-NOX provide more evidence to validate the “sliding scale rule” hypothesis. Like sGC, Vc H-NOX binds NO in multiple steps, forming first a 6-coordinate heme-NO complex with a rate of 1.1 × 109 M−1s−1, and then converts to a 5c heme-NO complex at a rate also dependent on [NO]. Although the formation of oxyferrous Vc H-NOX is not detectable under normal atmospheric oxygen level, ferrous Vc H-NOX is oxidized to ferric form at a rate of 0.06 s−1 when mixed with O2. Ferric Vc H-NOX exists as a mixture of high- and low-spin states and is influenced by binding to different ligands. Characterization of both ferric and ferrous Vc H-NOX and their complexes with various ligands lay the foundation for understanding the possible dual roles in gas and redox sensing of Vc H-NOX.
PMCID: PMC3999706  PMID: 24351060
15.  DNA Recognition of 5-Carboxylcytosine by a Zfp57 Mutant at an Atomic Resolution of 0.97 Å 
Biochemistry  2013;52(51):10.1021/bi401360n.
The Zfp57 gene encodes a KRAB (Krüppel-associated box) domain-containing C2H2 zinc finger transcription factor that is expressed in early development. Zfp57 protein recognizes methylated CpG dinucleotide within GCGGCA elements at multiple imprinting control regions. In the previously determined structure of the mouse Zfp57 DNA-binding domain in complex with DNA containing 5-methylcytosine (5mC), the side chains of Arg178 and Glu182 contact the methyl group via hydrophobic and van der Waals interactions. We examined the role of Glu182 in recognition of 5mC by mutagenesis. The majority of mutants examined lose selectivity of methylated (5mC) over unmodified (C) and oxidative derivatives, 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine (5caC), suggesting that the side chain of Glu182 (the size and the charge) is dispensable for methyl group recognition but negatively impacts the binding of unmodified cytosine as well as oxidized derivatives of 5mC to achieve 5mC selectivity. Substitution of Glu182 with its corresponding amide (E182Q) had no effect on methylated DNA binding but gained significant binding affinity for 5caC DNA, resulting in a binding affinity for 5caC DNA comparable to that of the wild-type protein for 5mC. We show structurally that the uncharged amide group of E182Q interacts favorably with the carboxylate group of 5caC. Furthermore, introducing a positively charged arginine at position 182 resulted in a mutant (E182R) having higher selectivity for the negatively charged 5caC.
PMCID: PMC3884566  PMID: 24236546
16.  Allele-Selective Inhibition of Huntingtin and Ataxin-3 Expression by RNA Duplexes Containing Unlocked Nucleic Acid (UNA) Substitutions 
Biochemistry  2013;52(51):9329-9338.
Unlocked nucleic acid (UNA) is an acyclic analog of RNA that can be introduced into RNA or DNA oligonucleotides. The increased flexibility conferred by the acyclic structure fundamentally affects the strength of base-pairing, createing opportunities for improved applications and new insights into molecular recognition. Here we test how UNA substitutions affect allele-selective inhibition of trinucleotide-repeat genes Huntingtin (HTT) and Ataxin-3 (ATX-3) expression. We find that the either the combination of mismatched bases and UNA substitutions or UNA substitutions alone can improve potency and selectivity. Inhibition is potent and selectivities of > 40-fold for inhibiting mutant versus wild-type expression can be achieved. Surprisingly, even though UNA preserves the potential for complete base-pairing, the introduction of UNA substitutions at central positions within fully complementary duplexes leads to >19-fold selectivity. Like mismatched bases, the introduction of central UNA bases disrupts the potential for cleavage of substrate by Argonaute 2 (AGO2) during gene silencing. UNA-substituted duplexes are as effective as other strategies for allele-selective silencing of trinucleotide repeat disease genes. Modulation of AGO2 activity by the introduction of UNA substitutions demonstrates that backbone flexibility is as important as base-pairing for catalysis of fully complementary duplex substrates. UNA can be used to tailor RNA silencing for optimal properties and allele-selective action.
PMCID: PMC3893079  PMID: 24266403
17.  Development of RNA aptamers targeting Ebola virus VP35 
Biochemistry  2013;52(47):8406-8419.
Viral protein 35 (VP35), encoded by filoviruses, are multifunctional dsRNA binding proteins that play important roles in viral replication, innate immune evasion and pathogenesis. The multifunctional nature of these proteins also presents opportunities to develop countermeasures that target distinct functional regions. However, functional validation and the establishment of therapeutic approaches toward such multifunctional proteins, particularly for non-enzymatic targets, are often challenging. Our previous work on filoviral VP35 proteins defined conserved basic residues located within its C-terminal dsRNA binding interferon (IFN) inhibitory domain (IID) as important for VP35 mediated IFN antagonism and viral polymerase co-factor functions. In the current study, we used a combination of structural and functional data to determine regions of Ebola virus (EBOV) VP35 (eVP35) to target for aptamer selection using SELEX. Select aptamers, representing two distinct classes, were further characterized based on their interaction properties to eVP35 IID. These results revealed that the aptamers bind to distinct regions of eVP35 IID with high affinity (10–50 nM) and specificity. These aptamers can compete with dsRNA for binding to eVP35 and disrupt the eVP35-nucleoprotein (NP) interaction. Consistent with the ability to antagonize eVP35-NP interaction, select aptamers can inhibit the function of the EBOV polymerase complex reconstituted by expression of select viral proteins. Taken together, our results support the identification of two aptamers that bind filoviral VP35 proteins with high affinity and specificity and have the capacity to potentially target filoviral VP35 proteins as a therapeutic target.
PMCID: PMC3909728  PMID: 24067086
RNA aptamers; filovirus; viral protein VP35; inhibitors; antivirals
18.  Kinetics of Presynaptic Filament Assembly in the Presence of SSB and Mediator Proteins† 
Biochemistry  2013;52(45):10.1021/bi401060p.
Enzymes of the RecA/Rad51 family catalyze DNA strand exchange reactions that are important for homologous recombination and for the accurate repair of DNA double-strand breaks. RecA/Rad51 recombinases are activated by their assembly into presynaptic filaments on single-stranded DNA (ssDNA), a process that is regulated by ssDNA-binding (SSB) and mediator proteins. Mediator proteins stimulate strand exchange by accelerating the rate-limiting displacement of SSB from ssDNA by the incoming recombinase. The use of mediators is a highly conserved strategy in recombination, but the precise mechanism of mediator activity is unknown. In this study, the well-defined bacteriophage T4 recombination system (UvsX recombinase, Gp32 SSB, and UvsY mediator) is used to examine the kinetics of presynaptic filament assembly on native ssDNA in vitro. Results indicate that the ATP-dependent assembly of UvsX presynaptic filaments on Gp32-covered ssDNA is limited by a salt-sensitive nucleation step in the absence of mediator. Filament nucleation is selectively enhanced and rendered salt-resistant by mediator protein UvsY, which appears to stabilize a pre-nucleation complex. This mechanism potentially explains how UvsY promotes presynaptic filament assembly under physiologically relevant conditions of ionic strength and Gp32 concentration. Other data suggest that presynaptic filament assembly involves multiple nucleation events, resulting in many short UvsX-ssDNA filaments or clusters, which may be the relevant form for recombination in vivo. Together, these findings provide the first detailed kinetic model for presynaptic filament assembly involving all three major protein components — recombinase, mediator, and SSB, on native ssDNA.
PMCID: PMC3864638  PMID: 24124995
Recombination; presynaptic filament; kinetics; fluorescence; mediator; ssDNA-binding protein
19.  Molecular Architecture of Bacterial Flagellar Motor in Cells 
Biochemistry  2014;53(27):4323-4333.
The flagellum is one of the most sophisticated self-assembling molecular machines in bacteria. Powered by the proton motive force, the flagellum rapidly rotates in either a clockwise or counterclockwise direction, which ultimately controls bacterial motility and behavior. Escherichia coli and Salmonella enterica have served as important model systems for extensive genetic, biochemical, and structural analysis of the flagellar motor, providing unparalleled insights into its structure, function, and gene regulation. Despite these advances, our understanding of flagellar assembly and rotational mechanisms remains incomplete, in part because of the limited structural information available regarding the intact rotor-stator complex and secretion apparatus. Cryo-electron tomography (cryo-ET) has become a valuable imaging technique capable of visualizing the intact flagellar motor in cells at molecular resolution. Because the resolution achievable by cryo-ET with large bacteria (such as E. coli and S. enterica) is limited, analysis of small,diameter bacteria (including Borrelia burgdorferi and Campylobacter jejuni) can provide additional insights into the in situ structure of the flagellar motor and other cellular components. This review is focused on the application of cryo-ET, in combination with genetic and biophysical approaches, to the study of flagellar structures and its potential for improving the understanding of rotor-stator interactions, the rotational switching mechanism, and the secretion and assembly of flagellar components.
PMCID: PMC4221660  PMID: 24697492
20.  Tribody: Robust Self-assembled Trimeric Targeting Ligands with High Stability and Significantly Improved Target-binding Strength 
Biochemistry  2013;52(41):10.1021/bi400716w.
The C-terminal coiled-coil region of mouse and human cartilage matrix protein (CMP) self-assembles into a parallel trimeric complex. Here, we report a general strategy for the development of highly stable trimeric targeting ligands (tribody), against epidermal growth factor receptor (EGFR) and prostate-membrane specific antigen (PSMA) as examples, by fusing a specific target-binding moiety with a trimerization domain derived from CMP. The resulting fusion proteins can efficiently self-assemble into a well-defined parallel homotrimer with high stability. Surface plasmon resonance (SPR) analysis of the trimeric targeting ligands demonstrated significantly enhanced target binding strength compared with the corresponding monomers. Cellular binding studies confirmed that the trimeric targeting ligands have superior binding strength towards their respective receptors. Significantly, EGFR-binding tribody was considerably accumulated in tumor in xenograft mice bearing EGFR positive tumors, indicating its effective cancer targeting feature under in vivo conditions. Our results demonstrate that CMP-based self-assembly of tribody can be a general strategy for the facile and robust generation of trivalent targeting ligands for a wide variety of in vitro and in vivo applications.
PMCID: PMC3851414  PMID: 24050811
Self-assembly; Targeting ligand; Trimerization; Trivalent; High stability; High affinity; EGFR; PSMA
21.  Post-Translational Modification by Cysteine Protects Cu/Zn-Superoxide Dismutase From Oxidative Damage 
Biochemistry  2013;52(36):10.1021/bi4006122.
Reactive oxygen species (ROS) are cytotoxic. To remove ROS, cells have developed ROS-specific defense mechanisms, including the enzyme Cu/Zn superoxide dismutase (SOD1), which catalyzes the disproportionation of superoxide anions into molecular oxygen and hydrogen peroxide. Although hydrogen peroxide is less reactive than superoxide, it is still capable of oxidizing, unfolding, and inactivating SOD1, at least in vitro. To explore the relevance of post-translational modification (PTM) of SOD1, including peroxide-related modifications, SOD1 was purified from post-mortem human nervous tissue. As much as half of all purified SOD1 protein contained non-native post-translational modifications (PTMs), the most prevalent modifications being cysteinylation and peroxide-related oxidations. Many PTMs targeted a single reactive SOD1 cysteine, Cys111. An intriguing observation was that unlike native SOD1, cysteinylated SOD1 was not oxidized. To further characterize how cysteinylation may protect SOD1 from oxidation, cysteine modified SOD1 was prepared in vitro and exposed to peroxide. Cysteinylation conferred nearly complete protection from peroxide-induced oxidation of SOD1. Moreover, SOD1 that has been cysteinylated and peroxide oxidized in vitro comprised a set of PTMs that bear a striking resemblance to the myriad of PTMs observed in SOD1 purified from human tissue.
PMCID: PMC3859700  PMID: 23927036
22.  The contribution of selenocysteine to the peroxidase activity of selenoprotein S 
Biochemistry  2013;52(33):10.1021/bi400741c.
Selenoprotein S (SelS, VIMP) is an intrinsically disordered enzyme that utilizes selenocysteine to catalyze the reduction of disulfide bonds and peroxides. Here it is demonstrated that selenocysteine is the residue oxidized by the peroxide substrate. It is possible to trap the reaction intermediate selenenic acid (SeOH) when the resolving cysteine is mutated. The selenocysteine allows SelS to rapidly reform its selenenylsulfide bond following its reduction, and to resist inactivation by H2O2. We propose that SelS’s peroxidase mechanism is similar to that of atypical 2-Cys peroxiredoxin and that selenocysteine allows SelS to sustain activity under oxidative stress.
PMCID: PMC3809988  PMID: 23914919
23.  Temporally Overlapped but Uncoupled Motions in Dihydrofolate Reductase Catalysis 
Biochemistry  2013;52(32):5332-5334.
Temporal correlations between protein motions and enzymatic reactions are often interpreted as evidence for catalytically important motions. Using dihydrofolate reductase as a model system, we show that there are many protein motions that temporally overlapped with the chemical reaction, and yet they do not exhibit the same kinetic behaviors (KIE and pH dependence) as the catalyzed chemical reaction. Thus, despite the temporal correlation, these motions are not directly coupled to the chemical transformation, and they might represent a different part of the catalytic cycle or simply be the product of the intrinsic flexibility of the protein.
PMCID: PMC3779903  PMID: 23883151
24.  The Structural Basis for Divergence of Substrate Specificity and Biological Function within HAD Phosphatases in Lipopolysaccharide and Sialic Acid Biosynthesis 
Biochemistry  2013;52(32):5372-5386.
The haloacid dehalogenase enzyme superfamily (HADSF) is largely composed of phosphatases, which, relative to members of other phosphatase families, have been particularly successful at adaptation to novel biological functions. Herein, we examine the structural basis for divergence of function in two bacterial homologs: 2-keto-3-deoxy-D-manno-octulosonate 8-phosphate phosphohydrolase (KDO8P phosphatase, KDO8PP) and 2-keto-3-deoxy-9-O-phosphonononic acid phosphohydrolase (KDN9P phosphatase, KDN9PP). KDO8PP and KDN9PP catalyze the final step of KDO and KDN synthesis, respectively, prior to transfer to CMP to form the activated sugar nucleotide. KDO8PP and KDN9PP orthologs derived from an evolutionarily diverse collection of bacterial species were subjected to steady-state kinetic analysis to determine their specificities towards catalyzed KDO8P and KDN9P hydrolysis. Although each enzyme was more active with its biological substrate, the degree of selectivity (as defined by the ratio of kcat/Km for KDO8P vs. KDN9P) varied significantly. High-resolution X-ray structure determination of Haemophilus influenzae KDO8PP bound to KDO/VO3− and Bacteriodes thetaiotaomicron KDN9PP bound to KDN/VO3− revealed the substrate-binding residues. Structures of the KDO8PP and KDN9PP orthologs were also determined to reveal the differences in active site structure that underlies the variation in substrate preference. Bioinformatic analysis was carried out to define the sequence divergence among KDN9PP and KDO8PP orthologs. The KDN9PP orthologs were found to exist as single domain proteins or fused with the pathway nucleotidyl transferases; fusion of KDO8PP with the transferase is rare. The KDO8PP and KDN9PP orthologs share a stringently conserved Arg residue, which forms a salt bridge with the substrate carboxylate group. The split of the KDN9PP lineage from the KDO8PP orthologs is easily tracked by the acquisition of a Glu/Lys pair that supports KDN9P binding. Moreover, independently evolved lineages of KDO8PP orthologs exist, separated by diffuse active-site sequence boundaries. We infer high tolerance of the KDO8PP catalytic platform to amino acid replacements that, in turn, influence substrate specificity changes and thereby facilitate divergence of biological function.
PMCID: PMC3966652  PMID: 23848398
2-keto-3-deoxyoctulosonic acid (KDO); 2-keto-3-deoxynononic acid (KDN); phosphohydrolases; enzyme evolution; ortholog boundaries
25.  Affinity, Kinetic, and Structural Study of the Interaction of 3-O-Sulfotransferase Isoform 1 with Heparan Sulfate† 
Biochemistry  2006;45(16):5122-5128.
The 3-O-sulfonation of glucosamine residues in heparan sulfate (HS) by 3-O-sulfotransferase (3-OST) is a key substitution that is present in HS sequences of biological importance, in particular HS anticoagulant activity. Six different isoforms of 3-OST have been identified that exhibit different substrate specificity. In this paper the affinity and kinetics of the interaction between 3-O-sulfotransferase isoform 1 (3-OST-1) and HS have been examined using surface plasmon resonance (SPR). 3-OST-1 binds with micomolar affinity to HS (KD= 2.79 µM), and this interaction is apparently independent of the presence of the coenzyme, 3′-phosphoadenosine 5′-phosphosulfate (PAPS). A conformational change in the complex has also been detected, supporting data from previous studies. Selected 3-OST-1 mutants have provided valuable information of amino acid residues that participate in 3-OST-1 interaction with HS substrate and its catalytic activity. The results from this study contribute to understanding the substrate specificity among the 3-OST isoforms and in the mechanism of 3-OST-1-catalyzed biosynthesis of anticoagulant HS.
PMCID: PMC4129659  PMID: 16618101

Results 1-25 (142)