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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.  Type II Kinase Inhibitors Show an Unexpected Inhibition Mode against Parkinson’s Disease-Linked LRRK2 Mutant G2019S 
Biochemistry  2013;52(10):1725-1736.
A number of well-known type II inhibitors (ATP non-competitive) that bind kinases in their DFG-out conformation were tested against wild-type LRRK2 and the most common Parkinson’s disease-linked mutation G2019S. We found that traditional type II inhibitors exhibit surprising variability in their inhibition mechanism between wild type (WT) and the G2019S mutant of LRRK2. The type II kinase inhibitors were found to work by an ATP-competitive fashion against the G2019S mutant, whereas they appear to follow the expected non-competitive mechanism against WT. Since the G2019S mutation lies in the DXG-motif (DYG in LRRK2 but DFG in most other kinases) of the activation loop, we explored the structural consequence of the mutation on loop dynamics using an enhanced sampling method called metadynamics. The simulations suggest that the G2019S mutation stabilizes the DYG-in state of LRRK2 through a series of hydrogen bonds, leading to an increase in the conformational barrier between the active and inactive forms of the enzyme and a relative stabilization of the active form. The conformational bias toward the active form of LRRK2 mutants has two primary consequences: 1) the mutant enzyme becomes hyperactive, a known contributor to the Parkinsonian phenotype, as a consequence of being “locked” into the activated state and 2) the mutation creates an unusual allosteric pocket that can bind type II inhibitors but in an ATP competitive fashion. Our results suggest that developing type II inhibitors, which are generally considered superior to type I inhibitors due to desirable selectivity profiles, might be especially challenging for the G2019S LRRK2 mutant.
PMCID: PMC3966205  PMID: 23379419
14.  A Conserved Amphipathic Ligand Binding Region Influences K-Path Dependent Activity of Cytochrome c Oxidase 
Biochemistry  2013;52(8):1385-1396.
A conserved, crystallographically-defined bile acid binding site was originally identified in the membrane domain of mammalian and bacterial cytochrome c oxidase (CcO). Current studies show other amphipathic molecules including detergents, fatty acids, steroids, and porphyrins bind to this site and affect the already 50% inhibited activity of the E101A mutant of Rhodobacter sphaeroides CcO, as well as altering the activity of wildtype and bovine enzymes. Dodecyl maltoside, Triton X100, C12E8, lysophophatidylcholine and CHOBIMALT detergents further inhibit RsCcO E101A, with lesser inhibition observed in wildtype. The detergent inhibition is overcome in the presence of μM concentrations of steroids and porphyrin analogs including deoxycholate, cholesteryl hemisuccinate, bilirubin, and protoporphyrin IX. In addition to alleviating detergent inhibition, amphipathic carboxylates including arachidonic, docosahexanoic, and phytanic acids stimulate the activity of E101A to wildtype levels by providing the missing carboxyl group. Computational modeling of dodecyl maltoside, bilirubin, and protoporphyrin IX into the conserved steroid site shows energetically favorable binding modes for these ligands and suggests that a groove at the interface of subunit I and II, including the entrance to the K-path and helix VIII of subunit I, mediates the observed competitive ligand interactions involving two overlapping sites. Spectral analysis indicates that ligand binding to this region affects CcO activity by altering the K path dependent electron transfer equilibrium between heme a and heme a3. The high affinity and specificity of a number of compounds for this region, and its conservation and impact on CcO activity, support its physiological significance.
PMCID: PMC3622084  PMID: 23351100
15.  Potent Mechanism-Based Inactivation Of Cytochrome P450 2B4 By 9-Ethynylphenanthrene: Implications For Allosteric Modulation Of Cytochrome P450 Catalysis1 
Biochemistry  2013;52(2):355-364.
The mechanism-based inactivation of cytochrome P450 2B4 (CYP2B4) by 9-ethynylphenanthrene (9EP) has been investigated. The partition ratio and kinact are 0.2 and 0.25 min−1, respectively. Intriguingly, the inactivation exhibits sigmoidal kinetics with a Hill coefficient of 2.5 and S50 of 4.5 μM indicative of homotropic cooperativity. Enzyme inactivation led to an increase in mass of the apo-CYP2B4 by 218 Da as determined by ESI-LC/MS, consistent with covalent protein modification. The modified CYP2B4 was purified to homogeneity and its structure determined by X-ray crystallography. The structure showed that 9EP is covalently attached to the Oγ of Thr 302 via an ester bond, which is consistent with the increased mass of the protein. The presence of the bulky phenanthrenyl ring resulted in inward rotations of Phe 297 and Phe 206 leading to a compact active site. Thus, binding of another molecule of 9EP in the active site is prohibited. However, results from the quenching of 9EP fluorescence by unmodified or 9EP-modified CYP2B4 revealed at least two binding sites with distinct affinities, with the low affinity site being the catalytic site and the high affinity site on the protein periphery. Computer-aided docking and MD simulations with one or two ligands bound revealed that the high affinity site is situated at the entrance of a substrate access channel surrounded by the F’ helix, β1/β2 loop and β4 loop and functions as an allosteric site to enhance the efficiency of activation of the acetylenic group of 9EP and subsequent covalent modification of Thr 302.
PMCID: PMC3568706  PMID: 23276288
16.  Critical Role in CXCR4 Signaling and Internalization of the Polypeptide Main Chain in the Amino Terminus of SDF-1α Probed by Novel N-Methylated Synthetically and Modularly Modified Chemokine Analogues 
Biochemistry  2012;51(30):5951-5957.
The replication of human immunodeficiency virus type 1 (HIV-1) can be profoundly inhibited by the natural ligands of two major HIV-1 coreceptors, CXCR4 and CCR5. Stromal cell-derived factor-1α (SDF-1α) is a natural ligand of CXCR4. We have recently developed a synthetic biology approach of using synthetically and modularly modified (SMM)-chemokines to dissect various aspects of the structure-function relationship of chemokines and their receptors. Here, we used this approach to design novel SMM-SDF-1α analogues containing unnatural N-methylated residues in the amino terminus to investigate whether the polypeptide main chain amide bonds in the N-terminus of SDF-1α play a role in SDF-1α signaling via CXCR4 and/or receptor internalization. The results show that SDF-1α analogues with a modified N-methylated main chain at position 2, 3, or 5 retain significant CXCR4 binding and yet completely lose signaling activities. Furthermore, a representative N-methylated analogue has been shown to be incapable of causing CXCR4 internalization. These results suggest that the ability of SDF-1α to activate CXCR4 signaling and internalization is dependent upon the main chain amide bonds in the N-terminus of SDF-1α. This study demonstrates the feasibility and value of applying a synthetic biology approach to chemically engineer natural proteins and peptide ligands as probes of important biological functions that are not addressed by other biological techniques.
PMCID: PMC3891921  PMID: 22779681
17.  NMR solution structure of an N2-guanine DNA adduct derived from the potent tumorigen dibenzo[a,l]pyrene: Intercalation from the minor groove with ruptured Watson-Crick base pairing 
Biochemistry  2012;51(48):9751-9762.
The most potent tumorigen identified among the polycyclic aromatic hydrocarbons (PAH) is the non-planar fjord region dibenzo[a,l]pyrene (DB[a,l]P). It is metabolically activated in vivo through the widely-studied diol epoxide (DE) pathway to form covalent adducts with DNA bases, predominantly guanine and adenine. The (+)-11S,12R,13R,14S DE enantiomer forms adducts via its C14-position with the exocyclic amino group of guanine. Here, we present the first NMR solution structure of a DB[a,l]P-derived adduct, the 14R (+)-trans-anti-DB[a,l]P–N2-dG (DB[a,l]P-dG) lesion in double-stranded DNA. In contrast to the stereochemically identical benzo[a]pyrene-derived N2-dG adduct (B[a]P-dG) in which the B[a]P rings reside in the B-DNA minor groove on the 3’-side of the modifed deoxyguanosine, in the DB[a,l]P-derived adduct the DB[a,l]P rings intercalate into the duplex on the 3’-side of the modified base from the sterically crowded minor groove. Watson-Crick base pairing of the modified guanine with the partner cytosine is broken, but these bases retain some stacking with the bulky DB[a,l]P ring system. This new theme in PAH DE - DNA adduct conformation differs from: (1) the classical intercalation motif where Watson-Crick base-pairing is intact at the lesion site, and (2) the base-displaced intercalation motif in which the damaged base and its partner are extruded from the helix . The structural considerations that lead to the intercalated conformation of the DB[a,l]P-dG lesion in contrast to the minor groove alignment of the B[a]P-dG adduct, and the implications of the DB[a,l]P-dG conformational motif for the recognition of such DNA lesions by the human nucleotide excision repair apparatus, are discussed.
PMCID: PMC3578312  PMID: 23121427
18.  Why does the G117H mutation considerably improve the activity of human butyrylcholinesterase against sarin? Insights from QM/MM-FE calculations 
Biochemistry  2012;51(44):8980-8992.
Human butyrylcholinesterase (BChE) is recognized as the most promising bioscavenger for organophosphorus (OP) warfare nerve agents. The G117H mutant of human BChE has been identified as a potential catalytic bioscavenger with a remarkably improved activity against OP nerve agents such as sarin, but it still does not satisfy the clinical use. For further design of the higher-activity mutants against OP nerve agents, it is essential to understand how the G117H mutation improves the activity. The reaction mechanisms and the free energy profiles for spontaneous reactivation of the wild-type BChE and its G117H mutant phosphorylated by sarin have been explored, in this study, by performing first-principles quantum mechanical/molecular mechanical free energy (QM/MM-FE) calculations, and the remarkable role of the G117H mutation on the activity has been elucidated. For both the wild-type and G117H mutant enzymes, H438 acts as a general base to initiate the spontaneous reactivation which consists of two reaction steps: the nucleophilic attack at the phosphorus by a water molecule and decomposition of the pentacoordinated phosphorus intermediate. The calculated overall free energy barriers, i.e. 30.2 and 23.9 kcal/mol for the wild-type and G117H mutant, respectively, are in good agreement with available experimental kinetic data. Based on the calculated results, the mutated residue (H117 in the G117H mutant) cannot initiate the spontaneous reactivation as a general base. Instead, it skews the oxyanion hole and makes the phosphorus more open to the nucleophilic water molecule, resulting in remarkable change of the rate-determining step and significantly improved catalytic activity of human BChE.
PMCID: PMC3499032  PMID: 23092211
19.  The Internal Dynamics of Mini c TAR DNA Probed by EPR of Nitroxide Spin Labels at the Lower Stem, the Loop, and the Bulge † 
Biochemistry  2012;51(43):8530-8541.
Electron paramagnetic resonance (EPR) at 236.6 GHz and 9.5 GHz probed the tumbling of nitroxide spin probes in the lower stem, the upper loop, and near the bulge of mini c TAR DNA. High frequency 236.6 GHz EPR, not previously applied to spin labeled oligonucleotides, was notably sensitive to fast, anisotropic, hindered local rotational motion of the spin probe, occurring approximately about the NO nitroxide axis. Labels attached to the 2′-amino cytidine sugar in the mini c TAR DNA showed such anisotropic motion, which was faster in the lower stem, a region previously suggested to be partially melted. More flexible labels attached to phosphorothioates at the end of the lower stem tumbled isotropically in mini c TAR DNA, mini TAR RNA, and ψ3 RNA, but at 5 °C the motion became more anisotropic for the labeled RNAs, implying more order within the RNA lower stems. As observed by 9.5 GHz EPR, the slowing of nanosecond motions of large segments of the oligonucleotide was enhanced by increasing the ratio of the nucleocapsid protein NCp7 to mini c TAR DNA from zero to two. The slowing was most significant at labels in the loop and near the bulge. At a 4:1 ratio of NCp7 to mini c TAR DNA all labels reported tumbling times > 5 ns, indicating a condensation of NCp7 and TAR DNA. At the 4:1 ratio, pulse dipolar EPR spectroscopy of bi-labels attached near the 3′ and 5′ terminals showed evidence for an NCp7-induced increase in the 3′ - 5 ′end-to-end distance distribution and a partially melted stem.
PMCID: PMC3549007  PMID: 23009298
EPR; spin label; NCp7; mini c TAR DNA
20.  Farnesyl Diphosphate Analogues with Aryl Moieties are Efficient Alternate Substrates for Protein Farnesyltransferase 
Biochemistry  2012;51(41):8307-8319.
Farnesylation is an important post-translational modification essential for proper localization and function of many proteins. Transfer of the farnesyl group from farnesyl diphosphate (FPP) to proteins is catalyzed by protein farnesyltransferase (FTase). We employed a library of FPP analogues with a range of aryl groups substituting for individual isoprene moieties to examine some of the structural and electronic properties of analogue transfer to peptide catalyzed by FTase. Analysis of steady-state kinetics for modification of peptide substrates revealed that the multiple turnover activity depends on the analogue structure. Analogues where the first isoprene is replaced by a benzyl group and an analogue where each isoprene is replaced by an aryl group are good substrates. In sharp contrast with the steady-state reaction, the single turnover rate constant for dansyl-GCVLS alkylation was found to be the same for all analogues, despite the increased chemical reactivity of the benzyl analogues and the increased steric bulk of other analogues. However, the single turnover rate constant for alkylation does depend on the Ca1a2X peptide sequence. These results suggest that the isoprenoid transition state conformation is preferred over the inactive E•FPP• Ca1a2X ternary complex conformation. Furthermore, these data suggest that the farnesyl binding site in the exit groove may be significantly more selective for the farnesyl diphosphate substrate than the active site binding pocket and therefore might be a useful site for design of novel inhibitors.
PMCID: PMC3562418  PMID: 22989235
FPP; substrate analogue; FTase; protein prenylation; enzymology
21.  Determining α-helical and β-sheet Secondary Structures via Pulsed EPR Spectroscopy 
Biochemistry  2012;51(38):7417-7419.
A new method has been developed to determine α-helical and β-sheet secondary structural components of aqueous and membrane-bound proteins using pulsed electron paramagnetic resonance (EPR) spectroscopy. The threepulse electron spin echo envelope modulation (ESEEM) technique was used to detect weakly coupled 2H-labeled nuclei on side chains in the proximity of a strategically placed nitroxide spin-label up to 8 Å away. Changes in the ESEEM spectra for different samples correlate directly to periodic structural differences between α-helical and β-sheet motifs. These distinct trends were demonstrated with α-helical (M2δ subunit of the acetylcholine receptor) and β-sheet (Ubiquitin) peptides in biologically relevant sample environments.
PMCID: PMC3535504  PMID: 22966895
22.  A Novel Synthetic Bivalent Ligand to Probe Chemokine Receptor CXCR4 Dimerization and Inhibit HIV-1 Entry 
Biochemistry  2012;51(36):7078-7086.
The chemokine receptor CXCR4 is one of two principal coreceptors for HIV-1 entry into target cells. CXCR4 is known to form homodimers. We previously demonstrated that the amino (N)-terminus of viral macrophage protein (vMIP)-II is the major determinant for CXCR4 recognition, and that V1 peptide derived from the N-terminus of vMIP-II (1-21 residues) showed significant CXCR4 binding. Interestingly, an all-D-amino acid analog of V1 peptide, DV1 peptide, displayed even higher binding affinity and strong antiviral activity in inhibiting the replication of CXCR4-dependent HIV-1 strains. In the present study, we synthetically linked two DV1 peptides with the formation of a disulfide bond between the two cysteine residues present in the peptide sequence to generate a dimeric molecule potentially capable of interacting with two CXCR4 receptors. DV1 dimer showed enhanced binding affinity and antiviral activity compared with DV1 monomer. Ligand binding site mapping experiments showed that DV1 dimer overlaps with HIV-1 gp120 on CXCR4 binding sites, including several transmembrane (TM) residues located close to the extracellular side and the N-terminus of CXCR4. This finding was supported by the molecular modeling of CXCR4 dimer–DV1 dimer interaction based on the crystal structure of CXCR4, which showed that DV1 dimer is capable of interacting with the CXCR4 dimeric structure by allowing the N-terminus of each DV1 monomer to reach into the binding pocket of CXCR4 monomer. The development of this bivalent ligand provides a tool to further probe the functions of CXCR4 dimerization and to study CXCR4 heterodimerization with other receptors.
PMCID: PMC3476724  PMID: 22897429
23.  A population-based experimental model for protein evolution: effects of mutation rate and selection stringency on evolutionary outcomes 
Biochemistry  2013;52(8):1490-1499.
Protein evolution is a critical component of organismal evolution and a valuable method for the generation of useful molecules in the laboratory. Few studies, however, have experimentally characterized how fundamental parameters influence protein evolution outcomes over long evolutionary trajectories or multiple replicates. In this work, we applied phage-assisted continuous evolution (PACE) as an experimental platform to study evolving protein populations over hundreds of rounds of evolution. We varied evolutionary conditions as T7 RNA polymerase evolved to recognize the T3 promoter DNA sequence and characterized how specific combinations of both mutation rate and selection stringency reproducibly result in different evolutionary outcomes. We observed significant and dramatic increases in the activity of the evolved RNA polymerase variants on the desired target promoter after 96 hours of selection, confirming positive selection occurred under all conditions. We used high-throughput sequencing to quantitatively define convergent genetic solutions, including mutational “signatures” and non-signature mutations that map to specific regions of protein sequence. These findings illuminate key determinants of evolutionary outcomes, inform the design of future protein evolution experiments, and demonstrate the value of PACE as a method to study protein evolution.
PMCID: PMC3641671  PMID: 23360105
24.  Direct Evidence for Spontaneous Branch Migration in Antiparallel DNA Holliday Junctions 
Biochemistry  2000;39(37):11514-11522.
The Holliday junction is a central intermediate in genetic recombination. It contains four strands of DNA that are paired into four double helical arms flanking a branch point. In naturally-occurring Holliday junctions, the sequence flanking the branch point contains twofold (homologous) symmetry. As a consequence of this symmetry, the junction can undergo a conformational isomerization known as branch migration, which relocates the site of branching. In the absence of proteins and in the presence of Mg2+, the four arms are known to stack in pairs, forming two helical domains whose orientations are antiparallel. Nevertheless, the mechanistic models proposed for branch migration are all predicated on a parallel alignment of helical domains. Here, we have used antiparallel DNA double crossover molecules to demonstrate that branch migration can occur in antiparallel Holliday junctions. We have constructed a DNA double crossover molecule with three crossover points. Two adjacent branch points in this molecule are flanked by symmetric sequences. The symmetric crossover points are held immobile by the third crossover point, which is flanked by asymmetric sequences. Restriction of the helices that connect the immobile junction to the symmetric junctions releases this constraint. The restricted molecule undergoes branch migration, even though it is constrained to an antiparallel conformation.
PMCID: PMC3748963  PMID: 10985798
Branch migration; DNA double crossover molecules; Branched junction conformational isomerizations; Hydroxyl radical autofootprinting; DNA topology
25.  Conformational Closure of the Catalytic Site of Human CD38 Induced by Calcium 
Biochemistry  2008;47(52):13966-13973.
First identified on the surface of lymphoids as a type II transmembrane protein, CD38 has now been established to have dual functions not only as a receptor but also as a multi-functional enzyme, catalyzing the synthesis of and hydrolysis of a general calcium messenger molecule, cyclic ADP-ribose (cADPR). The receptorial functions of CD38 include the induction of cell adhesion, differentiation, apoptosis, and cytokine production upon antibody ligation. Here we determined the crystal structure of calcium loaded human CD38 at 1.45 Å resolution which reveals that CD38 undergoes dramatic structural changes to an inhibited conformation in the presence of calcium. The structural changes are highly localized and occur in only two regions. The first region is part of the active site and consists of residues 121–141. In the presence of calcium, W125 moves 5 Å into the active site and forms hydrophobic interactions with W189. The movement closes the active site pocket and reduces entry of substrates, resulting in inhibition of the enzymatic activity. The structural role of calcium in inducing these conformational changes is readily visualized in the crystal structure. The other region that undergoes calcium induced changes is at the receptor region, where a highly ordered helix is unraveled to a random coil. The results suggest a novel conformational coupling mechanism, whereby protein interaction targeted at the receptor region can effectively regulate the enzymatic activity of CD38.
PMCID: PMC3732380  PMID: 19117080
Human CD38; calcium loaded structure; active site closure; inhibitory conformation

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