Interaction of antimalarial drug quinacrine with nucleic acids of variable sequence studied by spectroscopic methods

The interaction of antimalarial drug quinacrine (QA) with polynucleotides is studied by UV-visible absorption, fluorescence and surface-enhanced Raman spectroscopy (SERS). The polynucleotides employed for such a study were calf thymus DNA, poly(A).poly(T), poly(A).poly(U), poly(C).poly(G) and poly(dG-dC).poly(dG-dC). Absorption and fluorescence spectra of QA complexes indicate that an interaction with the biomolecule is taking place, although different interaction mechanisms are probable depending on the sequence. The SERS spectra also reflect spectral changes which depend on the polymer sequence and that can be correlated to those observed by fluorescence, with the advantage of the detailed structural information provided by this vibrational technique. QA interacts with polynucleotides through its diprotonated form and by ring stacking. The strength of such interaction is extremely sequence dependent, thus suggesting different interaction mechanisms in each case. The SERS technique allows the simultaneous study of those polynucleotide moieties that are directly involved in the interaction thanks to the short-range character of the SERS spectroscopy. The interaction of QA with the above nucleic acids lead to a different change in the chain stability and flexibility which is further related to the different denaturation tendency of the polymer in the presence of the metal surface.     

Interaction of Antimalarial Drug Quinacrine with Nucleic Acids of Variable Sequence Studied by Spectroscopic Methods

Abstract

The interaction of antimalarial drug quinacrine (QA) with polynucleotides is studied by UV- visible absorption, fluorescence and surface-enhanced Raman spectroscopy(SERS). The polynucleotides employed for such a study were calf thymus DNA, poly(A).poly(T), poly(A).poly(U), poly(C).poly(G) and poly(dG-dC).poly(dG-dC). Absorption and fluorescence spectra of QA complexes indicate that an interaction with the biomolecule is taking place, although different interaction mechanisms are probable depending on the sequence. The SERS spectra also reflect spectral changes which depend on the polymer sequence and that can be correlated to those observed by fluorescence, with the advantage of the detailed structural information provided by this vibrational technique. QA interacts with polynucleotides through its diprotonated form and by ring stacking. The strength of such interaction is extremely sequence dependent, thus suggesting different interaction mechanisms in each case. The SERS technique allows the simultaneous study of those polynucleotide moieties that are directly involved in the interaction thanks to the short-range character of the SERS spectroscopy. The interaction of QA with the above nucleic acids lead to a different change in the chain stability and flexibility which is further related to the different denaturation tendency of the polymer in the presence of the metal surface.     

Variations in the concentration of phenolic compounds in the seagrass Posidonia oceanica under conditions of competition

The concentration of phenolic compound was measured in the seagrass Posidonia oceanica when interacting with two Bryopsidophyceae, Caulerpa taxifolia and Caulerpa racemosa, between May 1999 and May 2000. These measurements were performed on adult and intermediate leaves and in sheaths of the seagrass. Sampling was carried out at three stations subject to increasing levels of interaction with Caulerpa. The number of tannin cells was also analysed. Five phenolic compounds were identified in P. oceanica, with a predominance of caffeic acid in the adult and intermediate leaves. For a given level of interaction (and for both caulerpa sp.), a significant seasonal variation in phenolic compounds was shown in the adult leaves (higher in November and lower in September and March for example for the interaction with C. taxifolia). Only for two compounds (corresponding to a mixture containing ferulic acid and the ester methyl 12-acetoxyricinoleate) were significant differences observed as a function of the level of interaction with C. taxifblia, and only in the adult leaves: higher concentrations of phenols were observed with increasing level of interaction. Thus,adult leaves gave values of 55.5 +/- 14.1 microg g(-1) dm without interaction (OCt) and 94.9 +/- 23.4 microg g(-l) dm with high interaction (2Ct),corresponding to an increase of 70%. No significant difference was observed with intermediate leaves and sheaths, or for interaction with C. racemosa. The number of tannin cells (supposed to produce the phenolic compounds) largely increased in the adult and intermediate leaves when the degree of interaction with C. taxifolia increased: 90 mm above the base of the sheath (in adult leaves), 16.7 +/- 10.6 tannin cells cm(-2) were found without interaction (OCt), and 57.8 + 21.2 tannin cells cm(-2) with high interaction (2Ct). No significant difference was found for C. racemosa interaction. It thus appears that when the seagrass P. oceanica is in interaction with C. taxifolia, it accelerates its production of secondary metabolites so as to limit invasion of the beds.     

Structural and mutational characterization of L-carnitine binding to human carnitine acetyltransferase

We report the crystal structure of a binary complex of human peroxisomal carnitine acetyltransferase and the substrate l-carnitine, refined to a resolution of 1.8 Angstrom with an R(factor) value of 18.9% (R(free)=22.3%). L-carnitine binds to a preformed pocket in the active site tunnel of carnitine acetyltransferase aligned with His(322). The quaternary nitrogen of carnitine forms a pi-cation interaction with Phe(545), while Arg(497) forms an electrostatic interaction with the negatively charged carboxylate group. An extensive hydrogen bond network also occurs between the carboxylate group and Tyr(431), Thr(444), and a bound water molecule. Site-directed mutagenesis and kinetic characterization reveals that Tyr(431), Thr(444), Arg(497), and Phe(545) are essential for high affinity binding of L-carnitine.     

Modulation of amyloid precursor protein metabolism by X11alpha /Mint-1. A deletion analysis of protein-protein interaction domains

Modulation of amyloid precursor protein (APP) metabolism plays a pivotal role in the pathogenesis of Alzheimer's disease. The phosphotyrosine-binding/protein interaction (PTB/PI) domain of X11alpha, a neuronal cytosolic adaptor protein, binds to the YENPTY sequence in the cytoplasmic carboxyl terminus of APP. This interaction prolongs the half-life of APP and inhibits Abeta40 and Abeta42 secretion. X11alpha/Mint-1 has multiple protein-protein interaction domains, a Munc-18 interaction domain (MID), a Cask/Lin-2 interaction domain (CID), a PTB/PI domain, and two PDZ domains. These X11alpha protein interaction domains may modulate its effect on APP processing. To test this hypothesis, we performed a deletion analysis of X11alpha effects on metabolism of APP(695) Swedish (K595N/M596L) (APP(sw)) by transient cotransfection of HEK 293 cells with: 1) X11alpha (X11alpha-wt, N-MID-CID-PTB-PDZ-PDZ-C), 2) amino-terminal deletion (X11alpha-DeltaN, PTB-PDZ-PDZ), 3) carboxyl-terminal deletion (X11alpha-DeltaPDZ, MID-CID-PTB), or 4) deletion of both termini (PTB domain only, PTB). The carboxyl terminus of X11alpha was required for stabilization of APP(sw) in cells. In contrast, the amino terminus of X11alpha was required to stimulate APPs secretion. X11alpha, X11alpha-DeltaN, and X11alpha-PTB, but not X11alpha-DeltaPDZ, were effective inhibitors of Abeta40 and Abeta42 secretion. These results suggest that additional protein interaction domains of X11alpha modulate various aspects of APP metabolism.     

Molecular examination of the transmembrane requirements of the platelet-derived growth factor beta receptor for a productive interaction with the bovine papillomavirus E5 oncoprotein

The small transmembrane E5 protein of bovine papillomavirus (BPV) transforms cells by forming a stable complex with and activating the platelet-derived growth factor beta receptor (PDGFbetaR). The E5/PDGFbetaR interaction is thought to involve specific physical contacts between the transmembrane domains of the two proteins. Lys(499) at the extracellular juxtamembrane position and Thr(513) within the transmembrane domain of the PDGFbetaR are required for the interaction and are predicted to contact analogously positioned residues in the E5 protein. Here, mutagenic analysis of the transmembrane region of the PDGFbetaR was performed to further characterize the nature of the E5/PDGFbetaR interaction. We show that the receptor transmembrane domain, with minimal extracellular and intracellular sequence, is sufficient for the interaction. In addition, we provide evidence that the polar nature of Thr(513) as well as its positioning along the transmembrane alpha-helix is important for the interaction. We also identify the receptor transmembrane amino acids Ile(506) and Leu(520) as additional requirements for the interaction. Because Lys(499), Thr(513), Ile(506), and Leu(520) all align along the same face of the predicted PDGFbetaR transmembrane alpha-helix, our data support the model that the PDGFbetaR contacts the E5 protein via multiple amino acids along a single alpha-helical interface.     

The interaction of ApoA-I and ABCA1 triggers signal transduction pathways to mediate efflux of cellular lipids

Reverse cholesterol transport (RCT) has been characterized as a crucial step for antiatherosclerosis, which is initiated by ATP-binding cassette A1 (ABCA1) to mediate the efflux of cellular phospholipids and cholesterol to lipid-free apolipoprotein A-I (apoA-I). However, the mechanisms underlying apoA-I/ABCA1 interaction to lead to the lipidation of apoA-I are poorly understood. There are several models proposed for the interaction of apoA-I with ABCA1 as well as the lipidation of apoA-I mediated by ABCA1. ApoA-I increases the levels of ABCA1 protein markedly. In turn, ABCA1 can stabilize apoA-I. The interaction of apoA-I with ABCA1 could activate signaling molecules that modulate posttranslational ABCA1 activity or lipid transport activity. The key signaling molecules in these processes include protein kinase A (PKA), protein kinase C (PKC), Janus kinase 2 (JAK2), Rho GTPases and Ca²⁺, and many factors also could influence the interaction of apoA-I with ABCA1. This review will summarize these mechanisms for the apoA-I interaction with ABCA1 as well as the signal transduction pathways involved in these processes.     

Identification of residues critical for Ras(17N) growth-inhibitory phenotype and for Ras interaction with guanine nucleotide exchange factors.

The Ras(17N) dominant negative antagonizes endogenous Ras function by forming stable, inactive complexes with Ras guanine nucleotide exchange factors (GEFs; e.g., SOS1). We have used the growth-inhibitory phenotype of Ras(17N) to characterize two aspects of Ras interaction with GEFs. First, we used a nonprenylated version of Ras(17N), designated Ras(17N/186S), which no longer associates with the plasma membrane and lacks the growth-inhibitory phenotype, to address the importance of Ras subcellular location and posttranslational modification for its interaction with GEFs. We observed that addition of an N-terminal myristylation signal to Ras(17N/186S) restored the growth-inhibitory activity of nonprenylated Ras(17N). Thus, membrane association, rather than prenylation, is critical for Ras interaction with Ras GEFs. Second, we used a biological selection approach to identify Ras residues which are critical for Ras(17N) growth inhibition and hence for interaction with Ras GEFs. We identified mutations at residues 75, 76, and 78 that abolished the growth-inhibitory activity of Ras(17N). Since GEF interaction is dispensable for oncogenic but not normal Ras function, our demonstration that single-amino-acid substitutions at these three positions impaired the transforming activity of normal but not oncogenic Ras provides further support for the role of these residues in Ras-GEF interactions. Finally, Ras(WT) proteins with mutations at these residues were no longer activated by mammalian SOS1. Altogether, these results suggest that the Ras intracellular location and Ras residues 75 to 78 are critical for Ras-GEF interaction.     

The Magnitude of Tobacco Smoking-Betel Quid Chewing-Alcohol Drinking Interaction Effect on Oral Cancer in South-East Asia. A Meta-Analysis of Observational Studies

Tobacco smoking, betel quid chewing and alcohol drinking are oral cancer risk factors. Observational studies unanimously report that oral cancer risk in smoking-drinking-chewing exposed subjects is exceptionally high. However, none of them assessed the fractions of this risk attributable to the three individual risk factors and to the smoking-drinking-chewing interaction. The present study sought to assess the magnitude of the smoking-drinking-chewing interaction effect on oral cancer. A meta-analysis of observational South-East Asian studies which reported oral cancer odds ratios (ORs) stratified for smoking-drinking-chewing exposures was performed. The pooled ORs were estimated and controlled for quality, heterogeneity, publication bias and inclusion criteria. The smoking-drinking-chewing interaction effect was estimated through the pooled Relative Excess Risk due to Interaction (RERI, excess risk in smoking-drinking-chewing exposed individuals with respect to the risk expected from the addition of the three individual risks of smoking, drinking and chewing). Fourteen studies were included with low between-study heterogeneity. The pooled ORs for smoking, drinking, chewing, smoking-drinking-chewing, respectively were 3.6 (95% confidence interval −95% CI, 1.9–7.0), 2.2 (95% CI, 1.6–3.0), 7.9 (95% CI, 6.7–9.3), 40.1 (95% CI, 35.1–45.8). The pooled RERI was 28.4 (95% CI, 22.9–33.7). Among smoking-drinking-chewing subjects, the individual effects accounted for 6.7% (smoking), 3.1% (drinking), 17.7% (chewing) of the risk, while the interaction effect accounted for the remaining 72.6%. These data suggest that 44,200 oral cancer cases in South-East Asia annually occur among smoking-drinking-chewing exposed subjects and 40,400 of these are exclusively associated with the interaction effect. Effective oral cancer control policies must consider concurrent tobacco smoking, alcohol drinking, betel quid chewing usages as a unique unhealthy lifestyle.     

A novel type of bifunctional inhibitor directed against proteolytic activity and receptor/ligand interaction. Cystatin with a urokinase receptor binding site

Cancer invasion and metastasis is a process requiring a coordinated series of (anti-)adhesive, migratory, and pericellular proteolytic events involving various proteases such as urokinase-type plasminogen activator (uPA)/plasmin, cathepsins B and L, and matrix metalloproteases. Novel types of double-headed inhibitors directed to different tumor-associated proteolytic systems were generated by substitution of a loop in chicken cystatin, which is nonessential for cysteine protease inhibition, with uPA-derived peptides covering the human uPA receptor binding sequence uPA-(19-31). The inhibition constants of these hybrids toward cysteine proteases are similar to those of wild-type cystatin (K(i), papain (pm), 1.9-2.4; K(i), cathepsin B (nm), 1.0-1.7; K(i), cathepsin L (pm), 0.12-0.61). FACS analyses revealed that the hybrids compete for binding of uPA to the cell surface-associated uPA receptor (uPAR) expressed on human U937 cells. The simultaneous interaction of the hybrid molecules with papain and uPAR was analyzed by surface plasmon resonance. The measured K(D) value of a papain-bound cystatin variant harboring the uPAR binding sequence of uPA (chCys-uPA-(19-31)) and soluble uPAR was 17 nm (K(D) value for uPA/uPAR interaction, 5 nm). These results indicate that cystatins with a uPAR binding site are efficient inhibitors of cysteine proteases and uPA/uPAR interaction at the same time. Therefore, these compact and small bifunctional inhibitors may represent promising agents for the therapy of solid tumors.     

Binding of bovine follicular fluid glycosaminoglycans to fibronectin, laminin and low-density lipoproteins

Interactions of bovine follicular fluid glycosaminoglycans (GAGs) with extracellular matrix (ECM) components fibronectin and laminin and with low-density lipoproteins (LDL) were examined using affinity chromatography. Glycosaminoglycans from small (diameter less than 5 mm) and large (diameter 11-20 mm) follicles were isolated from follicular fluid. The dermatan sulphate or heparan sulphate from small or large follicles was applied to Fn-, Lm- or LDL-Sepharose columns. Portions of each fraction of the bound or unbound GAG were then subjected to gel filtration h.p.l.c. for quantification. The binding interaction between dermatan sulphate and fibronectin was significantly greater than between heparan sulphate and fibronectin (P less than 0.05); the binding interaction between GAGs from small follicles and fibronectin was significantly greater than between GAGs from large follicles (P less than 0.05). The binding interaction between GAGs from small follicles and laminin was significantly greater than for GAGs from large follicles (P less than 0.05). Dermatan sulphate from small follicles bound to fibronectin (42%), laminin (36%) and LDL (14%) and that from large follicles bound to fibronectin (14%), laminin (23%) and LDL (14%). Heparan sulphate from small follicles bound to fibronectin (17%), laminin (15%) and that from large follicles bound to fibronectin (13%), laminin (10%) and LDL (6%). These results suggest that dermatan sulphate, but not heparan sulphate, from follicles at different stages of development exhibit a varied ability to interact with components of the ECM. Both substances bound to LDL comparably in small amounts.     

First-principles study of interaction of serine with nucleobases of DNA and RNA

The nature of interaction between serine—a vital molecule for cancer cell proliferation and nucleic acid bases—adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U) is investigated within the framework of Møller–Plesset perturbation theory (MP2) and density functional theory (DFT). To quantify the interaction strength between serine and nucleobases, the corresponding binding energies were computed, showing energetic ordering such that G?>?C?>?T?>?A?>?U. This shows that the interaction energy of serine with guanine is the highest, while with uracil it is the lowest. The amount of charge transferred is the lowest in case of the serine-guanine complex and highest in case of the serine-uracil complex. The results show the serine-guanine complex to be more stable and to have a salt bridge structure involving the -COOH group. Theoretical analysis based on MP2 and DFT shows that the interaction between the serine and nucleobases is mainly determined by hydrogen bonding.     

The length of the aminoacyl-acceptor stem of the selenocysteine-specific tRNA(Sec) of Escherichia coli is the determinant for binding to elongation factors SELB or Tu

Mutations in selC, which reduce the 8-base pair aminoacyl-acceptor helix to the canonical 7-base pair length (tRNA(Sec)(delAc] or which replace the extra arm of tRNA(Sec) by that of a serine acceptor tRNA species (tRNA(Sec)(ExS), block the function in selenoprotein synthesis in vivo (Baron, C., Heider, J., and B?ck, A. (1990) Nucleic Acids Res. 18, 6761-6766). tRNA(Sec), tRNA(Sec)(delAc), and tRNA(Sec)(ExS) were purified and analyzed for their interaction with purified seryl-tRNA synthetase, selenocysteine synthase and translation factors SELB and EF-Tu. It was found that seryl-tRNA synthetase displays 10-fold impaired Km and Kcat values for tRNA(Sec) in comparison to tRNA(Ser), decreasing the overall charging efficiency (Kcat/Km) of tRNA(Sec) to 1% of that characteristic for tRNA(Ser). tRNA(Sec)(ExS) was a less efficient substrate for the enzyme (Kcat/Km 0.2% of the tRNA(Ser) value) whereas the tRNA(Ser)(delAc) variant was charged with an approximately 2-3-fold improved rate compared to wild-type tRNA(Sec). Both mutant tRNA variants, when charged with L-serine, were able to interact with selenocysteine synthase to give rise to selenocysteyl-tRNA with tRNA(Sec)(ExS) being as efficient as wild-type tRNA(Sec). Seryl-tRNA(Sec)(delAc), on the other hand, was selenylated very slowly. Reduction of the length of the aminoacyl-acceptor stem to 7 base pairs prevented the interaction with translation factor SELB but allowed binding to EF-Tu, irrespective of whether tRNA(Sec)(delAc) was charged with serine or selenocysteine. The aminoacyl-acceptor helix of tRNA(Sec), therefore, is a major determinant directing binding to SELB and precluding interaction with EF-Tu.     

血清白蛋白与酪胺分子的体外相互作用分析

利用毛细管电泳 (capillary electrophoresis, CE)建立牛血清白蛋白(bovine serum albumin, BSA)-酪胺(tyramine, TA)分子作用机制的分析方法,构建TA-BSA相互作用模型,并研究其相互作用机理. 生理条件下,采用HD法(Hummel-Dreyer, HD),前沿分析法(frontal analysis, FA)和空峰法(vacant peak, VP)研究TA与BSA的结合机制,构建TA-BSA理论模型,获取TA和BSA相互作用参数,分析理论模型的适用度. 通过分子模拟,构建TA与BSA的结合模型,考察TA的BSA结合机制. 结果表明,HD法和VP法均适用于分析TA-BSA体系的相互作用,VP法最优. 模型适用度分析得出双对数方程最适合模拟TA-BSA相互作用,TA与BSA结合强度较弱,且只有单一类型的结合位点. 构建的TA与BSA结合模型表明,TA与BSA的相互作用力主要是氢键和范德华力,兼有疏水作用力. 本文结果可为分析生物胺-蛋白质分子作用机制研究提供有意义的参考.     

Molecular analysis of the interaction between the intracellular loops of the human serotonin receptor type 6 (5-HT6) and the alpha subunit of GS protein

The serotonin type 6 (5-HT(6)) receptor is a G-protein coupled receptor (GPCR) coupled to a stimulatory G-protein (G(S)). To identify the structural basis for the interaction of the 5-HT(6) receptor with the G(S) protein, we have dissected the interaction between GST-fusion proteins containing the second intracellular loop (iL2), the third intracellular loop (iL3), or the C-terminal tail of the 5-HT(6) receptor and the alpha subunit of G(S) (Galpha(S)). The direct interaction of iL3 and Galpha(S) was demonstrated by co-immunoprecipitation. Furthermore, the kinetic parameters of the interaction between iL3 and Galpha(S) were measured by surface plasmon resonance, and the apparent dissociation constant was determined to be 0.9 x 10(-6)M. In contrast, the second intracellular loop and C-terminal tail regions showed negligible affinity to Galpha(S). The critical residues within the iL3 region for the interaction with Galpha(S) were identified as conserved positively charged residues near the C-terminus of iL3 by measuring the cellular levels of cAMP produced in response to 5-HT stimulation of cells transfected with 5-HT(6) receptor mutants.     

Interactions of nuclear estrogen receptor with DNA and RNA

The interaction of the nuclear estrogen receptor from hen oviduct with nucleic acids were studied by competition assay using DNa-cellulose centrifugation. We demonstrated that the estradiol-receptor complex binds similarly well to poly(A) RNA and denatured DNA. The estrogen receptor was found to interact more strongly with poly(G), poly(U) than with poly(A), poly(C). The receptor complex binds similarly to poly(A) and poly(dA), and to poly(U) and poly(dU). However, the receptor complex shows stronger binding to poly(G) than to poly(dG) and to poly(C) than to poly(dC). Studies with heteropolyribonucleotides indicated that poly(U1G1) is more effective in competing for the estrogen receptor, and poly(AC) and poly(AUG) are moderately effective, whereas poly(ACU) is least effective. GMP and dGMP showed some competition for the nuclear receptor at 300-fold higher nucleotide concentrations than that of the synthetic poly(G). Observations that the nuclear estrogen receptor binds to poly(A) RNA and interacts selectively with polyribonucleotides suggest that the estrogen receptor-RNA interaction may play a role for the function of estrogens in gene regulation.     

The interaction of ethidium with synthetic double-stranded polynucleotides at low ionic strength.

The interaction of ethidium with synthetic DNA and RNA double-stranded polymers at 0.01 M ionic strength, pH 7.0, has been studied by fluorimetry at low drug to nucleotide ratios. Binding constants have been calculated assuming an excluded-neighbouring site model for the interaction of ethidium with double-stranded polymers. The values obtained are poly d(AT).poly d(AT), 9.5 X 10(6) M-1; poly dA.poly dT, 6.5 X 10(5) M-1; poly d(GC).poly d(GC), 9.9 X 10(6) M-1; poly dG,poly dC, 4.5 X 1-(6) M-1; poly d(AC); poly d(GT), 9.8 X 10(6) M-1; poly d(AG).poly d(CT), 1.3 X 10(6) M-1; poly rA.poly rU, 4.1 X 10(7) M-1. The displacement of ethidium from poly d(AT).poly d(AT) by 9-aminoacridine and an acridine-containing antitumor agent (NSC 156303; 4'-(9-acridinylamino)methanesulphon-m-anisidide) has also been examined.     

Trimer hydroxylated quinone derived from apocynin targets cysteine residues of p47phox preventing the activation of human vascular NADPH oxidase

Enzymatically derived oligophenols from apocynin can be effective inhibitors of human vascular NADPH oxidase (Nox). An isolated trimer hydroxylated quinone (IIIHyQ) has been shown to inhibit endothelial NADPH oxidase with an IC(50) ~30 nM. In vitro studies demonstrated that IIIHyQ is capable of disrupting the interaction between p47(phox) and p22(phox), thereby blocking the activation of the Nox2 isoform. Herein, we report the role of key cysteine residues in p47(phox) as targets for the IIIHyQ. Incubation of p47(phox) with IIIHyQ results in a decrease of ~80% of the protein free cysteine residues; similar results were observed using 1,2- and 1,4-naphthoquinones, whereas apocynin was unreactive. Mutants of p47(phox), in which each Cys was individually replaced by Ala (at residues 111, 196, and 378) or Gly (at residue 98), were generated to evaluate their individual importance in IIIHyQ-mediated inhibition of p47(phox) interaction with p22(phox). Specific Michael addition on Cys196, within the N-SH3 domain, by the IIIHyQ is critical for disrupting the p47(phox)-p22(phox) interaction. When a C196A mutation was tested, the IIIHyQ was unable to disrupt the p47(phox)-p22(phox) interaction. However, the IIIHyQ was effective at disrupting this interaction with the other mutants, displaying IC(50) values (4.9, 21.0, and 2.3μM for the C111A, C378A, and C98G mutants, respectively) comparable to that of wild-type p47(phox).     

Structural and mutational characterization of -carnitine binding to human carnitine acetyltransferase

We report the crystal structure of a binary complex of human peroxisomal carnitine acetyltransferase and the substrate l-carnitine, refined to a resolution of 1.8 Angstrom with an R(factor) value of 18.9% (R(free)=22.3%). L-carnitine binds to a preformed pocket in the active site tunnel of carnitine acetyltransferase aligned with His(322). The quaternary nitrogen of carnitine forms a pi-cation interaction with Phe(545), while Arg(497) forms an electrostatic interaction with the negatively charged carboxylate group. An extensive hydrogen bond network also occurs between the carboxylate group and Tyr(431), Thr(444), and a bound water molecule. Site-directed mutagenesis and kinetic characterization reveals that Tyr(431), Thr(444), Arg(497), and Phe(545) are essential for high affinity binding of L-carnitine.