Transcription factor SPZ1 promotes TWIST-mediated epithelial-mesenchymal transition and oncogenesis in human liver cancer.

The epithelial-mesenchymal transition (EMT) is an important process in the progression of cancer. However, its occurrence and mechanism of regulation are not fully understood. We propose a regulatory pathway in which spermatogenic leucine zipper 1 (SPZ1) promotes EMT through its transactivating ability in increasing TWIST1 expression. We compared the expression of SPZ1 and TWIST1 in specimens of hepatocarcinoma cells (HCCs) and non-HCCs. Expression of SPZ1 exhibited a tumor-specific expression pattern and a high correlation with patients' survival time, tumor size, tumor number and progression stage. Moreover, forced expression and knockdown of SPZ1 in hepatoma cells showed that SPZ1 was able to regulate the cellular proliferation, invasion, and tumorigenic activity in a TWIST1-dependent manner in vitro and in vivo. These data demonstrate that SPZ1, a newly dscribed molecule, transactivates TWIST1 promoters, and that this SPZ1-TWIST axis mediates EMT signaling and exerts significant regulatory effects on tumor oncogenesis.

Screening and characterization of LAB-produced bacteriocin-like substances from the intestine of grey mullet (Mugil cephalus L.) as potential biocontrol agents in aquaculture.

AIM: To isolate and characterize bacteriocin-like inhibitory substance (BLIS)-producing lactic acid bacteria from the intestine of grey mullet. METHODS AND RESULTS: Inhibitory activity against at least one or more indicator strains was observed in one Enterococcus thailandicus, one Enterococcus faecium and two Lactococcus garvieae strains. Enterococcus faecium B3-8 and Ent. thailandicus B3-22 showed the greatest inhibitory activities against Listeria monocytogenes ATCC 19111 and were therefore further characterized. The results suggested that the inhibitory substances from the two strains showed similar characteristics with respect to sensitivity to heat and proteolytic enzymes. BLIS from Ent. thailandicus B3-22 was characterized by a broader inhibitory spectrum than that from Ent. faecium B3-8. SDS-PAGE revealed that the molecular size of partially purified BLISs from Ent. faecium B3-8 and Ent. thailandicus B3-22 was c. 5 and 3 kDa, respectively. The molecular mass of purified bacteriocin from Ent. thailandicus B3-22 was further determined to be 6319 Da by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The results indicated that BLIS from Ent. thailandicus B3-22 can effectively inhibit the growth of all tested L. garvieae strains. CONCLUSIONS: The findings obtained in this study suggest the potential use of Ent. thailandicus B3-22 as a biocontrol agent against pathogenic L. garvieae in the aquaculture. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report describing the characteristics of BLIS from Ent. thailandicus that showed potential for use as a biocontrol agent in the aquaculture.

The HPV E6 oncoprotein targets histone methyltransferases for modulating specific gene transcription.

Expression of viral proteins causes important epigenetic changes leading to abnormal cell growth. Whether viral proteins directly target histone methyltransferases (HMTs), a key family enzyme for epigenetic regulation, and modulate their enzymatic activities remains elusive. Here we show that the E6 proteins of both low-risk and high-risk human papillomavirus (HPV) interact with three coactivator HMTs, CARM1, PRMT1 and SET7, and downregulate their enzymatic activities in vitro and in HPV-transformed HeLa cells. Furthermore, these three HMTs are required for E6 to attenuate p53 transactivation function. Mechanistically, E6 hampers CARM1- and PRMT1-catalyzed histone methylation at p53-responsive promoters, and suppresses the binding of p53 to chromatinized DNA independently of E6-mediated p53 degradation. p53 pre-methylated at lysine-372 (p53K372 mono-methylation) by SET7 protects p53 from E6-induced degradation. Consistently, E6 downregulates p53K372 mono-methylation and thus reduces p53 protein stability. As a result of the E6-mediated inhibition of HMT activity, expression of p53 downstream genes is suppressed. Together, our results not only reveal a clever approach for the virus to interfere with p53 function, but also demonstrate the modulation of HMT activity as a novel mechanism of epigenetic regulation by a viral oncoprotein.

Impact of model uncertainty on soil quality standards for cadmium in rice paddy fields.

At present, soil quality standards used for agriculture do not consider the influence of pH and CEC on the uptake of pollutants by crops. A database with 750 selected paired samples of cadmium (Cd) in soil and paddy rice was used to calibrate soil to plant transfer models using the soil metal content, pH, and CEC or soil Cd and Zn extracted by 0.01 M CaCl2 as explanatory variables. The models were validated against a set of 2300 data points not used in the calibration. These models were then used inversely to derive soil quality standards for Japonica and Indica rice cultivars based on the food quality standards for rice. To account for model uncertainty, strict soil quality standards were derived considering a maximum probability that rice exceeds the food quality standard equal to 10 or 5%. Model derived soil standards based on Aqua Regia ranged from less than 0.3 mg kg⁻¹ for Indica at pH 4.5 to more than 6 mg kg⁻¹ for Japonica-type cultivars in clay soils at pH 7. Based on the CaCl2 extract, standards ranged from 0.03 mg kg⁻¹ Cd for Indica cultivars to 0.1 mg kg⁻¹ Cd for Japonica cultivars. For both Japonica and Indica-type cultivars, the soil quality standards must be reduced by a factor of 2 to 3 to obtain the strict standards. The strong impact of pH and CEC on soil quality standards implies that it is essential to correct for soil type when deriving national or local standards. Validation on the remaining 2300 samples indicated that both types of models were able to accurately predict (> 92%) whether rice grown on a specific soil will meet the food quality standard used in Taiwan.

Changing microbial concentrations are associated with ventilation performance in Taiwan's air-conditioned office buildings.

UNLABELLED: Our study conducted serial environmental measurements in 12 large office buildings with two different ventilation designs to obtain airborne microbial concentrations in typical office buildings, and to examine the effects of occupant density, ventilation type and air exchange efficiency on indoor microbial concentrations. Duplicate samples of airborne fungi and bacteria, a total of 2477 measurements, were collected based on a scheme of conducting sampling three times a day for at least seven consecutive days at every study building. Air change rates (ACHs) were also estimated by tracer gas concentration decay method, and measured by continuous Multi-Gas monitor for each building. Most sampling sites were with total fungal and bacteria concentrations higher than 1000 CFU/m(3), an often-quoted guideline in earlier research. Significantly higher concentrations of fungi and bacteria, as well as higher indoor/outdoor (I/O) ratios across most groups of airborne microbes, were identified in buildings with fan coil unit (FCU) system than those with air-handling unit (AHU) system (Student's t test, P < 0.0001). Older buildings and higher air exchange rates were statistically associated with greater indoor bacteria levels in FCU ventilated buildings (R(2) = 0.452); a pattern not found in AHU buildings. Increasing ACH seemed to be the determinant factor for rising indoor fungal and Cladosporium concentrations in those FCU buildings (R(2) = 0.346; 0.518). Our data indicated that FCU ventilated buildings might have provided more outdoor matters into indoor environments through direct penetration of outdoor air. Results also demonstrated a quantitative association between rising numbers of occupants and increasing indoor levels of yeast in both FCU and AHU ventilated buildings. The regression model identified in this study may be considered a reference value for proposing an optimal ACH, while with adequate filtration of fresh air, as an effective strategy in lowering indoor microbial concentrations in air-conditioned buildings. PRACTICAL IMPLICATIONS: As control of indoor microbial contamination has become an increasing concern around the world, feasibility and effectiveness of adopting ventilation approach has attracted a significant interest. This field investigation demonstrated, quantitatively, critical variables to be taken into consideration while applying such a measure, including the kinds of microbes to be removed and the types of ventilation system already in place.

Risk assessment of formaldehyde in typical office buildings in Taiwan.

Our study conducts a series of investigations in five office buildings chosen according to the types of construction, ventilation, and building age. Formaldehyde was measured by continuous photoacoustic Multi-Gas monitor Type 1302 (Brüel & Kjaer). The 8-h average concentrations in working hours were used to estimate the lifetime cancer probability (LCP) and chronic non-carcinogenic hazard index (HI). The carcinogenic effect of formaldehyde estimate by LCP (70 years old) is about 2.06 x 10(-4) to 1.75 x 10(-3) after adjusting their working time. The levels of risk are 100-1000 times of the acceptable carcinogenic risk. A similar trend is observed for the levels of HI calculated. Many studies have suggested that exposure to high levels of formaldehyde may cause nasal cancer and other health effects. Therefore, promoting the labeling system for low emission materials to protect consumers from exposure to excessive emissions and helping the industry to develop low emission materials is evidently urgent and deserves greater efforts.

Reconstitution of recombinant TFIIH that can mediate activator-dependent transcription.

BACKGROUND: TFIIH is one of the general transcription factors required for accurate transcription of protein-coding genes by RNA polymerase II. TFIIH has helicase and kinase activities, plays a role in promoter opening and promoter escape, and is also implicated in efficient activator-dependent transcription. RESULTS: We have established a reconstitution system of recombinant TFIIH using a three-virus baculovirus expression system. The recombinant TFIIH was active in CTD kinase and DNA helicase assays, and showed both basal and activator-dependent transcriptional activities that were indistinguishable from those of HeLa cell-derived TFIIH. Further analyses using recombinant TFIIH confirmed a critical role of TFIIH in activator-dependent transcription. The dose response of TFIIH in activator-dependent transcription suggested that mere recruitment of TFIIH is not sufficient for transcriptional activation. The sensitivity of activator-dependent transcription to nonhydrolysable ATP analogues indicated the importance of the enzymatic activities of TFIIH in transcriptional activation. CONCLUSIONS: Our results raise a possibility that transcriptional activation by GAL4-VP16 requires enzymatic activities. Recombinant TFIIH reconstituted from this baculovirus system should be useful for analysis of the mechanisms of activation by GAL4-VP16.

TATA-binding protein-associated factors enhance the recruitment of RNA polymerase II by transcriptional activators.

Transcription factor (TF) IID, comprised of the TATA-binding protein (TBP) and TBP-associated factors (TAFs), is a general transcription factor required for RNA polymerase II (pol II) transcription on most eukaryotic genes. Recent findings that TAFs may not be globally required for activator-dependent transcription in vivo and in vitro and that both TAF-dependent and TAF-independent promoters are found in yeast suggest that transcriptional activation can occur through at least two different pathways, depending on the presence or absence of TAFs. Using order-of-addition and template challenge assays performed in a human cell-free transcription system reconstituted with recombinant general transcription factors (TFIIB, TBP, TFIIE, TFIIF), a recombinant general cofactor (PC4), and highly purified epitope-tagged multiprotein complexes (TFIID, TFIIH, pol II), we demonstrate that when TBP is used as the TATA-binding factor transcriptional activators such as Gal4-VP16 and human papillomavirus E2 mainly function by facilitating pol II entry to the promoter region. In contrast, when TFIID is used as the TATA-binding factor, promoter recognition by TFIID appears to be the rate-limiting step facilitated by transcriptional activators during preinitiation complex assembly. Using protein-protein pull-down and far-Western analyses, we further show that the presence of TAFs in TFIID facilitates the recruitment of pol II by transcriptional activators, thereby switching the rate-limiting step from pol II entry to promoter recognition. Our findings thus provide distinct molecular mechanisms for TAF-independent and TAF-dependent activation.

The intronless and TATA-less human TAF(II)55 gene contains a functional initiator and a downstream promoter element.

Human TAF(II)55 (hTAF(II)55) is a component of the multisubunit general transcription factor TFIID and has been shown to mediate the functions of many transcriptional activators via direct protein-protein interactions. To uncover the regulatory properties of the general transcription machinery, we have isolated the hTAF(II)55 gene and dissected the regulatory elements and the core promoter responsible for hTAF(II)55 gene expression. Surprisingly, the hTAF(II)55 gene has a single uninterrupted open reading frame and is the only intronless general transcription factor identified so far. Its expression is driven by a TATA-less promoter that contains a functional initiator and a downstream promoter element, as illustrated by both transfection assays and mutational analyses. Moreover, this core promoter can mediate the activity of a transcriptional activator that is artificially recruited to the promoter in a heterologous context. Interestingly, in the promoter-proximal region there are multiple Sp1-binding sites juxtaposed to a single AP2-binding site, indicating that Sp1 and AP2 may regulate the core promoter activity of the hTAF(II)55 gene. These findings indicate that a combinatorial regulation of a general transcription factor-encoding gene can be conferred by both ubiquitous and cell type-specific transcriptional regulators.

Origin of hepatitis delta virus mRNA.

Hepatitis delta virus (HDV) is unique relative to all known animal viruses, especially in terms of its ability to redirect host RNA polymerase(s) to transcribe its 1,679-nucleotide (nt) circular RNA genome. During replication there accumulates not only more molecules of the genome but also its exact complement, the antigenome. In addition, there are relatively smaller amounts of an 800-nt RNA of antigenomic polarity that is polyadenylated and considered to act as mRNA for translation of the single and essential HDV protein, the delta antigen. Characterization of this mRNA could provide insights into the in vivo mechanism of HDV RNA-directed RNA transcription and processing. Previously, we showed that the 5' end of this RNA was located in the majority of species, at nt 1630. The present studies show that (i) at least some of this RNA, as extracted from the liver of an HDV-infected woodchuck, behaved as if it contained a 5'-cap structure; (ii) in the infected liver there were additional polyadenylated antigenomic HDV RNA species with 5' ends located at least 202 nt and even 335 nt beyond the nt 1630 site, (iii) the 5' end at nt 1630 was not detected in transfected cells, following DNA-directed HDV RNA transcription, in the absence of genome replication, and (iv) nevertheless, using in vitro transcription with purified human RNA polymerase II holoenzyme and genomic RNA template, we did not detect initiation of template-dependent RNA synthesis; we observed only low levels of 3'-end addition to the template. These new findings support the interpretation that the 5' end detected at nt 1630 during HDV replication represents a specific site for the initiation of an RNA-directed RNA synthesis, which is then modified by capping.

Alleviation of human papillomavirus E2-mediated transcriptional repression via formation of a TATA binding protein (or TFIID)-TFIIB-RNA polymerase II-TFIIF preinitiation complex.

Transcription in human papillomaviruses (HPVs) is mainly regulated by cellular transcription factors and virus-encoded E2 proteins that act as sequence-specific DNA-binding proteins. Although the functions of E2 as a transcriptional activator and a repressor have been well documented, the role of cellular factors involved in E2-mediated regulation of the HPV promoters and the mechanism by which E2 modulates viral gene expression remain unclear. Using reconstituted cell-free transcription systems, we found that cellular enhancer-binding factors and general cofactors, such as TAF(II)s, TFIIA, Mediator, and PC4, are not required for E2-mediated repression. Unlike other transcriptional repressors that function through recruitment of histone deacetylase or corepressor complexes, HPV E2 is able to directly target components of the general transcription machinery to exert its repressor activity on the natural HPV E6 promoter. Interestingly, preincubation of TATA binding protein (TBP) or TFIID with HPV template is not sufficient to overcome E2-mediated repression, which can be alleviated only via formation of a minimal TBP (or TFIID)-TFIIB-RNA polymerase II-TFIIF preinitiation complex. Our data therefore indicate that E2 does not simply work by displacing TBP or TFIID from binding to the adjacent TATA box. Instead, E2 appears to function as an active repressor that directly inhibits HPV transcription at steps after TATA recognition by TBP or TFIID.

Isolation of mouse TFIID and functional characterization of TBP and TFIID in mediating estrogen receptor and chromatin transcription.

TFIID is a general transcription factor required for the assembly of the transcription machinery on most eukaryotic promoters transcribed by RNA polymerase II. Although the TATA-binding subunit (TBP) of TFIID is able to support core promoter and activator-dependent transcription under some circumstances, the roles of TBP-associated factors (TAF(II)s) in TFIID-mediated activation remain unclear. To define the evolutionarily conserved function of TFIID and to elucidate the roles of TAF(II)s in gene activation, we have cloned the mouse TAF(II)55 subunit of TFIID and further isolated mouse TFIID from a murine FM3A-derived cell line that constitutively expresses FLAG-tagged mouse TAF(II)55. Both mouse and human TFIIDs are capable of mediating transcriptional activation by Gal4 fusions containing different activation domains in a highly purified human cell-free transcription system devoid of TFIIA and Mediator. Although TAF(II)-independent activation by Gal4-VP16 can also be observed in this highly purified human transcription system with either mouse or yeast TBP, TAF(II)s are strictly required for estrogen receptor-mediated activation independently of the core promoter sequence. In addition, TAF(II)s are necessary for transcription from a preassembled chromatin template. These findings clearly demonstrate an essential role of TAF(II)s as a transcriptional coactivator for estrogen receptor and in chromatin transcription.

Immunoaffinity purification and functional characterization of human transcription factor IIH and RNA polymerase II from clonal cell lines that conditionally express epitope-tagged subunits of the multiprotein complexes.

Purification of multiprotein complexes such as transcription factor (TF) IIH and RNA polymerase II (pol II) has been a tedious task by conventional chromatography. To facilitate the purification, we have developed an effective scheme that allows human TFIIH and pol II to be isolated from HeLa-derived cell lines that conditionally express the FLAG-tagged p62 subunit of human TFIIH and the RPB9 subunit of human pol II, respectively. An approximate 2000-fold enrichment of FLAG-tagged TFIIH and a 1000-fold enhancement of total pol II are achieved by a one-step immunoaffinity purification. The purified complexes are functional in mediating basal and activated transcription, regardless of whether TATA-binding protein or TFIID is used as the TATA-binding factor. Interestingly, repression of basal transcription by the positive cofactor PC4 is alleviated by increasing amounts of TFIID, TFIIH, and pol II holoenzyme, suggesting that phosphorylation of PC4 by these proteins may cause a conformational change in the structure of PC4 that allows for preinitiation complex formation and initiation of transcription. Furthermore, pol II complexes with different phosphorylation states on the carboxyl-terminal domain of the largest subunit are selectively purified from the inducible pol II cell line, making it possible to dissect the role of carboxyl-terminal domain phosphorylation in the transcription process in a highly defined in vitro transcription system.

Expression of gonadotropin-releasing hormone (GnRH) gene in human uterine endometrial tissue.

Many peripheral reproductive tissues have been found to contain gonadotrophin-releasing hormone (GnRH) and express the GnRH gene at low levels, presumably because the hormone functions in a paracrine/autocrine fashion. This study was designed to investigate and characterize GnRH gene expression in human endometrial tissue at different stages of the endometrial cycle. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis together with Southern blot assay demonstrated that human endometrial tissue expresses the proGnRH gene. RNA samples from endometrial tissue were analysed with two pairs of oligonucleotide primers. Both gave a doublet 870 bases apart at the expected sizes, indicating that both the upstream and downstream transcriptional start sites of the GnRH gene are used in endometrial tissue and that transcripts with and without intron 1 were produced. Our data also demonstrated that utilization of the two promoters varies with the stage of the endometrial cycle. The largest difference came from the mRNA transcribed from the downstream promoter and without intron 1. This mRNA was expressed at a very low level during the proliferative phase and dramatically increased almost 10-fold (P < 0.01) during the early secretory phase, and subsequently decreased 5-fold during the late secretory stage. The presence of GnRH mRNA in the endometrium, as well as the differential expression of the GnRH gene during the early secretory phase provides physiological evidence that human GnRH may play a paracrine/autocrine function in the human uterus.

Effect of D57N mutation on membrane activity and molecular unfolding of cobra cardiotoxin.

Cobra cardiotoxins (CTXs) are able to adopt a three-fingered beta-strand structure with continuous hydrophobic patch that is capable of interacting with zwitterionic phospholipid bilayer. In addition to the four disulfide bonds that form the rigid core of CTXs, Asp57 near the C-terminus interacts electrostatically with Lys2 near the N-terminus (Chiang et al. 1996. Biochemistry. 35:9177-9186). We indicate herein, using circular dichroism and the time-resolved polarized tryptophan fluorescence measurement, that Asp57 to Asn57 (D57N) mutation perturbs the structure of CTX molecules at neutral pH. The structural stability of the D57N mutant was found to be lower, as evidenced by the reduced effective concentration of the 2,2,2-trifluoethanol (TFE)-induced beta-sheet to alpha-helix transition. Interestingly, the single mutation also allows a greater degree of molecular unfolding, because the rotational correlation time of the TFE-induced unfolding intermediate is larger for the D57N mutant. It is suggested that the electrostatic interaction between N- and C-termini also contributes to the formation of the functionally important continuous hydrophobic stretch on the distant end of CTX molecules, because both the binding to anilinonaphthalene fluorescent probe and the interaction with phospholipid bilayer were also reduced for D57N mutant. The result emphasizes the importance of the hydrophobic amino acid residues near the tip of loop 3 as a continuous part of the three-fingered beta-strand CTX molecule and indicates how a distant electrostatic interaction might be involved. It is also implicated that electrostatic interaction plays a role in expanding the radius of gyration of the folding/unfolding intermediate of proteins.

TAFII-independent activation mediated by human TBP in the presence of the positive cofactor PC4.

TFIID is a multiprotein complex comprised of the TATA-binding protein (TBP) and an array of TBP-associated factors (TAFIIs). Whereas TBP is sufficient for basal transcription in conjunction with other general transcription factors and RNA polymerase II, TAFIIs are additionally required for activator-dependent transcription in mammalian cell-free transcription systems. However, recent in vivo studies carried out in yeast suggest that TAFIIs are not globally required for activator function. The discrepancy between in vivo yeast studies and in vitro mammalian cell-free systems remains to be resolved. In this study, we describe a mammalian cell-free transcription system reconstituted with only recombinant proteins and epitope-tagged multiprotein complexes. Transcriptional activation can be recapitulated in this highly purified in vitro transcription system in the absence of TAFIIs. This TBP-mediated activation is not induced by human mediator, another transcriptional coactivator complex potentially implicated in activator response. In contrast, general transcription factors TFIIH and TFIIA play a significant role in TBP-mediated activation, which can be detected in vitro with Gal4 fusion proteins containing various transcriptional activation domains. Our data, therefore, suggest that TFIIH and TFIIA can mediate activator function in the absence of TAFIIs.

Properties of PC4 and an RNA polymerase II complex in directing activated and basal transcription in vitro.

A human RNA polymerase II (pol II) complex was isolated from a HeLa-derived cell line that conditionally expresses an epitope-tagged RPB9 subunit of human pol II. The isolated FLAG-tagged pol II complex (f:pol II) contains a subset of general transcription factors but is devoid of TFIID and TFIIA. In conjunction with TATA-binding protein (TBP) or TFIID, f:pol II is able to mediate both basal and activated transcription by Gal4-VP16 when a transcriptional coactivator PC4 is also provided. Interestingly, PC4, in the absence of a transcriptional activator, actually functions as a repressor to inhibit basal transcription. Remarkably, TBP is able to mediate activator function in this transcription system. The presence of TBP-associated factors, however, helps overcome PC4 repression and further enhance the level of activation mediated by TBP. Alleviation of PC4 repression can also be achieved by preincubation of the transcriptional components with the DNA template. Sarkosyl disruption of preinitiation complex formation further illustrates that PC4 can only inhibit transcription prior to the assembly of a functional preinitiation complex. These results suggest that PC4 represses basal transcription by preventing the assembly of a functional preinitiation complex, but it has no effect on the later steps of the transcriptional process.

Analysis of binding of cobra cardiotoxins to heparin reveals a new beta-sheet heparin-binding structural motif.

Heparin and heparan sulfate have recently been shown to bind to snake cardiotoxin (CTX) and to potentiate its penetration into phospholipid monolayer under physiological ionic conditions. Herein we analyze the heparin-binding domain of CTX using 10 CTXs from Taiwan and African cobra venom. We also performed computer modeling to obtain more information of the binding at molecular level. The results provide a molecular model for interaction of CTX-heparin complex where the cationic belt of the conserved residues on the concave surface of three finger beta-sheet polypeptides initiates ionic interaction with heparin-like molecules followed by specific binding of Lys residues near the tip of loop 2 of CTX. The dissociation constants of CTXs differ by as much as 4 orders of magnitude, ranging from approximately 140 microM for toxin gamma to approximately 20 nM for CTX M3, depending on the presence of Lys residues near the tip of loop 2. High affinity heparin binding becomes possible due to the presence of Arg-28, Lys-33, or the so-called consensus heparin binding sequence of XKKXXXKRX near the tip of the loop. The well defined three-finger loop structure of CTX provides an interesting template for the design of high affinity heparin-binding polypeptides with beta-sheet structure. The finding that several cobra CTXs and phospholipase A2 bind to heparin with different affinity may provide information on the synergistic action of the two venom proteins.

Crystal structure of cardiotoxin V from Taiwan cobra venom: pH-dependent conformational change and a novel membrane-binding motif identified in the three-finger loops of P-type cardiotoxin.

The crystal structure of cardiotoxin V from Taiwan cobra venom (CTX A5) has been solved at pH 8.5 and refined to an R-factor of 20.7% for 7013 reflections [>2sigma(F)] between 8- and 2.19-A resolution. The refined model shows that CTX A5 exists as a dimer. The assembly consists of 974 non-hydrogen atoms from 124 residues and 73 water molecules. The global monomeric structure is similar to that determined by NMR at pH 3.7, characterized by a core formed by two beta-sheets connected with three-finger loops. However, local conformational differences are detected in two functionally important regions, loops I and II. A disparity between the NMR and X-ray structure of CTX A5 is detected near the tip of loop I and can be attributed to the difference in the protonation state of His4 at different pH, resulting in a reorientation of the His4 imidazole ring. A concerted motion of amino acid side chains located near His4 is detected and possibly contributes to the pH-dependent binding ability of CTX A5 to phospholipid model membranes. The second difference, detected at the tip of loop II, is due to the hydrophobic contact between CTX dimers in the crystal packing and the interaction of water molecules with amino acid residues in the loop II region of the CTX containing Pro31 (P-type CTX). This interaction forces loop II into a more rigid omega shape bridging the main chain at positions 27 and 34, contradictory to the flexible, tapering shape detected by NMR. Thus, a novel continuous hydrophobic column capable of binding to and possibly penetrating the membrane lipid bilayer is formed by the tips of the three-finger loops. In this respect, the X-ray crystal structure of CTX A5 may represent the CTX structure in the membrane-binding mode.