Main-Chain Dynamics of Cardiotoxin II from Taiwan Cobra (<i>Naja naja atra</i>) as Studied by Carbon-13 NMR at Natural Abundance:  Delineation of the Role of Functionally Important Residues

Lee, Chang Shin; Kumar, Thallapuranam Krishnaswamy Suresh; Lian, Lu; Cheng, Jya Wei; Yu, Chao

doi:10.1021/bi971979c

Cited by 23 publications

(26 citation statements)

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“…Tyrosine residues located at positions 11 and 22 are lodged in the double-and triple-stranded ␤-sheet segments, respectively. Tyr 22 is one of the most well conserved residue found in all the cardiotoxin isoforms (17)(18)(19). This residue (Tyr 22 ) is the locus of a prominent hydrophobic core that plays an important role in maintaining the structural integrity of the three-dimensional structure of cardiotoxins.…”

Section: Resultsmentioning

confidence: 99%

Binding of Nucleotide Triphosphates to Cardiotoxin Analogue II from the Taiwan Cobra Venom (Naja naja atra)

Jayaraman

Krishnaswamy

Kumar

et al. 1999

Journal of Biological Chemistry

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Snake venom cardiotoxins have been recently shown to block the enzymatic activity of phospholipid protein kinase and Na ؉ ,K ؉ -ATPase. To understand the molecular basis for the inhibitory effects of cardiotoxin on the action of these enzymes, the nucleotide triphosphate binding ability of cardiotoxin analogue II (CTX II) from the Taiwan cobra (Naja naja atra) venom is investigated using a variety of spectroscopic techniques such as fluorescence, circular dichroism, and two-dimensional NMR. CTX II is found to bind to all the four nucleotide triphosphates (ATP, UTP, GTP, and CTP) with similar affinity. Detailed studies of the binding of dATP to CTX II indicated that the toxin molecule is significantly stabilized in the presence of the nucleotide. Molecular modeling, based on the NOEs observed for the dATP⅐CTX II complex, reveals that dATP binds to the CTX II molecule at the groove enclosed between the Nand C-terminal ends of the toxin molecule. Based on the results obtained in the present study, a molecular mechanism to account for the inhibition of the enzymatic activity of the phospholipid-sensitive protein kinase and Na ؉ ,K ؉ -ATPase is also proposed.Snake venom cardiotoxins are small molecular mass (ϳ7 kDa), highly basic proteins cross-linked by four disulfide bridges (1-3). These toxins exhibit a wide variety of biological activities such as contraction of cardiac muscle cells, lysis of erythrocytes, and selective toxicity to certain types of tumor cells (4, 5). More recently, cardiotoxins have been demonstrated to selectively inhibit the action of certain key enzymes such as Na ϩ ,K ϩ -ATPase and phospholipid-sensitive protein kinase (2, 6 -8). Although the general molecular mechanism underlying the enzyme inhibitory action of cardiotoxins is still an enigma, it is contemplated that cardiotoxin could effectively block the enzymatic activities of Na ϩ ,K ϩ -ATPase and phospholipid-sensitive protein kinase by competitively binding (in the present study) to adenosine triphosphate (ATP), which is a key substrate for the functioning of these enzymes. However, to date, the evidence for the binding of a nucleotide triphosphate to cardiotoxin has not been reported.In the present study, for the first time, we demonstrate that the cardiotoxin analogue II (CTX II) 1 isolated from the Taiwan cobra venom (Naja naja atra) bind to nucleotide triphosphates. Herein, we also propose a molecular model of the CTX II⅐dATP complex and suggest a reasonable molecular mechanism to explain the reported inhibitory action of snake venom cardiotoxin on phospholipid-sensitive protein kinase and Na ϩ ,K ϩ -ATPase. MATERIALS AND METHODSCTX II was purified as described by Yang et al. (9). The concentration of the protein was estimated from its absorbance at 280 nm (⑀ 1 mM ϭ 4.08). The nucleotides were purchased from Sigma. The concentration of the nucleotides was estimated (10) from their extinction coefficient values (⑀ 259 ϭ 15,400 for AMP, ADP, ATP, and dATP; ⑀ 271 ϭ 9,000 for CTP; ⑀ 253 ϭ 13,700 for GTP; and ⑀ 260 ϭ 1,000 for UTP). Al...

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Section: Resultsmentioning

confidence: 99%

Binding of Nucleotide Triphosphates to Cardiotoxin Analogue II from the Taiwan Cobra Venom (Naja naja atra)

Jayaraman

Krishnaswamy

Kumar

et al. 1999

Journal of Biological Chemistry

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“…S5). 32,33 The average S 2 values of Den overall and the b-sheet were 0.84 and 0.90, respectively. Compared to the rest of the Den backbone, only the S 2 values of the residue R1 were lower than that of G48.…”

Section: Dynamics Analysis Of Denmentioning

confidence: 98%

Dynamics and functional differences between dendroaspin and rhodostomin: Insights into protein scaffolds in integrin recognition

et al. 2012

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Dendroaspin (Den) and rhodostomin (Rho) are snake venom proteins containing a PRGDMP motif. Although Den and Rho have different 3D structures, they are highly potent integrin inhibitors. To study their structure, function, and dynamics relationships, we expressed Den and Rho in Pichia pastoris. The recombinant Den and Rho inhibited platelet aggregation with the K I values of 149.8 and 83.2 nM. Cell adhesion analysis showed that Den was 3.7 times less active than Rho when inhibiting the integrin aIIbb3 and 2.5 times less active when inhibiting the integrin avb3. In contrast, Den and Rho were similarly active when inhibiting the integrin a5b1 with the IC 50 values of 239.8 and 256.8 nM. NMR analysis showed that recombinant Den and Rho have different 3D conformations for their arginyl-glycyl-aspartic acid (RGD) motif. However, the comparison with Rho showed that the docking of Den into integrin avb3 resulted in a similar number of contacts. Analysis of the dynamic properties of the RGD loop in Den and Rho showed that they also had different dynamic properties. These results demonstrate that protein scaffolds affect the function, structure, and dynamics of their RGD motif.

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“…m values signify the rate of overall tumbling of the protein molecule in solution and it ( m ) bears a near-linear relationship with the molecular weight of the protein (Lee et al 1998). In this context, we measured the longitudinal (T 1 ) and transverse relaxation (T 2 ) rates of the backbone 15 N nucleus using isotope-( 15 N) enriched nFGF-1 sample (Suppl.…”

Section: Sos Does Not Oligomerize Nfgf-1mentioning

confidence: 99%

Oligomerization of acidic fibroblast growth factor is not a prerequisite for its cell proliferation activity

et al. 2002

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Oligomerization of fibroblast growth factors (FGFs) induced on binding to heparin or heparan sulfate proteoglycan is considered to be crucial for receptor activation and initiation of biological responses. To gain insight into the mechanism of activation of the receptor by FGFs, in the present study we investigate the effect(s) of interaction of a heparin analog, sucrose octasulfate (SOS), on the structure, stability, and biological activities of a recombinant acidic FGF from Notophthalmus viridescens (nFGF-1). SOS is found to bind to nFGF-1 and significantly increase the thermodynamic stability of the protein. Using a variety of techniques such as size-exclusion chromatography, sedimentation velocity, and multidimensional nuclear magnetic resonance (NMR) spectroscopy, it is shown that binding of SOS to nFGF-1 retains the protein in its monomeric state. In its monomeric state (complexed to SOS), n-FGF-1 shows significant cell proliferation activity. 15N and 1 H chemical shift perturbation and the intermolecular nuclear Overhauser effects (NOEs) between SOS and nFGF-1 reveal that the ligand binds to the dense, positively charged cluster located in the groove enclosed by ␤-strands 10 and 11. In addition, molecular modeling based on the NOEs observed for the SOS-nFGF-1 complex, indicates that SOS and heparin share a common binding site on the protein. In conclusion, the results of the present study clearly show that heparin-induced oligomerization of nFGF-1 is not mandatory for its cell proliferation activity.Keywords: nFGF-1; ␤-barrel; stability; oligomerization; heparin binding; receptor Supplemental material: See www.proteinscience.org Fibroblast growth factors (FGFs) are a large family of monomeric growth factors that control proliferation, differentiation, and migration of a variety of cell types and can act as potent stimulators of angiogenesis and promote wound healing (Bikfalvi 1997;Conrad 1998;Friesel and Maciag 1999). At the cell surface, FGFs bind to two receptor classes

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Main-Chain Dynamics of Cardiotoxin II from Taiwan Cobra (Naja naja atra) as Studied by Carbon-13 NMR at Natural Abundance: Delineation of the Role of Functionally Important Residues

Cited by 23 publications

References 50 publications

Binding of Nucleotide Triphosphates to Cardiotoxin Analogue II from the Taiwan Cobra Venom (Naja naja atra)

Binding of Nucleotide Triphosphates to Cardiotoxin Analogue II from the Taiwan Cobra Venom (Naja naja atra)

Dynamics and functional differences between dendroaspin and rhodostomin: Insights into protein scaffolds in integrin recognition

Oligomerization of acidic fibroblast growth factor is not a prerequisite for its cell proliferation activity

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