Activation of Ca2+-independent membrane-damaging activity in Lys49–phospholipase A2 promoted by amphiphilic molecules

Bortoleto-Bugs, Raquel Kely; Neto, Augusto Agostinho; Ward, Richard J.

doi:10.1016/j.bbrc.2004.06.181

Cited by 10 publications

(8 citation statements)

References 54 publications

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“…This interaction may either directly perturb the membrane structure itself or exert its effects via interaction with an as yet unidentified membrane receptor (85). This hypothesis is also consistent with recent experimental data on bothropstoxin-I, suggesting that its membrane-damaging effects are dependent on the binding of SDS in the case of artificial membranes and in the presence of an intact hydrophobic channel (86,87).…”

Section: Ligand-induced Conformational Change In Lys 49supporting

confidence: 87%

A Molecular Mechanism for Lys49-Phospholipase A2 Activity Based on Ligand-induced Conformational Change

Ambrósio

Nonato²,

Araújo

et al. 2005

Journal of Biological Chemistry

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Agkistrodon contortrix laticinctus myotoxin is a Lys49 -phospholipase A 2 (EC 3.1.1.4) isolated from the venom of the serpent A. contortrix laticinctus (broad-banded copperhead). We present here three monomeric crystal structures of the myotoxin, obtained under different crystallization conditions. The three forms present notable structural differences and reveal that the presence of a ligand in the active site (naturally presumed to be a fatty acid) induces the exposure of a hydrophobic surface (the hydrophobic knuckle) toward the C terminus. The knuckle in A. contortrix laticinctus myotoxin involves the side chains of Phe 121 and Phe 124 and is a consequence of the formation of a canonical structure for the main chain within the region of residues 118 -125. Comparison with other Lys49 -phospholipase A 2 myotoxins shows that although the knuckle is a generic structural motif common to all members of the family, it is not readily recognizable by simple sequence analyses. An activation mechanism is proposed that relates fatty acid retention at the active site to conformational changes within the C-terminal region, a part of the molecule that has long been associated with Ca 2؉ -independent membrane damaging activity and myotoxicity. This provides, for the first time, a direct structural connection between the phospholipase "active site" and the C-terminal "myotoxic site," justifying the otherwise enigmatic conservation of the residues of the former in supposedly catalytically inactive molecules.

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Section: Ligand-induced Conformational Change In Lys 49supporting

confidence: 87%

A Molecular Mechanism for Lys49-Phospholipase A2 Activity Based on Ligand-induced Conformational Change

Ambrósio

Nonato²,

Araújo

et al. 2005

Journal of Biological Chemistry

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“…Spectroscopic analyses of the tryptophan environment were performed over a range of heparin/protein ratios. Each BthTx-I monomer contains a single tryptophan residue located in the dimer interface region, which is a sensitive probe of the microenvironment at the interface [10,11]. The effect of heparins on the ITFE of BthTx-I is presented in Fig.…”

Section: Structural Interaction Of Bthtx-i With Heparinmentioning

confidence: 99%

“…Bothropstoxin-I was purified from whole B. jararacussu venom using a combination of cation exchange and subsequent size exclusion chromatography [11] and stored at 4 • C in 25 mM Tris-HCl, 150 mM of NaCl at pH 7 until further use. Protein purity was routinely evaluated by silver staining of SDS-PAGE gels.…”

Section: Protein Purification Gel Filtration and Sample Preparationmentioning

confidence: 99%

“…The flexibility at the interface 0141-8130/$ -see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2005.08.003 region permits a quaternary structural transition between "open" and "closed" dimer conformations, and a model for the Ca 2+ -independent membrane-damaging mechanism has been suggested in which a transition between these two conformations in the membrane-bound protein results in the perturbation and disruption of the membrane bilayer [10,11].…”

Section: Introductionmentioning

confidence: 99%

“…With the aim of further understanding the structural interaction of protein/heparin, we have studied the bothropstoxin-I (BthTx-I), a Lys49-PLA 2 isolated from the venom of Bothrops jararacussu that forms homodimers both in solution and in the crystalline state [9][10][11]. The flexibility at the interface 0141-8130/$ -see front matter © 2005 Elsevier B.V. All rights reserved.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

The interaction between heparin and Lys49 phospholipase A2 reveals the natural binding of heparin on the enzyme

Bugs

Bortoleto-Bugs

Cornélio

2005

International Journal of Biological Macromolecules

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Photoacoustic spectroscopy of aromatic amino acids in proteins

2007

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This paper concerns the use of photoacoustic spectroscopy (PAS) to study the presence of aromatic amino acid in proteins. We examined the aromatic amino acids in six proteins with well-known structures using absorption spectra of near ultraviolet PAS over the wavelength range 240-320 nm. The fundamental understanding of the physical and chemical properties that govern the absorption of light and a subsequent release of heat to generate a transient pressure wave was used to test the concept of monitoring aromatic amino acids with this method. Second derivative spectroscopy in the ultraviolet region of proteins was also used to study the regions surrounding the aromatics and the percentage area in each band was related in order to determine the contribution in function of the respective molar extinction coefficients for each residue. Further investigation was conducted into the interaction between sodium dodecyl sulphate (SDS) and bothropstoxin-I (BthTx-I), with the purpose of identifying the aromatics that participate in the interaction. The clear changes in the second derivative and curve-fitting procedures suggest that initial SDS binding to the tryptophan located in the dimer interface and above 10 SDS an increased intensity between 260 and 320 nm, demonstrating that the more widespread tyrosine and phenylalanine residues contribute to the SDS/BthTx-I interactions. These results demonstrate the potential of near UV-PAS for the investigation of membrane proteins/detergent complexes in which light scattering is significant.

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Activation of Ca2+-independent membrane-damaging activity in Lys49–phospholipase A2 promoted by amphiphilic molecules

Cited by 10 publications

References 54 publications

A Molecular Mechanism for Lys49-Phospholipase A2 Activity Based on Ligand-induced Conformational Change

A Molecular Mechanism for Lys49-Phospholipase A2 Activity Based on Ligand-induced Conformational Change

The interaction between heparin and Lys49 phospholipase A2 reveals the natural binding of heparin on the enzyme

Photoacoustic spectroscopy of aromatic amino acids in proteins

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