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.

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.

[Relapsing polychondritis: report of a case and review of the literature].

Relapsing polychondritis (RP) is a rare disease of unknown etiology, characterized by episodic and progressive chondritis, ocular and audiovestibular involvement, and occasional cardiovascular abnormalities. The inflammation typically involves the cartilage of the ears, nose, trachea, larynx, ribs, joints, and Eustachian tubes. The major clinical features include auricular chondritis, arthritis, nasal chondritis, ocular inflammation, chondritis of the respiratory tract, audiovestibular damage, and cardiovascular disorders. RP occurs predominantly in Caucasians and is uncommon in Oriental patients. Two cases of RP have previously been reported in Taiwan. We report one Taiwanese patient who was diagnosed with RP according to the criteria proposed by Damiani and Levine. The patient has bilateral auricular chondritis, episcleritis, and uveitis. Successful treatment was accomplished with systemic corticosteroid and topical corticosteroid eye drops. At an outpatient visit one year and ten months after discharge, there was no evidence of recurrence.