A helical capping motif in ShK toxin and its role in helix stabilization.
ShK toxin, a 35-residue polypeptide cross-linked by three disulfides, is a potent blocker of voltage-gated potassium channels and is of interest as a lead in the development of new immunosuppressant agents. ShK toxin contains two short stretches of alpha-helix, the first of which is preceded by a putative N-capping box encompassing residues Thr13 and Gln16. (1)H and (13)C NMR data support the presence of this structural motif, but the hydrogen bonds involving residues 13 and 16 in the solution structure of ShK toxin do not match the pattern expected for a conventional N-cap motif. They do, however, fit the pattern for the recently described ST-motif, class 4a (Wan and Milner-White (1999) Journal of Molecular Biology, 1999, Vol. 286, pp. 1651-1662). The (1)H NMR chemical shifts, nuclear Overhauser effects, and amide exchange rates of native ShK toxin are compared with those of three synthetic analogues with the substitutions Thr13 to Ala and Gln16 to Glu and Ala in order to determine the contribution of this motif to the structure and stability of ShK toxin. Disruption of the capping interactions destabilizes the helices, with the Thr13 to Ala substitution being much more disruptive than Gln16 to Ala, consistent with the lack of hydrogen bonding to the side chain of residue i + 4 in a class 4a ST-motif. Mutation of residues 13 and 16 has only a minor effect on potassium channel binding, probably because the disulfide bonding network minimizes the effect of loss of the capping motif on the overall structure. The implications of these findings for the design of ShK analogues are discussed.
Lanigan
M D
MD
Biomolecular Research Institute, Parkville 3052, Australia.
Tudor
J E
JE
Pennington
M W
MW
Norton
R S
RS
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D003064:Cnidarian Venoms / Q000138:chemical synthesis / Q000737:chemistry* / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D015221:Potassium Channels / Q000737:chemistry; D017433:Protein Structure, Secondary
D003064:Cnidarian Venoms; D010455:Peptides; D015221:Potassium Channels; C095391:ShK neurotoxin