Role of zinc in the structure and toxic activity of botulinum neurotoxin.
Zn2+-protease activity of botulinum neurotoxin causes the blockage of neurotransmitter release resulting in botulism disease. We have investigated the role of Zn2+ in the biological activity of type A botulinum neurotoxin by removing the bound Zn2+ by EDTA treatment, followed by monitoring its structure in terms of secondary and tertiary folding (second derivative UV, FT-IR, and circular dichroism spectroscopy) and function in terms of its effect on the release of norepinephrine from PC12 cells. The single Zn2+ bound to each neurotoxin molecule was reversibly removed by EDTA treatment, whereas the biological activity of the neurotoxin was irreversibly lost. Based on the Amide III IR spectral analysis, the alpha-helical content of neurotoxin increased from 29% to 42% upon removal of Zn2+, which reverted to 31% upon treatment with 1:5 molar excess of exogenous Zn2+. Second derivative UV spectroscopy revealed no change in surface topography of Tyr residues with removal of Zn2+. However, near-UV circular dichroism signals suggested significant alterations in the topography of Phe and Tyr residues that could be buried in the protein matrix. Thermal unfolding experiments suggested that removal of Zn2+ results in the formation of the molten globule-like structure of type A botulinum neurotoxin. Tertiary structural changes introduced by Zn2+ removal were irreversible, which correlated well with the irreversibility of the biological activity of the neurotoxin. On the basis of these results, we suggest that Zn2+ plays a significant structural role in addition to its catalytic role in Zn2+-protease activity of type A botulinum neurotoxin.
Fu
F N
FN
Department of Chemistry and Biochemistry, University of Massachusetts Dartmouth, Dartmouth, Massachusetts 02747, USA.
Lomneth
R B
RB
Cai
S
S
Singh
B R
BR
D000818:Animals; D001059:Apoproteins / Q000737:chemistry / Q000633:toxicity; D019274:Botulinum Toxins, Type A / Q000737:chemistry* / Q000633:toxicity; D002118:Calcium / Q000378:metabolism; D002463:Cell Membrane Permeability; D018678:Cholinergic Agents / Q000737:chemistry* / Q000633:toxicity; D002942:Circular Dichroism; D006358:Hot Temperature; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000633:toxicity; D009474:Neurons / Q000187:drug effects; D009638:Norepinephrine / Q000378:metabolism; D016716:PC12 Cells; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D015032:Zinc
D001059:Apoproteins; D018678:Cholinergic Agents; D008666:Metalloendopeptidases; D019274:Botulinum Toxins, Type A; D015032:Zinc; D002118:Calcium; D009638:Norepinephrine