X-ray absorption spectroscopy study of zinc coordination in tetanus neurotoxin, astacin, alkaline protease and thermolysin.
Tetanus and botulinum neurotoxins constitute a new group of Zn-endopeptidases which has been recently actively investigated with the purpose of correlating their biochemical properties to their neurobiocytosis inhibitory capacity. Crystallographic data show that Zn-endopeptidases are characterized by an active site with a Zn atom coordinated to two histidines and glutamate-bound water molecule. The two histidines and glutamate resides belong to the HEXXH motif which is characteristic of most Zn-endopeptidases. A forth metal ligand is a glutamate in thermolysin-like proteinases, but it is an histidine in the astacin family of proteinases and in alkaline protease. Astacin and alkaline protease possess a tyrosine as fifth Zn ligand, whose position in the case of alkaline protease could not be determined by X-ray crystallography. Not much is known about the atom arrangement around the active site in tetanus neurotoxin. In this work X-ray absorption spectroscopy has been used to obtain information on the Zn coordination mode in tetanus neurotoxin. The near-edge and extended fine-structure absorption spectra of this toxin are compared with those of astacin, alkaline protease and thermolysin. The present data and sequence information suggest a new pattern of Zn coordination in tetanus neurotoxin with one water molecule and three aromatic residues as metal ligands. These residues are the two histidines of the characteristic motif and a tyrosine which is tentatively identified with Tyr242, on the basis of sequence comparison and mutagenesis experiments. The mean distances of the Zn from the nearest coordinated atoms is reported. Our results indicate that alkaline protease, like astacin, also possesses a tyrosine as a fifth ligand.
Morante
S
S
Dipartimento di Fisica, Universita' degli Studi di Roma 'Tor Vergata', Roma, Italy.
Furenlid
L
L
Schiavo
G
G
Tonello
F
F
Zwilling
R
R
Montecucco
C
C
D000595:Amino Acid Sequence; D000818:Animals; D003400:Astacoidea; D010450:Endopeptidases / Q000737:chemistry*; D005583:Fourier Analysis; D008666:Metalloendopeptidases / Q000737:chemistry*; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D013057:Spectrum Analysis; D013744:Tetanus Toxin / Q000737:chemistry*; D013820:Thermolysin / Q000737:chemistry*; D015032:Zinc / Q000737:chemistry*
D013744:Tetanus Toxin; D010450:Endopeptidases; D008666:Metalloendopeptidases; C071288:astacin; D013820:Thermolysin; D015032:Zinc