The first strain of Clostridium perfringens isolated from an avian source has an alpha-toxin with divergent structural and kinetic properties.
Clostridium perfringens alpha-toxin is a 370-residue, zinc-dependent, phospholipase C that is the key virulence determinant in gas gangrene. It is also implicated in the pathogenesis of sudden death syndrome in young animals and necrotic enteritis in chickens. Previously characterized alpha-toxins from different strains of C. perfringens are almost identical in sequence and biochemical properties. We describe the cloning, nucleotide sequencing, expression, characterization, and crystal structure of alpha-toxin from an avian strain, SWan C. perfringens (SWCP), which has a large degree of sequence variation and altered substrate specificity compared to these strains. The structure of alpha-toxin from strain CER89L43 has been previously reported in open (active site accessible to substrate) and closed (active site obscured by loop movements) conformations. The SWCP structure is in an open-form conformation, with three zinc ions in the active site. This is the first example of an open form of alpha-toxin crystallizing without the addition of divalent cations to the crystallization buffer, indicating that the protein can retain three zinc ions bound in the active site. The topology of the calcium binding site formed by residues 269, 271, 336, and 337, which is essential for membrane binding, is significantly altered in comparison with both the open and closed alpha-toxin structures. We are able to relate these structural changes to the different substrate specificity and membrane binding properties of this divergent alpha-toxin. This will provide essential information when developing an effective vaccine that will protect against C. perfringens infection in a wide range of domestic livestock.
Justin
Neil
N
School of Crystallography, Birkbeck College, Malet Street, London WC1E 7HX, United Kingdom.
Walker
Nicola
N
Bullifent
Helen L
HL
Songer
Glenn
G
Bueschel
Dawn M
DM
Jost
Helen
H
Naylor
Claire
C
Miller
Julie
J
Moss
David S
DS
Titball
Richard W
RW
Basak
Ajit K
AK
D000595:Amino Acid Sequence; D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity; D001715:Bird Diseases / Q000382:microbiology; D001717:Birds / Q000382:microbiology*; D002104:Cadmium / Q000378:metabolism; D002135:Calcium-Binding Proteins; D002417:Cattle; D003001:Cloning, Molecular; D003016:Clostridium perfringens / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D003460:Crystallization; D018360:Crystallography, X-Ray; D004761:Enterocolitis, Pseudomembranous / Q000382:microbiology / Q000662:veterinary; D005798:Genes, Bacterial; D006461:Hemolysis / Q000187:drug effects; D007700:Kinetics; D008297:Male; D008563:Membrane Lipids / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D010738:Type C Phospholipases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity; D015032:Zinc / Q000378:metabolism
D001427:Bacterial Toxins; D002135:Calcium-Binding Proteins; D008563:Membrane Lipids; D002104:Cadmium; D010738:Type C Phospholipases; C040649:alpha toxin, Clostridium perfringens; D015032:Zinc