Independent interaction of the acyltransferase HlyC with two maturation domains of the Escherichia coli toxin HlyA

Abstract

The apparently unique fatty acylation mechanism that underlies activation (maturation) of Escherichia coli haemolysin and related toxins is further clarified by investigation of the interaction of protoxin with the specific acyltransferase HlyC. Using deleted protoxin variants and protoxin peptides as substrates in an in vitro maturation reaction dependent upon HlyC and acyl-acyl carrier protein, two independent HlyC recognition domains were identified on the 1024-residue protoxin, proA, and they were shown to span the two target lysine residues K564 (KI) and K690 (KII) that are fatty acylated. Each domain required 15-30 amino acids for basal recognition and 50-80 amino acids for wild-type acylation. The two domains (FAI and FAII) competed with each other in cis and in trans for HlyC. The affinity of FAI for HlyC is approximately four times greater than that of FAII resulting in an overall 80% acylation at KI and 20% acylation at KII in both whole toxin and peptide derivatives. No other proA sequences were required for toxin maturation, and excess Ca2+ prevented acylation of both lysines. The lack of primary sequence identity between FAI and FAII domains in proA and among corresponding sites on related protoxins currently precludes an explanation of the basis of HlyC recognition by proA.

Structure-function studies of the adenylate cyclase toxin of Bordetella pertussis and the leukotoxin of Pasteurella haemolytica by heterologous C protein activation and construction of hybrid proteins

“…One group, which includes the hemolysin (HlyA) of Escherichia coli, the hemolysins (ApxIA and ApxIIA) of Actinobacillus pleuropneumoniae, and the adenylate cyclase toxin (CyaA) of Bordetella pertussis, have a low specificity and are active on a wide range of mammalian cell types. A second group, which includes the leukotoxins of Pasteurella haemolytica (LktA), Actinobacillus actinomycetemcomitans (LtxA), and A. pleuropneumoniae (ApxIIIA), have high specificity and are able to lyse only leukocytes of ruminant, primate, or porcine origin, respectively.The RTX toxins have a common domain structure: a hydrophobic domain in the N-terminal half of the protein which is required for pore formation and forms a putative membranespanning region (1); the GD-rich repeats forming a calciumbinding domain towards the C terminus of the protein, which is involved in target cell binding (3); a domain at the extreme C terminus of the protein which functions as a signal for secretion, a process dependent on the products of the B and D genes and which occurs without a periplasmic intermediate (14); and a region between the pore-forming domain and the calcium-binding domain which is highly conserved within the RTX family and which was recently implicated in protoxin activation (27,28). All RTX toxins are synthesized as protoxins encoded by the A gene.…”

E. coli Alpha Hemolysin and Properties

“…Indeed, in vitro, Ca +2 ions prevent acylation at both sites (Stanley et al, 1996). The extreme sensitivity of the proHlyA activation reaction to free Ca +2 supports the view that intracellular Ca +2 levels in E. coli are too low to affect toxin activity and that Ca +2 binding does not occur until the toxin is outside the cell.…”

“…Both acylation sites in the HlyA genome function independently of one another with respect to the kinetics of their interaction with acylHlyC (Langston et al, 2004). By using deleted protoxin variants and protoxin peptides as substrates in an in-vitro maturation reaction dependent on only HlyC and acyl-ACP, two independent HlyC-recognition domains were identified on the HlyA protoxin, each of which spanned one of the target lysine residues (Stanley et al, 1996). Each domain required 15 to 30 amino acids for basal recognition and 50 to 80 for full wild-type acylation, but HlyC recognized a large topology rather than a linear sequence.…”

In Vivo Proteolytic Degradation of the Escherichia coli Acyltransferase HlyC

“…4, lanes d and c). Moreover, HlyC was detected in cell lysates from a strain encoding HlyC and another form of HlyA (HlyA ⌬722-724, Table I), lacking part of a putative HlyC binding domain (26,27) (Fig. 4, lane a).…”

“…The unique acylation reaction catalyzed by HlyC does not involve covalent attachment of fatty acid molecules to the acyltransferase but a direct interaction between acylACP-HlyC and pro-HlyA through a non-covalent ternary complex (22). A HlyC binding domain in pro-HlyA has been proposed (26,27), and some highly conserved amino acid residues in the carboxyl and amino termini of HlyC essential for the acylation process have been identified (17, 28 -30).…”