Secretion and Circular Dichroism Analysis of the C-Terminal Signal Peptides of HlyA and LktA

Abstract

The secretion of the 107 kDa hemolysin A (HlyA) from Escherichia coli is mediated by membrane proteins hemolysin B (HlyB) and hemolysin D (HlyD). The signal for transport has been mapped to the C-terminal 60 amino acids of the HylA molecule. We have shown previously that the C-terminal 70 amino acids of leukotoxin (LktA) from Pasteurella hemolytica can substitute functionally for the HlyA signal sequence. This 70 amino acid peptide contains little primary sequence similarity to the HlyA signal sequence, and we have hypothesized that these signal sequences assume a similar higher-order structure which is recognized by the HlyB/D transporter. In the present study, we have expressed and purified small peptides containing the C-terminal 61 amino acids of HlyA and the C-terminal 70 amino acids of LktA. We show that these signal peptides are sufficient for secretion from E. coli in a HlyB/D dependent manner. Circular dichroism analyses show that both molecules exhibit common biophysical properties. In aqueous solution, they appear to be mainly unstructured, but in a membrane mimetic environment they assume a helical secondary structure. The conformational change observed for both peptides going from an aqueous to a membrane mimetic environment may be an important feature of these signal sequences necessary for their recognition and transport.

Random and directed mutagenesis to elucidate the functional importance of helix II and F-989 in the C-terminal secretion signal of Escherichia coli hemolysin

“…Moreover, these data suggest, as we previously proposed, that the functional secretion signal could be unfolded when it is presented to the translocation apparatus. The unfolded nature of the signal region in vivo indicated by genetic studies is supported by a recent circular dichroism analysis of the C-terminal, 60-residue peptide of HlyA, which indicates that the peptide adopts a predominantly random conformation in aqueous solution (27). The same study indicated that this peptide adopts a helical secondary structure in the presence of sodium dodecyl sulfate or anionic phospholipids, and a more recent nuclear magnetic resonance analysis (25) detected ␣-helical structures close to the position of the predicted ␣-helices I and II.…”

“…Such blocks of repeats, which define the so-called RTX toxins (24), can occur variously from 70 to 150 residues from the C terminus, and the number of RTX repeats can also vary from 5 to 7 to up to at least 33. In several cases, in addition to HlyA, direct evidence for the presence of a C-terminal secretion signal has been obtained (3,8,17,21,26,27). Moreover, albeit with various degrees of efficiency, translocators can be exchanged and secretion of heterologous toxins or proteases can be demonstrated, with the E. coli HlyBD translocator being particularly promiscuous in this respect (see, for example, references 3, 4, 11, 20, and 26).…”

“…Subsequent deletion studies indicated that a maximum of 53 to 60 residues at the C termini of closely related hemolysins may encompass the signal sequence (12,16). Moreover, Jarchau et al (13) and Zhang et al (27) have shown that the C-terminal 60 to 62 residues of HlyA alone can be recognized by the translocator and that such peptides are efficiently secreted to the medium. However, other regions of HlyA implicated in targeting the molecule to the medium have not been excluded.…”

Preface: the concept and consequences of multidrug resistance

“…Biochemical experiments showed that the signal sequences, known to interact with the transporter [60], formed amphiphilic helices only in the presence of lipids. This suggested that HlyB might recognise its substrate within the inner leaflet of the membrane [76]. We were eventually able to replace the HlyA signal entirely with random synthetic sequences and showed that only a resemblance to the native signal in charge and amphiphilicity, not in primary sequence, was required [24].…”

Secretion Signal and Protein Targeting in Bacteria: a Biological Puzzle

“…An obvious signal for targeting was also believed to exist in the T1SS, whose substrates carry a noncleavable C-terminal signal able to bind the ABC component of the T1SS. This signal does not have a conserved amino acid sequence but has a helical secondary structure spanning about 30 to 60 residues that, in principle, cannot be removed without loss of targeting/secretion (27). However, studies in the laboratory of Cécile Wandersman have previously shown that the T1SS-dependent HasA substrate from Serratia marcescens can be efficiently targeted to the secretion machinery despite lacking 14 C-terminal amino acids (6).…”