Characterization of the type III export signal of the flagellar hook scaffolding protein FlgD of Escherichia coli

Abstract

Transport of flagellar structural proteins beyond the cytoplasmic membrane is accomplished by a type III secretory pathway [flagellar type III secretion system (fTTSS)]. The mechanism of substrate recognition by the fTTSS is still enigmatic. Using the hook scaffolding protein FlgD of Escherichia coli as a model substrate, it is demonstrated that the export signal is contained within the N-terminal 71 amino acids of FlgD. Analysis of frame-shift mutations and alterations of the nucleotide sequence suggest a proteinaceous nature of the signal. Furthermore, the physicochemical properties of the first about eight amino acids are crucial for export.

Application of a Short, Disordered N-Terminal Flagellin Segment, a Fully Functional Flagellar Type III Export Signal, to Expression of Secreted Proteins

“…Certain studies suggested that the signal sequence is found at the mRNA level (1,25) or that the combination of an Nterminal signal and a structural element in the mRNA is required (3). On the other hand, a growing number of data support that the signal is contained at the protein level, and it is located at or near the N terminus (6,12,17,19,22,36,39,40). The identified signal sequences of different flagellar proteins, however, do not show any significant sequence similarity, which indicates that determinants should be at higher than the primary structural level.…”

“…For example, residues 38 to 58 of Caulobacter crescentus flagellar hook protein were found to be essential for secretion (19). Similarly, it has been demonstrated that the export signal of the hook scaffolding protein FlgD of Escherichia coli is located exclusively within the N-terminal 71 amino acids (40). Single amino acid substitutions within the N-terminal region of the anti-sigma factor FlgM (6), which is also exported by the flagellum-specific export apparatus, severely impair its export.…”

“…The nature of the signal directing flagellar protein secretion is still debated because the protein substrates have no cleavable signal sequences or do not share any obvious consensus sequence (12). It has been suggested that the recognition of flagellar export substrates may involve mRNA signals (25), but a growing amount of evidence indicates that the signal is located in the disordered N-terminal region of the secreted proteins (6,11,12,19,33,36,40). For example, residues 38 to 58 of Caulobacter crescentus flagellar hook protein were found to be essential for secretion (19).…”

Crystal Structure of the C-Terminal Domain of a Flagellar Hook-Capping Protein from Xanthomonas campestris

“…XC1894 is a flagellar hook-capping protein from X. campestris Figure 2 shows the multiple sequence alignment of X. campestris XC1894 with the two well-studied FlgD sequences from E. coli 17,18 and Salmonella typhimurium. 7 As can be seen from the alignment, XC1894 exhibits high sequence identity (36.9% and 37.6%) and similarity (49.0% and 48.6%) with the E. coli and S. typhimurium FlgD sequences, respectively.…”

Structural Characterization at the Atomic Level of a Molecular Nano-Machine: The State of the Art of <i>Helicobacter Pylori</i> Flagellum Organization

“…A similar behavior occurs in H. pylori FlgD, whose crystals have been grown by our group. A study of FlgD from E. coli (Weber-Sparenberg et al, 2006) indicated that the first 71 N-terminal residues represent a signal for the export into the flagellar channel. The superimposition of FlgD structures is given within Fig.…”