Identification of amino acids in the leader peptide of Methanococcus voltae preflagellin that are important in posttranslational processing

Abstract

Archaeal flagellins are made initially as preproteins with short, positively charged leader peptides. Analysis of all available archaeal preflagellin sequences indicates that the -1 position is always held by a glycine while the -2 and -3 positions are almost always held by charged amino acids. To evaluate the importance of these and other amino acids in the leader peptides of archaeal flagellins for processing by a peptidase, Methanococcus voltae mutant FlaB2 preflagellin genes were generated by PCR and the proteins tested in a methanogen preflagellin peptidase assay that detects the removal of the leader peptide from preflagellin. When the -1 position was changed from glycine to other amino acids tested, no cleavage was observed by the peptidase, with the exception of a change to alanine at which poor, partial processing was observed. Amino acid substitutions at the -2 lysine position resulted in a complete loss of processing by the peptidase, while changes at the -3 lysine resulted in partial processing. A mutant preflagellin with a leader peptide shortened from 12 amino acids to 6 amino acids was not processed. When the invariant glycine residue present at position +3 was changed to a valine, no processing of this mutant preflagellin was observed. The identification of critical amino acids in FlaB2 required for proper processing suggests that a specific preflagellin peptidase may cleave archaeal flagellins by recognition of a conserved sequence of amino acids.

Characterization of Flagellum Gene Families of Methanogenic Archaea and Localization of Novel Flagellum Accessory Proteins

“…Although the overall leader sequence is not highly conserved, a net positive charge is present, and the three amino acids preceding the cleavage site tend to be KKG, KRG, or RRG. It has been demonstrated that a positively charged amino acid at position Ϫ2 and a small amino acid (glycine or alanine) at position Ϫ1 are important for preflagellin processing in M. voltae (46). Furthermore, that study identified other amino acids involved in processing that are also found in the flagellins identified in this study.…”

Archaeal Homolog of Bacterial Type IV Prepilin Signal Peptidases with Broad Substrate Specificity

“…Jarrell and colleagues (19) suggested that the archaeal type IV prepilin-like signal peptidase homologs are in fact preflagellin peptidases, i.e., enzymes with a dedicated role in the processing of flagellin precursors. According to these authors' hypothesis, other pro- (41). However, S. solfataricus PibD seems to be equipped with a much broader specificity.…”

“…However, S. solfataricus PibD seems to be equipped with a much broader specificity. It processes preGlcS*, even though the signal sequence lacks a lysine or arginine at position Ϫ3, whereas an M. voltae flagellin mutant with alanine or glutamate at the same position was cleaved only partially (41). Moreover, a lysine-to-alanine mutation of the GlcS* signal sequence at position Ϫ2 did not affect the cleavage reaction, and glycine at position Ϫ1 could be changed to alanine (Fig.…”

Type IV Pilin Proteins: Versatile Molecular Modules

“…Archaeal flagellins are more similar to T4 pilins than to eubacterial flagellins, including a requirement for processing of their type III secretion signal by dedicated peptidases prior to assembly (388). However, archaeal flagellar filaments are functionally similar to bacterial flagella, providing a propulsive force through rotation, rather than retraction, of the fibers.…”