Statistical characterization of the GxxxG glycine repeats in the flagellar biosynthesis protein FliH and its Type III secretion homologue YscL.
BACKGROUND
FliH is a protein involved in the export of components of the bacterial flagellum and we herein describe the presence of glycine-rich repeats in FliH of the form AxxxG(xxxG)mxxxA, where the value of m varies considerably in FliH proteins from different bacteria. While GxxxG and AxxxA patterns have previously been described, the long glycine repeat segments in FliH proteins have yet to be characterized. The Type III secretion system homologue to FliH (YscL, AscL, PscL, etc.) also contains a similar GxxxG repeat, and hence the presence of the repeat is evolutionarily conserved in these proteins, suggesting an important structural role or biological function.


RESULTS
A set of FliH and YscL protein sequences was downloaded from GenBank, and then filtered to reduce redundancy, to ensure the soundness of the sequences, and to eliminate, as much as possible, confounding phylogenetic signal between individual sequences by implementing a pairwise 25% sequence identity cut-off. The general features of the glycine-rich repeats in these proteins were examined, and it was found that the length of these repeat segments varied substantially among FliH proteins but was fairly consistent for the Type III (YscL) homologue sequences, with values of m ranging from 0 to 12 for FliH and 0 to 2 for YscL. The amino acid sequence distribution of each of the three positions in the GxxxG repeats was found to differ significantly from the overall amino acid composition of the FliH/YscL proteins. The high frequency of Glu, Gln, Lys and Ala residues in the repeat positions, which is not likely indicative of any contaminating phylogenetic signal, suggests an alpha-helical structure for this motif. In addition, we sought to determine whether certain pairs of amino acids, in certain pairs of positions, were found together significantly more often than would be predicted by chance. Several statistically significant correlations were uncovered, which may be important for maintaining helical stability or for forming helix-helix interactions. These correlations are likely not of a phylogenetic origin as the originating sequences for the pair correlations are derived from a low similarity set and the individual incidences of the pair correlations do not cluster in any obvious phylogenetic sense, nor is there much evidence of strict sequence conservation outside the positions of the glycine residues. Finally, the alpha-helices from a non-redundant set of proteins from the Protein Data Bank were searched for GxxxG repeats similar in length to those found in FliH, however there were no helices containing more than three contiguous glycine repeat segments; thus, long glycine repeats similar to those found in FliH are presumably quite rare in nature.


CONCLUSIONS
The glycine repeats in YscL and particularly FliH represent an intriguing amino acid sequence motif that is very rare in nature. Although we do not attempt to offer a mechanism whereby these repeats may have evolved, we do place the existence of the motif and some residue pairings within a rational structural context. While crystal structures of these proteins are necessary to fully elucidate the structural and functional significance of these repeats, the characterization reported here represents a first step in understanding this unique sequence feature.
Trost
Brett
B
Department of Computer Science, University of Saskatchewan, 110 Science Place, Saskatoon, S7N 5C9, Canada. brett.trost@usask.ca
Moore
Stanley A
SA
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000597:Amino Acids, Branched-Chain / Q000032:analysis* / Q000737:chemistry; D001419:Bacteria / Q000737:chemistry / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry*; D019295:Computational Biology; D003627:Data Interpretation, Statistical; D005407:Flagella / Q000378:metabolism*; D005998:Glycine / Q000737:chemistry*; D008969:Molecular Sequence Data; D016415:Sequence Alignment
D000597:Amino Acids, Branched-Chain; D001426:Bacterial Proteins; C069250:fliH protein, Bacteria; D005998:Glycine