Sequence analysis of the gliding protein Gli349 in Mycoplasma mobile

Abstract

The motile mechanism of Mycoplasma mobile remains unknown but is believed to differ from any previously identified mechanism in bacteria. Gli349 of M. mobile is known to be responsible for both adhesion to glass surfaces and mobility. We therefore carried out sequence analyses of Gli349 and its homolog MYPU2110 from M. pulmonis to decipher their structures. We found that the motif "YxxxxxGF" appears 11 times in Gli349 and 16 times in MYPU2110. Further analysis of the sequences revealed that Gli349 contains 18 repeats of about 100 amino acid residues each, and MYPU2110 contains 22. No sequence homologous to any of the repeats was found in the NCBI RefSeq non-redundant sequence database, and no compatible fold structure was found among known protein structures, suggesting that the repeat found in Gli349 and MYPU2110 is novel and takes a new fold structure. Proteolysis of Gli349 using chymotrypsin revealed that cleavage positions were often located between the repeats, implying that regions connecting repeats are unstructured, flexible and exposed to the solvent. Assuming that each repeat folds into a structural domain, we constructed a model of Gli349 that fits well the shape and size of images obtained with electron microscopy.

Keywords: Gli349; Mycoplasma mobile; YGF motif; motility; repeat sequence; sequence analysis.

Figure 1

Multiple sequence alignment of 10 subsequences of Gli349 containing a YGF motif is shown. The start and end positions of each repeat are denoted in the first column. Colors on the sequences denote as follows: yellow, 100% conserved residues; cyan, >50% conserved residues; orange, sites that are >70% conserved for D, N, S and T; green, sites that are >70% conserved for A, I, L and V.

Figure 2

Procedure for detecting repeats using the hidden Markov model.

Figure 3

Alignment scores of subsequences of 120 residues against the profile of repeat Set #3. Plotted are the scores at the center position of the subsequences of Gli349 (a) and MYPU2110 (b). Scores were calculated using HMMER. The unit on the vertical axis is the negative logarithm of the E-value of the alignment. The bars above the line denote repeats detected by HMMER. Most of the repeats were found to be in tandem form. For Gli349, experimentally determined chymotrypsin susceptible sites are shown by asterisks (a).

Figure 4

The degree of residue conservation at each position in the repeat is shown as information content. The information content of amino acid residue “a” at position “i” is calculated by the equation, I(a, i)=−p(a, i) log₂ p(a, i), where p(a, i) is the fractional content comprised by an amino acid residue. $\sum _{a}p(a,i)=1$ at each position. As $\left({\text{log}}_{2}20-\sum _{a=1}^{20}I(a,i)\right)$ becomes larger, the position “i” is regarded as more conserved. The three black bars indicate well conserved regions.

Figure 5

MSA of repeat Set #3. Residues are colored according to the predicted secondary structures: red, α-helix; blue, β-strand; yellow, coil; and white, ambiguous. The consensus secondary structure is βββα shown at the bottom. The subsequences are listed in order of their E-values. The YGF motif is denoted by a black bar on the top line. GLI and MYPU denote Gli349 and MYPU2110, respectively, and the following number denotes the repeat start position in each subsequence. Residue numbering is the same as in Fig. 4.

Figure 6

Model of Gli349. Low resolution image of Gli349 obtained with electron microscopy (EM) is shown in gray shade. Repeat regions shown in ovals connected by lines are assigned into the EM image of Gli349. The N-terminus is placed at the far right side in (a), and at the far left side in (b). The length of each rod and angles between two rods are the average values over EM images (unpublished data).