However, not all observed pairwise residue correlations in adjacent repeats are entirely well-explained within the context of the presented structural CP-868596 mw model. In addition we have no plausible explanation for why only FliH proteins, and no other sequences, contain these unique GxxxG repeats. There is also no obvious reason or explanation for the highly variable number of repeats in different FliH sequences. However, sequence deletions in Salmonella FliH that affect

in vitro ATPase hydrolysis assays for a FliI:FliH complex (either by enhancing or reducing FliI’s ATPase activity) overlap with one or more of the Salmonella FliH GxxxG repeats (see introduction) [17]. This suggests that secondary interactions

between FliI and FliH, in addition to the well-known interaction between the C-domain of FliH and the N-terminal 15 residues of FliI, may depend critically on the presence of the GxxxG motif [15, 18]. Studies on the ATPase activities and/or export capability of FliI:FliH pairs from other motile bacteria with engineered deletions in the FliH GxxxG repeats would likely shed light on the importance of the GxxxG repeats in flagellar protein export. While the extremely long length of the repeats in some FliH proteins implies that the repeats may cooperate to perform an important functional or structural role, the fact that other FliH sequences have short repeats segments, or even no repeat segment at all, would suggest otherwise. NSC 683864 molecular weight Alternately, another unidentified protein involved in the flagellum export pathway may be able to compensate for deletion of the GxxxG motifs in Suplatast tosilate FliH. Given the likely structural constraints on FliH participating in the flagellar export pathway via interactions with FliI, FliN and other proteins at the base of the flagellar export pore, it will be interesting to see if more

than one protein participates in interactions with the FliH GxxxG motifs. It is also interesting that extremely long glycine repeats evolved in FliH, but not in its Type III secretion homologue YscL, and this may actually tell us something, albeit cryptically, about differences in the two export systems. The extremely biased amino acid composition of the glycine repeats suggests that these regions may adopt nonstandard helix-helix tertiary or quaternary interactions that will be of interest for structural biologists to elucidate. Lastly, and perhaps most interestingly, the extreme rarity of this motif in other proteins is very surprising given that nature tends to find similar structural solutions to a biological problem multiple times. Crystal structures and careful biochemical/biological analysis of these proteins should ultimately be able to address these fascinating issues. Methods Acquiring the set of FliH proteins We endeavored to acquire FliH proteins from as many different bacterial species as possible.