Comparing and contrasting Escherichia coli and Mycobacterium tuberculosis mechanosensitive channels (MscL) - New gain of function mutations in the loop region

Sequence analysis of 35 putative MscL homologues was used to develop an opt imal alignment for Escherichia coli and Mycobacterium tuberculosis MscL and to place these homologues into sequence subfamilies, By using this alignme nt, previously identified E, coli MscL mutants that displayed severe and ve ry severe gain of function phenotypes were mapped onto the M. tuberculosis MscL sequence. Not all of the resulting M, tuberculosis mutants displayed a gain of function phenotype; for instance, normal phenotypes were noted for mutations at Ala(20), the analogue of the highly sensitive Gly(22) site in E, coli, A previously unnoticed intersubunit hydrogen bond in the extracel lular loop region of the M, tuberculosis MscL crystal structure has been an alyzed. Cross-linkable residues were substituted for the residues involved in the hydrogen bond, and cross-linking studies indicated that these sites are spatially close under physiological conditions. In general, mutation at these positions results in a gain of function phenotype, which provides st rong evidence for the importance of the loop region in MscL channel functio n. No analogue to this interesting interaction could be found in E. coli Ms cL by sequence alignment. Taken together, these results indicate that cauti on should be exercised in using the M. tuberculosis MscL crystal structure to analyze previous functional studies off. coli MscL.