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 optimal alignment for Escherichia coli and Mycobacterium tuberculosis MscL and to place these homologues into sequence subfamilies. By using this alignment, previously identified E. coli MscL mutants that displayed severe and very 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 extracellular loop region of the M. tuberculosis MscL crystal structure has been analyzed. 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 strong evidence for the importance of the loop region in MscL channel function. No analogue to this interesting interaction could be found in E. coli MscL by sequence alignment. Taken together, these results indicate that caution should be exercised in using the M. tuberculosis MscL crystal structure to analyze previous functional studies of E. coli MscL.
Maurer
J A
JA
Divisions of Chemistry and Chemical Engineering and Biology, California Institute of Technology, Pasadena, California 91125, USA.
Elmore
D E
DE
Lester
H A
HA
Dougherty
D A
DA
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D007473:Ion Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000378:metabolism*; D011487:Protein Conformation; D016415:Sequence Alignment; D017421:Sequence Analysis; D013314:Stress, Mechanical
D001426:Bacterial Proteins; D007473:Ion Channels