Structure of the MscL homolog from Mycobacterium tuberculosis: A gated mechanosensitive ion channel

Science

Volumn 282, Issue 5397, 1998, Pages 2220-2226

  Chang, Geoffrey ^a   Spencer, Robert H ^a   Lee, Allen T ^a   Barclay, Margaret T ^a   Rees, Douglas C ^a  

^a HOWARD HUGHES MEDICAL INSTITUTE   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ION CHANNEL;

AMINO ACID SEQUENCE; ARTICLE; CELLULAR DISTRIBUTION; ELECTROCHEMICAL ANALYSIS; MYCOBACTERIUM TUBERCULOSIS; NONHUMAN; NUCLEOTIDE SEQUENCE; OSMOTIC PRESSURE; PRIORITY JOURNAL; PROTEIN STRUCTURE; X RAY CRYSTALLOGRAPHY;

AMINO ACID SEQUENCE; BACTERIAL PROTEINS; BINDING SITES; CELL MEMBRANE; CLONING, MOLECULAR; CRYSTALLIZATION; CRYSTALLOGRAPHY, X-RAY; ESCHERICHIA COLI PROTEINS; ION CHANNEL GATING; ION CHANNELS; LIGANDS; MODELS, MOLECULAR; MOLECULAR SEQUENCE DATA; MOLECULAR WEIGHT; MYCOBACTERIUM TUBERCULOSIS; PROTEIN CONFORMATION; PROTEIN FOLDING; PROTEIN STRUCTURE, SECONDARY; TEMPERATURE;

BACTERIA (MICROORGANISMS); MYCOBACTERIUM; MYCOBACTERIUM TUBERCULOSIS; PROKARYOTA;

EID: 0032545321     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.282.5397.2220     Document Type: Article

References (46)

1. H. Sackin, Annu. Rev. Physiol. 57, 333 (1995).
2. S. I. Sukharev, P. Blount, B. Martinac, F. R. Blattner, C. Kung, Nature 368, 265 (1994).
3. P. C. Moe, P. Blount, C. Kung, Mol. Microbiol. 28, 583 (1998).
4. S. I. Sukharev, P. Blount, B. Martinac, C. Kung, Annu. Rev. Physiol. 59, 633 (1997).
5. P. Blount et al., EMBO J. 15, 4798 (1996).
6. C. C. Hase, R. F. Minchin, A. Kloda, B. Martinac, Biochem. Biophys. Res. Commun. 232, 777 (1997).
7. M. Schleyer, R. Schmid, E. P. Bakker, Arch. Microbiol. 160, 424 (1993).
8. N. Saint et al., J. Biol. Chem. 273, 14667 (1998).
9. MscL homologs were identified during BLAST searches of unfinished sequences from various genome sequencing projects including the following species: Actinobacillus actinomycetemcomitans, Bordetella pertussis, Chlorobium tepidum, Deinococcus radiodurans, Enterococcus faecalis, Mycobacterium leprae, Porphyromonas gingivalis, Pseudomonas aeruginosa, Streptococcus pyogenes, Vibrio cholerae, and Yersinia pestis (R. H. Spencer and G. Chang, unpublished material).
10. D. A. Doyle et al., Science 280, 69 (1998).
11. We identified the Tb-MscL homolog based upon a National Center for Biotechnology Information BLAST search of the M. tuberculosis genome project and isolated the coding region from genomic DNA (kind gift from S. Gordon, Institut Pasteur, Paris, France) using polymerase chain reaction-based techniques. The respective mscL genes from E. coli, Synechocystis sp., Haemophilus influenzae, and Bacillus subtilis were also cloned from genomic DNA. Plasmid clones containing the mscL genes from E. carotovora, P. fluorescens, and Staphylococcus aureus were obtained as a gift from C. Kung and P. C. Moe, University of Wisconsin-Madison, Madison, WI, and mscL from Clostridium perfringens was obtained as a gift from A. Okabe, Kagawa Medical School, Kagawa, Japan.
12. 2-terminus of MscL including a decahistidine repeat and a consensus enterokinase cleavage site. Additionally, protein was expressed in E. coli using a mscL knockout mutant (lysogenized with γDE3) and induced via the addition of 0.1% 1-β-D-thiogalactoside (Anatrace, Maumee, OH) and 1% lactose. MscL was solubilized from 1- to 2-kg batches of cells using 1.0% dodecyl-β-D-maltoside (DDM; Anatrace, Maumee, OH) and purified with 0.1% DDM using nickel-chelate (Qiagen, Chatsworth, CA), ion exchange, and gel filtration chromatography.
13. 2-terminal end.
14. 4 sites, and the correct hand for this space group was established from anomalous difference Fourier maps. The initial experimentally phased electron density maps revealed a pentameric structure with a real-space correlation coefficient of 45% between subunits. Fivefold noncrystallographic symmetry averaging, solvent flattening, and incremental phase extension from 4.5 to 3.5 Å resolution were accomplished using locally written software (G. Chang, unpublished) to yield maps of excellent quality for model building. The final inversion R factor of 28% reflects the relatively large solvent content, the intensity distribution of the data, and the presence of partially ordered detergent in the crystal.
15. W. Furey and S. Swaminathan, Methods Enzymol. 277, 590 (1997).
16. J. S. Sacks, J. Mol. Graphics 6, 224 (1988).
17. J. P. Abrahams and A. C. W. Leslie, Acta Crystallogr. D 52, 30 (1996).
18. M. Pellegrini, N. Gronbech-Jensen, J. A. Kelly, G. M. Pfluegl, T. O. Yeates, Proteins 29, 426 (1997).
19. P. Gros, W. F. van Gunsteren, W. G. J. Hol, Science 249, 1149 (1990).
20. J. Kuriyan et al., Proteins 10, 340 (1991).
21. A. T. Brünger, J. Kuriyan, M. Karplus, Science 235, 458 (1987).
22. free of 35% against the native data. Residues 10 to 118 of each subunit are included in the final model.
23. J. Deisenhofer, O. Epp, K. Miki, R. Huber, H. Michel, Nature 318, 618 (1985).
24. M. S. Weiss et al., Science 254, 1627 (1991).
25. E. G. Hutchinson and J. M. Thornton, Protein Sci. 5, 212 (1996).
26. S. Numa, Harvey Lect. 83, 121 (1989).
27. J. P. Changuex, J. L Galzi, A. Devillers-Thiery, D. Bertrand, Q. Rev. Biophys. 25, 395 (1992).
28. S. M. Soltis, M. H. B. Stowell, M. C. Weiner, G. N. Phillips, D. C. Rees, J. Appl. Crystallogr. 30, 190 (1997).
29. G. Chang, R. Spencer, A. Lee, M. Barclay, D. Rees, data not shown.
30. A. Kreusch, P. J. Pfaffinger, C. F. Stevens, S. Choe, Nature 392, 945 (1998).
31. X. R. Ou, P. Blount, R. J. Hoffman, C. Kung, Proc. Natl. Acad. Sci. U.S.A. 95, 11471 (1998).
32. P. Blount, S. I. Sukharev, M. J. Schroeder, S. K. Nagle, C. Kung, ibid. 93, 11652 (1996).
33. C. C. Hase, A. C. LeDain, B. Martinac, J. Membr. Biol. 157, 17 (1997).
34. _, J. Biol. Chem. 270, 18329 (1995).
35. W. J. Sigurdson, S. I. Sukharev, C. Kung, F. Sachs, Biophys. J. 72, WP104 (1997).
36. H. A. Lester, Annu. Rev. Biophys. Biomol. Struct. 21, 267 (1992).
37. N. Unwin, Nature 373, 37 (1995).
38. _, J. Mol. Biol. 257, 586 (1996).
39. S. Bailey, CCP4 Project, Acts Crystallogr. D 50, 760 (1994).
40. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G. Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R. Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
41. J. D. Thompson, T. J. Gibson, F. Plewniak, F. Jeanmougin, D. G. Higgins, Nucleic Acids Res. 25, 4876 (1997).
42. R. M. Esnouf, J. Mol. Graphics 15, 132 (1997).
43. P. J. Kraulis, J. Appl. Crystallogr. 24, 946 (1991).
44. E. A. Merritt and D. J. Bacon, Methods Enzymol. 277, 505 (1997).
45. B. Honig and A. Nicholls, Science 268, 1144 (1995).
46. We thank A. Chirino for advice and computer support, J. G. Spencer for technical assistance, S. Gordon of the Institut Pasteur (Paris, France) for his kind gift of genomic DNA from M. tuberculosis, C. Kung and P. C. Moe from the University of Wisconsin-Madison for several cloned mscL homologs and for the E. coli knockout mutant for mscL, A. Okabe of the Kagawa Medical School (Kagawa, Japan) for providing cloned DNA of mscL from C. perfringens, and D. Dougherty and H. Lester for helpful discussions. We also thank the staff at the Stanford Synchrotron Radiation Laboratory (SSRL) and the Advanced Light Source (ALS) for their help in data collection. The synchrotron rotation camera facilities are supported by the U.S. Department of Energy (ALS and SSRL) and NIH (SSRL). G.C. and R.H.S. were supported by NIH postdoctoral fellowship grant GM18486 and an Amgen postdoctoral fellowship, respectively, during the initial stages of this project. Supported by the Howard Hughes Medical Institute. Protein Data Bank identifier for Tb-MscL is 1MSL.