Mutants of FtsZ Targeting the Protofilament Interface: Effects on Cell Division and GTPase Activity
10.1128/jb.187.8.2727-2736.2005
Crossref journal-article
American Society for Microbiology
Journal of Bacteriology (235)
Abstract

ABSTRACT The bacterial cell division protein FtsZ assembles into straight protofilaments, one subunit thick, in which subunits appear to be connected by identical bonds or interfaces. These bonds involve the top surface of one subunit making extensive contact with the bottom surface of the subunit above it. We have investigated this interface by site-directed mutagenesis. We found nine bottom and eight top mutants that were unable to function for cell division. We had expected that some of the mutants might poison cell division substoichiometrically, but this was not found for any mutant. Eight of the bottom mutants exhibited dominant negative effects (reduced colony size) and four completely blocked colony formation, but this required expression of the mutant protein at four to five times the wild-type FtsZ level. Remarkably, the top mutants were even weaker, most showing no effect at the highest expression level. This suggests a directional assembly or treadmilling, where subunit addition is primarily to the bottom end of the protofilament. Selected pairs of top and bottom mutants showed no GTPase activity up to 10 to 20 μM, in contrast to the high GTPase activity of wild-type FtsZ above 1 μM. Overall, these results suggest that in order for a subunit to bind a protofilament at the 1 μM K d for elongation, it must have functional interfaces at both the top and bottom. This is inconsistent with the present model of the protofilament, as a simple stack of subunits one on top of the other, and may require a new structural model.

Bibliography

Redick, S. D., Stricker, J., Briscoe, G., & Erickson, H. P. (2005). Mutants of FtsZ Targeting the Protofilament Interface: Effects on Cell Division and GTPase Activity. Journal of Bacteriology, 187(8), 2727–2736.

Authors 4
  1. Sambra D. Redick (first)
  2. Jesse Stricker (additional)
  3. Gina Briscoe (additional)
  4. Harold P. Erickson (additional)
References 39 Referenced 89
  1. Anders, K. R., and D. Botstein. 2001. Dominant-lethal alpha-tubulin mutants defective in microtubule depolymerization in yeast. Mol. Biol. Cell12:3973-3986. (10.1091/mbc.12.12.3973) / Mol. Biol. Cell (2001)
  2. 10.1128/jb.172.10.5602-5609.1990
  3. Chen, Y., K. Bjornson, S. D. Redick, and H. P. Erickson. 2005. A rapid fluorescence assay for FtsZ assembly indicates cooperative assembly with a dimer nucleus. Biophys. J.88:505-514. (10.1529/biophysj.104.044149) / Biophys. J. (2005)
  4. Clackson, T., and J. A. Wells. 1995. A hot spot of binding energy in a hormone-receptor interface. Science267:383-386. (10.1126/science.7529940) / Science (1995)
  5. Cordell, S. C., E. J. Robinson, and J. Lowe. 2003. Crystal structure of the SOS cell division inhibitor SulA and in complex with FtsZ. Proc. Natl. Acad. Sci. USA100:7889-7894. (10.1073/pnas.1330742100) / Proc. Natl. Acad. Sci. USA (2003)
  6. Cunningham, B. C., and J. A. Wells. 1993. Comparison of a structural and a functional epitope. J. Mol. Biol.234:554-563. (10.1006/jmbi.1993.1611) / J. Mol. Biol. (1993)
  7. 10.1128/jb.173.11.3500-3506.1991
  8. DeLano, W. L. 2002. Unraveling hot spots in binding interfaces: progress and challenges. Curr. Opin. Struct. Biol.12:14-20. (10.1016/S0959-440X(02)00283-X) / Curr. Opin. Struct. Biol. (2002)
  9. Erickson, H. P. 1998. Atomic structures of tubulin and FtsZ. Trends Cell Biol.8:133-137. (10.1016/S0962-8924(98)01237-9) / Trends Cell Biol. (1998)
  10. Erickson, H. P., D. W. Taylor, K. A. Taylor, and D. Bramhill. 1996. Bacterial cell division protein FtsZ assembles into protofilament sheets and minirings, structural homologs of tubulin polymers. Proc. Natl. Acad. Sci. USA93:519-523. (10.1073/pnas.93.1.519) / Proc. Natl. Acad. Sci. USA (1996)
  11. Geladopoulos, T. P., T. G. Sotiroudis, and A. E. Evangelopoulos. 1991. A malachite green colorimetric assay for protein phosphatase activity. Anal. Biochem.192:112-116. (10.1016/0003-2697(91)90194-X) / Anal. Biochem. (1991)
  12. Gonzalez, J. M., M. Jimenez, M. Velez, J. Mingorance, J. M. Andreu, M. Vicente, and G. Rivas. 2003. Essential cell division protein FtsZ assembles into one monomer-thick ribbons under conditions resembling the crowded intracellular environment. J. Biol. Chem.278:37664-37671. (10.1074/jbc.M305230200) / J. Biol. Chem. (2003)
  13. Huecas, S., and J. M. Andreu. 2003. Energetics of the cooperative assembly of cell division protein FtsZ and the nucleotide hydrolysis switch. J. Biol. Chem.278:46146-46154. (10.1074/jbc.M307128200) / J. Biol. Chem. (2003)
  14. Khlebnikov, A., K. A. Datsenko, T. Skaug, B. L. Wanner, and J. D. Keasling. 2001. Homogeneous expression of the P(BAD) promoter in Escherichia coli by constitutive expression of the low-affinity high-capacity AraE transporter. Microbiology147:3241-3247. (10.1099/00221287-147-12-3241) / Microbiology (2001)
  15. Löwe, J., and L. A. Amos. 1998. Crystal structure of the bacterial cell-division protein FtsZ. Nature391:203-206. (10.1038/34472) / Nature (1998)
  16. Löwe, J., and L. A. Amos. 1999. Tubulin-like protofilaments in Ca2+-induced FtsZ sheets. EMBO J.18:2364-2371. (10.1093/emboj/18.9.2364) / EMBO J. (1999)
  17. Lu, C., J. Stricker, and H. P. Erickson. 1998. FtsZ from Escherichia coli, Azotobacter vinelandii, and Thermotoga maritima—quantitation, GTP hydrolysis, and assembly. Cell Motility Cytoskel.40:71-86. (10.1002/(SICI)1097-0169(1998)40:1<71::AID-CM7>3.0.CO;2-I) / Cell Motility Cytoskel. (1998)
  18. Lu, C., J. Stricker, and H. P. Erickson. 2001. Site-specific mutations of FtsZ—effects on GTPase and in vitro assembly. BMC Microbiol.1:7. (10.1186/1471-2180-1-7) / BMC Microbiol. (2001)
  19. 10.1128/jb.179.21.6788-6797.1997
  20. Mukherjee, A., C. Cao, and J. Lutkenhaus. 1998. Inhibition of FtsZ polymerization by SulA, an inhibitor of septation in Escherichia coli. Proc. Natl. Acad. Sci. USA95:2885-2890. (10.1073/pnas.95.6.2885) / Proc. Natl. Acad. Sci. USA (1998)
  21. Mukherjee, A., and J. Lutkenhaus. 1998. Dynamic assembly of FtsZ regulated by GTP hydrolysis. EMBO J.17:462-469. (10.1093/emboj/17.2.462) / EMBO J. (1998)
  22. Nogales, E., K. H. Downing, L. A. Amos, and J. Lowe. 1998. Tubulin and FtsZ form a distinct family of GTPases. Nat. Struct. Biol.5:451-458. (10.1038/nsb0698-451) / Nat. Struct. Biol. (1998)
  23. Nogales, E., M. Whittaker, R. A. Milligan, and K. H. Downing. 1999. High-resolution model of the microtubule. Cell96:79-88. (10.1016/S0092-8674(00)80961-7) / Cell (1999)
  24. Nogales, E., S. G. Wolf, and K. H. Downing. 1998. Structure of the αβ tubulin dimer by electron crystallography. Nature391:199-203. (10.1038/34465) / Nature (1998)
  25. Oliva, M. A., S. C. Cordell, and J. Lowe. 2004. Structural insights into FtsZ protofilament formation. Nat. Struct. Mol. Biol.11:1243-1250. (10.1038/nsmb855) / Nat. Struct. Mol. Biol. (2004)
  26. Oliva, M. A., S. Huecas, J. M. Palacios, J. Martin-Benito, J. M. Valpuesta, and J. M. Andreu. 2003. Assembly of archaeal cell division protein FtsZ and a GTPase-inactive mutant into double-stranded filaments. J. Biol. Chem.278:33562-33570. (10.1074/jbc.M303798200) / J. Biol. Chem. (2003)
  27. Phelps, K. K., and R. A. Walker. 2000. NEM tubulin inhibits microtubule minus end assembly by a reversible capping mechanism. Biochemistry39:3877-3885. (10.1021/bi992200x) / Biochemistry (2000)
  28. Reijo, R. A., E. M. Cooper, G. J. Beagle, and T. C. Huffaker. 1994. Systematic mutational analysis of the yeast β-tubulin gene. Mol. Biol. Cell5:29-43. (10.1091/mbc.5.1.29) / Mol. Biol. Cell (1994)
  29. Richards, K. L., K. R. Anders, E. Nogales, K. Schwartz, K. H. Downing, and D. Botstein. 2000. Structure-function relationships in yeast tubulins. Mol. Biol. Cell.11:1887-1903. (10.1091/mbc.11.5.1887) / Mol. Biol. Cell. (2000)
  30. Romberg, L., M. Simon, and H. P. Erickson. 2001. Polymerization of FtsZ, a bacterial homolog of tubulin. Is assembly cooperative? J. Biol. Chem.276:11743-11753. (10.1074/jbc.M009033200) / J. Biol. Chem. (2001)
  31. 10.1128/JB.185.11.3344-3351.2003
  32. Salimnia, H., A. Radia, S. Bernatchez, T. J. Beveridge, and J. R. Dillon. 2000. Characterization of the ftsZ cell division gene of Neisseria gonorrhoeae: expression in Escherichia coli and N. gonorrhoeae. Arch. Microbiol.173:10-20. (10.1007/s002030050002) / Arch. Microbiol. (2000)
  33. Scheffers, D., J. G. de Wit, T. den Blaauwen, and A. J. Driessen. 2001. Substitution of a conserved aspartate allows cation-induced polymerization of FtsZ. FEBS Lett.494:34-37. (10.1016/S0014-5793(01)02310-9) / FEBS Lett. (2001)
  34. Scheffers, D. J., J. G. de Wit, T. den Blaauwen, and A. J. Driessen. 2002. GTP hydrolysis of cell division protein FtsZ: evidence that the active site is formed by the association of monomers. Biochemistry41:521-529. (10.1021/bi011370i) / Biochemistry (2002)
  35. Schreiber, G., and A. R. Fersht. 1995. Energetics of protein-protein interactions: analysis of the barnase-barstar interface by single mutations and double mutant cycles. J. Mol. Biol.248:478-486. / J. Mol. Biol. (1995)
  36. 10.1128/JB.185.16.4796-4805.2003
  37. Wang, X., and J. Lutkenhaus. 1993. The FtsZ protein of Bacillus subtilis is localized at the division site and has GTPase activity that is dependent upon FtsZ concentration. Mol. Microbiol.9:435-442. (10.1111/j.1365-2958.1993.tb01705.x) / Mol. Microbiol. (1993)
  38. Wegner, A. 1976. Head to tail polymerization of actin. J. Mol. Biol.108:139-150. (10.1016/S0022-2836(76)80100-3) / J. Mol. Biol. (1976)
  39. Yu, X. C., and W. Margolin. 1997. Ca2+-mediated GTP-dependent dynamic assembly of bacterial cell division protein FtsZ into asters and polymer networks in vitro. EMBO J.16:5455-5463. (10.1093/emboj/16.17.5455) / EMBO J. (1997)
Dates
Type When
Created 20 years, 4 months ago (April 1, 2005, 5:33 p.m.)
Deposited 4 years ago (July 29, 2021, 1:27 p.m.)
Indexed 4 weeks ago (July 30, 2025, 8:24 p.m.)
Issued 20 years, 4 months ago (April 15, 2005)
Published 20 years, 4 months ago (April 15, 2005)
Published Print 20 years, 4 months ago (April 15, 2005)
Funders 0

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@article{Redick_2005, title={Mutants of FtsZ Targeting the Protofilament Interface: Effects on Cell Division and GTPase Activity}, volume={187}, ISSN={1098-5530}, url={http://dx.doi.org/10.1128/jb.187.8.2727-2736.2005}, DOI={10.1128/jb.187.8.2727-2736.2005}, number={8}, journal={Journal of Bacteriology}, publisher={American Society for Microbiology}, author={Redick, Sambra D. and Stricker, Jesse and Briscoe, Gina and Erickson, Harold P.}, year={2005}, month=apr, pages={2727–2736} }