1 Abbott Bioresearch Center, 100 Research Drive, Worcester, MA 01605, USA
2 Drug Discovery Division, Southern Research Institute, Birmingham, AL 35205, USA
Correspondence
David W. Borhani
(david.borhani{at}alum.mit.edu)
Mycobacterium tuberculosis FtsZ (MtbFtsZ) exhibits unusually slow polymerization kinetics in vitro (White et al., 2000). Recently, Anand et al. (2004)
hypothesized that removal of the non-conserved C terminus would bestow rapid polymerization to MtbFtsZ, comparable to the much faster-polymerizing Escherichia coli FtsZ (EcFtsZ) (Mukherjee & Lutkenhaus, 1999
). Indeed, MtbFtsZ(
C169), in which the last 169 residues were deleted (
211379; Fig. 1
), was found to polymerize much faster than wild-type MtbFtsZ (Anand et al., 2004
). It also formed polymers that were 1·5-times thicker and that exhibited an altered morphology, compared with wild-type MtbFtsZ protofilaments (White et al., 2000
). We show here, however, that the three-dimensional structure of MtbFtsZ(
C169) is so perturbed the truncation exposes a large hydrophobic patch that is normally buried (Fig. 2
) that polymerization of MtbFtsZ(
C169) is unlikely to be physiologically relevant. The cause(s) of the slow polymerization of wild-type MtbFtsZ must therefore be sought elsewhere.
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Removal of MtbFtsZ residues 211379 therefore removes both the conserved C-terminal domain and the non-conserved C-terminal tail (Fig. 3). Whereas a tail truncation mutant [i.e. MtbFtsZ(
C68)] may serve as an appropriate probe of the slow polymerization characteristics of MtbFtsZ, removal of the C-terminal domain as well changes the overall structure of FtsZ so as to render results obtained with MtbFtsZ(
C169) artefactual. Indeed, the polymerization characteristics of MtbFtsZ(
C169) are likely non-physiological for three reasons.
First, removal of the C-terminal domain exposes a large hydrophobic patch on the remaining N-terminal domain in MtbFtsZ(C169), and would therefore be expected, on structural and biophysical grounds, to create a non-physiological protein prone to aggregation. Notably, MtbFtsZ(
C169) had to be purified under denaturing conditions and then refolded (Anand et al., 2004
), unlike wild-type MtbFtsZ (White et al., 2000
). The N- and C-terminal domains are connected by an extensive buried, hydrophobic interface, as illustrated in Fig. 2
. Association of these two domains, as in wild-type MtbFtsZ, buries a total of 2382 Å2 of solvent-accessible surface area, a value typical for domaindomain interactions (Jones et al., 2000
). The interface is composed of 73 % non-polar atoms, and 50 % of the buried surface area comes from the side chains of 32 hydrophobic residues 10 Ile, 8 Leu, 6 Val, 3 Met, 3 Pro and 2 Phe out of 60 residues in the interface. (These same side chain types contribute just 17 % of the total surface area for the intact protein.) In short, these are not the characteristics of a dissociable (dispensable) domain interface.
Second, removal of the C-terminal domain would destabilize the conformation of the conserved loop T7. Loop T7 is critical to proper GTP hydrolysis and FtsZ polymerization (Lu et al., 2001; Scheffers et al., 2002
; Wang et al., 1997
). Of the only ten hydrogen bonds that link the N- and C-terminal domains, seven cluster tightly between the tip of loop T7 and the C-terminal domain. Furthermore, in MtbFtsZ(
C169), N-terminal domain residues 211217, which form helix H11 and the link to the C-terminal domain, are also deleted. It is unclear how the conformation of T7 can be maintained in the absence of its connections with both the N- and C-terminal domains.
Third, the tubulin protofilament EM structure (Nogales et al., 1998a) strongly suggests that the C-terminal domain makes substantial intersubunit longitudinal contacts in the FtsZ polymer (Fig. 1
). These contacts may not be critical to polymerization, but their loss would likely affect the kinetics of polymerization.
Anand et al. (2004) appear to have omitted several key control experiments. First, how do the polymerization kinetics and polymer morphology of MtbFtsZ(
C169) compare with the analogous EcFtsZ C-terminal deletion mutant? Second, GTP hydrolysis experiments would have differentiated between aggregation and true (proto)filament formation.
Rational mutagenesis of proteins is a powerful tool to help understand their function. With FtsZ, however, it appears that a gentler approach, e.g. MtbFtsZ(C68), is needed to ensure the physiological relevance of the results obtained.
Figures were generated with Ribbons (Carson, 1991), GRASP (Nicholls et al., 1991
), ESPript (Gouet et al., 1999
) and Pov4Grasp (Nicolas Calimet, University of Strasbourg, France; http://pov4grasp.free.fr). Surface areas were calculated with GRASP and the ProteinProtein Interaction Server (http://www.biochem.ucl.ac.uk/bsm/PP/server/; Jones et al., 2000
).
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