Journal of Bacteriology, August 2002, p . 4313-4315, Vol . 184, No . 15

Structural Evidence that the P/Q Domain of ZipA Is an Unstructured, Flexible Tether between the Membrane and the C-Terminal FtsZ-Binding Domain

Tomoo Ohashi,¹ Cynthia A . Hale,² Piet A . J . de Boer,² and Harold P . Erickson^1*

Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710-3709,¹ Department of Molecular Biology and Microbiology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4960²

Received 27 February 2002/ Accepted 6 May 2002

Purified ZipA derivatives (5) were subjected to sedimentation through a glycerol gradient and then examined by rotary shadowing electron microscopy (EM) as previously described (2) . Figure 1a shows a micrograph of ZipA(23-328), which contains the complete charged domain, the P/Q domain, and the C-terminal globular domain of native ZipA (5) . Figure 1b shows ZipA(186-328), which comprises only the C-terminal globular domain . These two constructs were described previously (4) . The C-terminal domain appears as small globular particles about 3.5 to 4 nm in diameter (the diameter was estimated after subtracting 2 nm for the thickness of the platinum shell), consistent with the size (2.5 by 3.5 by 5 nm) of the C-terminal domain determined by X-ray crystallography (10) and nuclear magnetic resonance analysis (11) . ZipA(23-328) showed globular particles that were indistinguishable from the C-terminal domain alone . Occasionally, a very small globular particle or a thin extension could be seen close to the main globular particle but, for the most part, there was no visible structure corresponding to the charged and P/Q domains in ZipA(23-328) . This suggested that the P/Q domain may be a largely unfolded polypeptide and invisible in the rotary shadowed specimen, consistent with the previous prediction from the sequence (3) . We suggest that the charged domain may be unstructured as well because, in our experience, a 60-aa globular domain should be visible in rotary shadowing . This is supported by the lack of predicted secondary structure in the charged domain (13) .

 
To investigate the structure in more detail, we constructed a ZipA variant [green fluorescent protein (GFP)-ZipA(39-328)] in which a GFP domain was fused to the N terminus of ZipA(39-328) . EM of GFP-ZipA(39-328) showed globular particles in closely spaced pairs (Fig . 1c) . The larger globular domain should correspond to the 238-aa GFP, and the smaller one should correspond to the 140-aa ZipA C-terminal domain . The charged-plus-P/Q domain is interpreted to form an invisible link connecting the two globular domains .

An unfolded polypeptide can be modeled as a worm-like chain characterized by a contour length L and a persistence length p . Rivetti et al . (15) provided an elegant analysis of DNA on mica, modeled as a worm-like chain, and concluded that the molecules rearranged by diffusion on the two-dimensional mica substrate . For our analysis, we assume that the flexible polypeptide can also assume a random two-dimensional distribution on mica (see reference 15 for a detailed discussion of the two-dimensional structure versus the three-dimensional structure of the worm-like chain) . The distribution of end-to-end lengths of the worm-like chains is given by the formula <R²> = 4pL (for the case in which L >> p), where R is the distance between the ends of the chain, <R²> is the average value of R², L is the contour length, and p is the persistence length .

The R value needed for the above formula is the distance between the ends of the flexible chain, but the best estimate we can make is to measure the center-to-center distance between the two globular domains . A histogram of center-to-center distances is shown in Fig . 2 . We considered subtracting 4 nm, the sum of the predicted radii of the N- and C-terminal globular domains, from the measured center-to-center distance . This correction would be appropriate if the exit points of the polypeptide from the two globular domains were facing each other (as illustrated in Fig . 3b, c, and e) . However, it is also possible that these exit points face away from each other and the polypeptide crosses over or around the domains (as in Fig . 3d), in which case we should add 4 nm . If we assume that the P/Q segment is quite flexible and the orientation of the globular domains is random, the simple center-to-center distance seems to be the best estimate of the end-to-end length of the charged-plus-P/Q segment . Note, however, that each measurement includes an up to ±5-nm error due to the unknown orientation of the globular domains .

 
A histogram of the center-to-center distances reveals a wide distribution, ranging from 7.8 to 19.5 nm, with an average of 12.4 nm (Fig . 2) . The average value of R² was determined from the data in this histogram, giving <R²> = 159 nm² . The presumed unstructured domain includes the 12-aa spacer followed by the 146-aa charged-plus-P/Q domain (from R39 to M185) . This gives a predicted contour length L = 60 nm ([0.38 nm/aa] x 158 aa) . The persistence length is now calculated to be p = <R²>/4L = 0.66 nm . This is about twice the length of a peptide bond and close to the values of 0.4 to 0.44 nm estimated from atomic force microscope measurements for unfolded polypeptides of titin and tenascin (12, 14) . It is also similar to the p = 0.95 nm calculated by the above equation for the presumably unstructured PEVK domain of titin (7) .

We also characterized the ZipA(23-328) and GFP-ZipA(39-328) proteins by sedimentation analysis . Determination of sedimentation coefficients by analytical ultracentrifugation (20,000 rpm in the ANL rotor, Beckman XLA analytical ultracentrifuge) yielded values of 2.2S for ZipA(23-328) and 3.2S for GFP-ZipA(39-328) . To estimate the shape of the proteins, we calculated S_max/S, where S_max is the sedimentation coefficient of an unhydrated sphere of protein of the same mass . S_max/S was calculated to be 1.73 for ZipA(23-328) and 1.75 for GFP-ZipA(39-328) . These values are indicative of moderately elongated proteins (16) and are consistent with the EM studies described above, suggesting that the P/Q domain of ZipA is largely unfolded .

Figure 3 presents molecular models of the GFP-ZipA structure showing the charged-plus-P/Q domains as unstructured polypeptide . Note that the GFP in our construct is at the position of the transmembrane segment . We propose, on the basis of our EM analyses, that the tether is flexible and can span a maximum distance of 60 nm . In the more relaxed conformations, FtsZ subunits that are bound to the C-terminal FtsZ binding domain of ZipA could be an average of 12 nm from the membrane . Since the FtsZ protofilaments are only 3 to 4 nm thick, the tether is long enough to loop around and bind the C terminus of FtsZ, even if it is facing away from the membrane (see reference 1 for a diagram) .

This study was supported by National Institutes of Health grants CA47056 (H.P.E.) and GM-57059 (P.D.B.) .

1. Erickson, H . P. 2001 . The FtsZ protofilament and attachment of ZipA-structural constraints on the FtsZ power stroke . Curr . Opin . Cell Biol . 13:55-60.
2. Fowler, W . E., and H . P . Erickson. 1979 . Trinodular structure of fibrinogen: confirmation by both shadowing and negative stain electron microscopy . J . Mol . Biol . 134:241-249.
3. Hale, C . A., and P . A . J . de Boer. 1997 . Direct binding of FtsZ to ZipA, an essential component of the septal ring structure that mediates cell division in E . coli . Cell 88:175-185.
4. Hale, C . A., and P . A . J . de Boer. 1999 . Recruitment of ZipA to the septal ring of Escherichia coli is dependent on FtsZ and independent of FtsA . J . Bacteriol . 181:167-176.
5. Hale, C . A., A . C . Rhee, and P . A . J . de Boer. 2000 . ZipA-induced bundling of FtsZ polymers mediated by an interaction between C-terminal domains . J . Bacteriol . 182:5153-5166.
6. Haney, S . A., E . Glasfeld, C . Hale, D . Keeney, Z . He, and P . A . de Boer. 2001 . Genetic analysis of the E . coli FtsZ-ZipA interaction in the yeast two-hybrid system: characterization of FtsZ residues essential for the interactions with ZipA and with FtsA . J . Biol . Chem . 276:11980-11987.
7. Li, H., A . F . Oberhauser, S . D . Redick, M . Carrion-Vazquez, H . P . Erickson, and J . M . Fernandez. 2001 . Multiple conformations of PEVK proteins detected by single-molecule techniques . Proc . Natl . Acad . Sci . USA 98:10682-10686.
8. Liu, Z., A . Mukherjee, and J . Lutkenhaus. 1999 . Recruitment of ZipA to the division site by interaction with FtsZ . Mol . Microbiol . 31:1853-1861.
9. Ma, X., and W . Margolin. 1999 . Genetic and functional analyses of the conserved C-terminal core domain of Escherichia coli ftsZ . J . Bacteriol . 181:7531-7544.
10. Mosyak, L., Y . Zhang, E . Glasfeld, S . Haney, M . Stahl, J . Seehra, and W . S . Somers. 2000 . The bacterial cell-division protein ZipA and its interaction with an FtsZ fragment revealed by X-ray crystallography . EMBO J . 19:3179-3191.
11. Moy, F . J., E . Glasfeld, L . Mosyak, and R . Powers. 2000 . Solution structure of ZipA, a crucial component of Escherichia coli cell division . Biochemistry 39:9146-9156.
12. Oberhauser, A . F., P . E . Marszalek, H . P . Erickson, and J . M . Fernandez. 1998 . The molecular elasticity of the extracellular matrix protein tenascin . Nature 393:181-185.
13. RayChaudhuri, D. 1999 . ZipA is a MAP-Tau homolog and is essential for structural integrity of the cytokinetic FtsZ ring during bacterial cell division . EMBO J . 18:2372-2383.
14. Rief, M., M . Gautel, F . Oesterhelt, J . M . Fernandez, and H . E . Gaub. 1997 . Reversible unfolding of individual titin immunoglobulin domains by AFM . Science 276:1109-1112.
15. Rivetti, C., M . Guthold, and C . Bustamante. 1996 . Scanning force microscopy of DNA deposited onto mica: equilibration versus kinetic trapping studied by statistical polymer chain analysis . J . Mol . Biol . 264:919-932.
16. Schürmann, G., J . Haspel, M . Grumet, and H . P . Erickson. 2001 . Cell adhesion molecule L1 in folded (horseshoe) and extended conformations . Mol . Biol . Cell 12:1765-1773.

Free Online Full-text Article

What Is Water Purification?, What Is Prokaryote?, What Is MIC?, What Is Anthrax?, What Is Pcr?, n, Bacterium, c, Microbe, s, Bacteriology, o, Bacteria, c, Microorganism, r, Sepsis, a, Thermophile, s, Salmonella, a, Microorganism, i, Bacillus subtilis, n, Cephalosporin, s, S. cerevisiae, a, Bioremediation, c, Salmonella typhimurium, o, Bacteria, o, Biological filters, e, Salmonella typhimurium, a, Antibiotics, o, Escherichia coli, n, Bacteroides, e, Thermophile, c, Ps. putida, o, Cryptococci, s, Neisseria, i, Growth media, o, Thermophile




 

© 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com
 

Last modified: May 25, 2005