4 0 ,6-Diamidino-2-phenylindole (DAPI) induces bundling of Escherichia coli FtsZ polymers inhibiting the GTPase activity Esteban Nova a,1 , Felipe Montecinos a,1 , Juan E. Brunet b , Rosalba Lagos a , Octavio Monasterio a, * a Departamento de Biologı ´a, Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago, Chile b Instituto de Quı ´mica, Facultad de Ciencias Ba ´ sicas y Matema ´ ticas, Universidad Cato ´ lica de Valparaı ´so, Casilla 4059, Valparaı ´so, Chile Received 31 January 2007, and in revised form 23 June 2007 Available online 10 July 2007 Abstract FtsZ (F ilamentous t emperature s ensitivity Z ) cell division protein from Escherichia coli binds the fluorescence probe DAPI. Bundling of FtsZ was facilitated in the presence of DAPI, and the polymers in solution remained polymerized longer time than the protofilaments formed in the absence of DAPI. DAPI decreased both the maximal velocity of the GTPase activity and the Michaelis–Menten constant for GTP, indicating that behaves like an uncompetitive inhibitor of the GTPase activity favoring the GTP form of FtsZ in the polymers. The results presented in this work support a cooperative polymerization mechanism in which the binding of DAPI favors protofilament lateral interactions and the stability of the resulting polymers. Ó 2007 Elsevier Inc. All rights reserved. Keywords: EcFtsZ; Bundling; Polymerization; DAPI; GTPase Escherichia coli FtsZ (EcFtsZ), 2 an essential protein for the division of the bacterial cell, is a monomeric protein of 40.3 kDa that self-assembles in vitro in the presence of GTP and magnesium to form protofilaments (for reviews see [1– 3]). It is thought that these polymers interact to form a ring associated with the cytoplasmic membrane at the division site responsible for bacterial septation [4]. It has been established that the in vitro polymerization is cooperative, with a critical concentration and a lag time in the polymer- ization kinetics [5,6]. A nucleation mechanism with a criti- cal concentration has been described for Methanococcus jannaschii FtsZ (MjFtsZ) [7], and a variation of this process involving an isodesmic assembly coupled with preferential cyclization of long polymers under crowding conditions has also been proposed [8]. FtsZ polymerization depends on the presence of magne- sium and GTP [9], therefore it belongs to the guanine nucleotide binding protein family. It has intrinsic GTPase activity that is stimulated by polymerization [10,11]. FtsZ, in the presence of GDP at high protein concentrations, forms various polymer structures such as arcs and rings [12]. During polymerization, the GTPase activity is stimu- lated and the polymer disassembles as the GTP is spent [13]. This behavior is different from that of microtubules where tubulin-GDP remains bound to other molecules of tubulin in the body of the microtubule protected by caps of GTP-tubulin at both ends of this assembly [14]. Divalent cations [13], DEAE-dextran [15] and ruthenium red [16] induce bundling of FtsZ protofilaments inhibiting the GTPase activity. The remarkable functional and structural similarity between tubulin and FtsZ suggest that FtsZ could be the ancestral homologue of tubulin [17] and as such, the 0003-9861/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2007.06.032 * Corresponding author. Fax: +56 2 276 3870. E-mail address: monaster@uchile.cl (O. Monasterio). 1 These authors contributed equally to this work. 2 Abbreviations used: DAPI, 4 0 ,6-diamidino-2-phenylindole; DTT, dithi- othreitol; EDTA, ethylenediamidine-tetraacetic acid; GTP, guanosine 5 0 - triphosphate; EcFtsZ, Escherichia coli FtsZ; MjFtsZ, Methanococcus jannaschhii FtsZ; MES, 2-[N-Morpholino]ethanesulfonic acid; PIPES 1,4- piperazinebis ethanesulfonic acid; IPTG, isopropyl- b - D - thiogalactopyranoside. www.elsevier.com/locate/yabbi ABB Archives of Biochemistry and Biophysics 465 (2007) 315–319