Structural characterization of microcin E492 amyloid formation: Identification of the precursors Rocío Arranz a,1 , Gabriela Mercado b,1 , Jaime Martín-Benito a , Rafael Giraldo c , Octavio Monasterio b , Rosalba Lagos b,⇑ , José M. Valpuesta a,⇑ a Centro Nacional de Biotecnología (CNB-CSIC), Campus de Cantoblanco, Madrid, Spain b Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago, Chile c Centro de Investigaciones Biológicas (CIB-CSIC), C/ Ramiro de Maeztu, 9. 28040 Madrid, Spain article info Article history: Received 7 December 2011 Received in revised form 26 February 2012 Accepted 27 February 2012 Available online 7 March 2012 Keywords: Microcin Amyloid Electron microscopy Image processing abstract Microcin E492 is a low-molecular weight, channel-forming bacteriotoxin that generates amyloid struc- tures. Using electron microscopy and image processing techniques several structural conformations can be observed. Prior to the conditions that induce amyloid formation and at its initial stage, microcin E492 molecules can be found in two main types of oligomers: a pentameric, pore-like structure consisting of globular monomers of 25 Å diameter, and long filaments made up of stacked pentamers. The equi- librium between these structures depends on the properties of the solvent, because samples kept in methanol mainly show the pentameric structure. Amyloid induction in aqueous solvent reveals the pres- ence, together with the above mentioned structures, of several amyloid structures such as flat and helical filaments. In addition, X-ray diffraction analysis demonstrated that the fibrils formed by microcin E492 presented cross-b structure, a distinctive property of amyloid fibrils. Based on the study of the observed structures we propose that microcin E492 has two conformations: a native one that assembles mainly into a pentameric structure, which functions as a pore, and an amyloid conformation which results in the formation of different types of amyloid filaments. Ó 2012 Elsevier Inc. All rights reserved. 1. Introduction Microcin E492 (MccE492) is a low-molecular mass (7.9 kDa) bacteriocin produced by Klebsiella pneumoniae RYC492 (de Lorenzo, 1984), which functions by forming pores in the mem- brane of the target cells thus decreasing their membrane potential (de Lorenzo and Pugsley, 1985; Lagos et al., 1993). It has previously been shown that, contrary to what occurs with other microcin sys- tems, MccE492 activity is higher during the exponential phase of growth, and then practically ceases during the stationary phase (de Lorenzo, 1984). Surprisingly, the amount of MccE492 secreted is similar in both phases (Corsini et al., 2002), which has led to the discovery that in the stationary phase most of MccE492 converts from a soluble to an insoluble conformation consisting of amy- loid-like fibrils, a process related with the loss of antibacterial activity (Bieler et al., 2005). Amyloid formation has usually been associated with protein misfolding and aggregation in eukaryotes, leading in many cases to diseases such as Parkinson’s (PD), Alzheimer’s (AD) and others (Dobson, 1999). Amyloid structures are not exclusive for eukary- otes since they have also been found in bacteria (Bieler et al., 2005; Chapman et al., 2002). Formation of MccE492 amyloids seems to modulate in vivo its antibacterial activity (Bieler et al., 2005), which suggests that amyloid formation is a conserved mechanism in all organisms that may play a role not only in dis- ease but also in other biological processes. Amyloid formation has been characterized mostly in eukaryotic systems. However, due to the complexity of these systems, the use of a model such as MccE492 for both in vivo and in vitro amyloid formation constitutes a very good alternative for the characteriza- tion of this process. To this end, we sought to study MccE492 amy- loid formation by electron microscopy and X-ray diffraction. Our results show that MccE492 has a globular, soluble conformation that forms pore-like, pentameric structures consistent with the role of MccE492 in channel formation. The transformation of the soluble conformation into non-soluble filamentous structures is accompanied by an increase in b-sheet structure, similar to that described in other amyloid formation processes (Bieler et al., 2005). These structures coexist with those formed by the soluble conformation. Our results pave the way for the structural charac- terization of conformational changes that give rise to amyloid 1047-8477/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.jsb.2012.02.015 ⇑ Corresponding authors. Fax: +56 2 2712983 (R. Lagos), +34 915854506 (J.M. Valpuesta). E-mail addresses: rolagos@uchile.cl (R. Lagos), jmv@cnb.csic.es (J.M. Valpuesta). 1 These authors contributed equally to the work. Journal of Structural Biology 178 (2012) 54–60 Contents lists available at SciVerse ScienceDirect Journal of Structural Biology journal homepage: www.elsevier.com/locate/yjsbi