Investigating interactions of the Bacillus subtilis spore coat proteins CotY and CotZ using single molecule force spectroscopy Huiqing Liu a,b , Daniela Krajcikova c , Zhe Zhang a , Hongda Wang a , Imrich Barak c , Jilin Tang a,⇑ a State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, PR China b University of Chinese Academy of Sciences, Beijing 100049, PR China c Institute of Molecular Biology, Slovak Academy of Sciences, Dubravska cesta 21, 84551 Bratislava, Slovakia article info Article history: Received 8 May 2015 Received in revised form 30 July 2015 Accepted 1 September 2015 Available online xxxx Keywords: Atomic force microscopy Single molecule force spectroscopy Protein–protein interaction CotY CotZ abstract Spores formed by Bacillus subtilis are surrounded by a protective and multilayered shell, termed the coat, which grants the spores resistance to various environmental stresses and facilitates spore germination. The spore coat consists of more than seventy different proteins, arranged into at least four distinct struc- tural layers: the undercoat, inner coat, outer coat and crust. However, how these proteins, especially the morphogenetic proteins, interact to establish the organized, functional coat layers remains poorly under- stood. CotY and CotZ as the components of the crust, play a morphogenetic role in the crust assembly around the spore. In this study, the single molecule force spectroscopy was used to investigate the inter- action and dynamics between CotY and CotZ at the single-molecule level. The results show that homo- typic interactions of CotY and CotZ and the heterotypic interaction between CotY and CotZ exist. Furthermore, the dissociation kinetics of the complexes were studied by monitoring the relationship between the unbinding forces and the loading rates at different pulling velocities. In this way, a series of kinetic parameters regarding the three different complexes were obtained. It revealed the strong inter- actions between CotY and CotZ, CotY and CotY, and a relatively weak interaction of CotZ and CotZ. Ó 2015 Elsevier Inc. All rights reserved. 1. Introduction Bacillus subtilis produces a metabolically dormant and highly resistant cellular form called the spore when it faces environmen- tal stress factors. The spores are extremely resistant to a vast range of adverse conditions such as toxic chemicals, high temperature, radiation and lytic enzymes (Higgins and Dworkin, 2012; Laaberki and Dworkin, 2008), and survive for long periods of time (Vreeland et al., 2000). Resistance characteristics of spores to envi- ronmental assaults is attributed mainly to the presence of the spore coat, a protective and multilayered protein structure that encases the spore (Henriques and Moran, 2007; McKenney et al., 2013). The coat is formed by more than 70 proteins of varying size deposited onto the spore surface (Kuwana et al., 2002). Electron microscopy as well as other means show the coat is arranged into at least four distinct structural layers: a diffuse undercoat, a thin lamellar inner coat, a thick electron-dense outer coat, and an outer- most layer named the crust (McKenney et al., 2010; Plomp et al., 2014). In addition to protecting the spore against physical and chemical insults, the coat is also essential for the spore to germi- nate and return to the vegetative cell when nutrients become available (Setlow, 2003; Troiano et al., 2015). In B. subtilis, lots of details about sporulation have been eluci- dated (de Hoon et al., 2010; Stragier and Losick, 1996). Spore for- mation begins with an asymmetric cell division producing two cellular compartments: a larger mother cell and a smaller fores- pore which initially lie side by side. Then the mother cell continues to engulf the forespore in a phagocytic-like process producing a forespore embosomed by the mother cell which nurtures the resulting forespore. This creates an organelle encased by a double membrane. In the final stages, the mother cell lyses, and the mature spore is released into the surrounding medium. The coat is the final spore structure to be completed, however, its assembly starts early at the onset of spore development. Although the coat consists of large numbers of proteins, only a small subset of them have a crucial role in proper assembly of indi- vidual layers of the coat. Those proteins are referred to as the mor- phogenetic proteins (Henriques and Moran, 2007; Kim et al., 2006), including SpoIVA, SafA, SpoVID, CotE and CotZ. At the early stages of coat development, a small protein SpoVM is anchored through its amphipathic helix to the outer forespore membrane. This pro- tein then attracts the morphogenetic protein SpoIVA to the surface http://dx.doi.org/10.1016/j.jsb.2015.09.001 1047-8477/Ó 2015 Elsevier Inc. All rights reserved. ⇑ Corresponding author. E-mail address: jltang@ciac.ac.cn (J. Tang). Journal of Structural Biology xxx (2015) xxx–xxx Contents lists available at ScienceDirect Journal of Structural Biology journal homepage: www.elsevier.com/locate/yjsbi Please cite this article in press as: Liu, H., et al. Investigating interactions of the Bacillus subtilis spore coat proteins CotY and CotZ using single molecule force spectroscopy. J. Struct. Biol. (2015), http://dx.doi.org/10.1016/j.jsb.2015.09.001