Nanodiscs as a tool to study interactions between bacterial division Proteins

Abstract

In Escherichia coli, cell division involves the assembly of a macromolecular complex, the divisome, formed by several proteins, 10 of them essential (Vicente et al. 2006). The nature of a large part of the divisome components is known, as it is their assembly pathway and some of the biochemical activities and protein-protein interactions involved (Harry et al. 2006, Vicente and Rico 2006). The elements of the divisome follow an assembly pathway in which both sequential and concerted stages are involved. Initially, three proteins, FtsZ, FtsA, and ZipA, assemble together, forming a proto-ring into which the other components are added (Vicente and Rico 2006). The Z-ring is highly dynamic, and its position is regulated by two negative control systems that inhibit ring formation at wrong places (Yu et al. 1999). Initial complex subsequently recruits the rest of division proteins (FtsK, FtsQ, FtsB, FtsL, FtsW, FtsI, FtsN), all of them membrane proteins (Adams et al. 2009). FtsZ is a 40 kDa soluble GTPase homolog of eukaryotic tubulin, whose GTP-linked assembly-disassembly cycle is important for cell division (Erickson et al. 2010, Mingorance et al. 2010). FtsZ is anchored to the inner cell membrane by two proteins, ZipA and FtsA, thus forming the proto-ring complex, which initiates cell division. FtsA is more widely conserved than ZipA, though both protein are essential for cell division in E. coli and other gram negative bacteria (Geissler et al. 2003).

As most of the available knowledge on the interaction of cell division proteins has been derived from the behavior of mutants, in which one or more components of the divisome have been genetically modified or impaired, it becomes essential to corroborate the commonly accepted hypothesis using a bottom-up synthetic approach. Thus, in this thesis we have used an in vitro membrane model, nanodiscs, to study two membrane-attached division proteins, ZipA and FtsN. ZipA is a 36.4 kDa protein with a single, N-terminal transmembrane helix followed by a large, flexible domain and small C-terminal globular domain that binds to FtsZ in the cytoplasm (Ohashi et al. 2002). On the other hand, has a reversed topology, with small N-terminal unstructured and charged domain in the cytoplasm, followed by a single transmembrane domain, a large flexible domain and a small SPOR domain in the periplasmic that interacts with peptidoglycan (Dai et al. 1996). It has been reported that FtsN is able to interact with FtsA through its small charged cytoplasmic region, as well as with other late division proteins such as FtsQ and FtsI (Di Lallo et al. 2003, Karimova et al. 2005, Bertsche et al. 2006, D'Ulisse et al. 2007, Busiek et al. 2012). As stated above, both proteins were incorporated into nanodiscs, a membrane model consisting of a discoidal patch of lipid bilayer surrounded by two copies of a Membrane Scaffold Protein (MSP), an apoA-I analogue, which stabilize the membrane (Segrest et al. 1999). The whole structure is water-soluble, monodisperse, and has a diameter of 10 nm. Nanodiscs provide a topologically restricted environment to membrane proteins while remaining in solution, allowing the use of a broad arsenal of biochemical and biophysical tools to quantitatively characterize the embedded protein and its interactions (Nath et al. 2007).

The first part of this thesis describes the hydrodynamic characterization of nanodiscs by analytical ultracentrifugation (AUC) and dynamic light scattering (DLS), with the aim of serving as a reference for more complex experiments including a membrane protein in nanodiscs. The second part, describes the incorporation of full-length ZipA into nanodiscs, the biophysical characterization of the structures by the AUC, DLS and fluorescence correlation spectroscopy (FCS), and the structural characterization by transmission electron microscopy (TEM). Finally, the interaction of ZipA nanodiscs with FtsZ, both in its oligomeric GDP-bound form and in its polymeric GTP-form, was quantitatively studied using AUC and FCS and FtsZ/nandosics complexes were visualized using TEM. Finally, the third part describes the purification, characterization and incorporation of FtsN in nanodiscs, using the same techniques as described for ZipA.

Objectives The main objective of this thesis was to use and characterize the nanodisc system as support for the study of interactions between proteins of E. coli divisome. This included (1) the biophysical characterization of nanodiscs with biophysical techniques available in the laboratory for the analysis of interactions (AUC, DLS, TEM); (2), the reconstitution of ZipA in nanodiscs and the quantitative study of its interaction with FtsZ; and (3), the purification, reconstitution and characterization of FtsN in nanodiscs.