Towards the structural characterization of proteins involved in peptidoglycan biosynthesis

I. Nikolaidis

Research output: ThesisDoctoral thesis 1 (Research UU / Graduation UU)

Abstract

The cell wall is an essential structure for bacterial survival and unique to bacteria. It is responsible for maintenance of cellular shape and allows the bacterium to withstand high differences in osmotic pressure between the inner and outer leaflet of the cell. Consequently, the bacterial cell wall has been an optimal target for the development of antibiotics for over half a century, particularly the assembly of its major structural component, the peptidoglycan. Despite the progress in understanding the structure and function of several enzymes involved in the biosynthesis of peptidoglycan, however, the mechanism and regulation of their intermolecular assembly is still not fully understood.

In this thesis, we use several biochemical and structural approaches to gain more insight into the complex process of peptidoglycan biosynthesis and its regulation. The first part of this thesis describes the characterization of an interaction between proteins that exert antagonistic functions during cell wall growth. By surface plasmon resonance we show that Pseudomonas aeruginosa PBP2 and SltB1 interact in a highly Ca2+-dependent manner. Structural determination of SltB1 revealed an EF-hand motif that might be required for the interaction of the two proteins.

The second part of this thesis not only presents strategies to express and purify multi-spanning membrane proteins of the SEDS protein family, but also provides the first biochemical evidence that Streptococcus pneumoniae RodA functions as Lipid II flippase, a role which has to date only been reported for Escherichia coli FtsW. In addition, we crystallized RodA and obtained initial diffraction data. Determination of its three-dimensional structure will not only provide insight into the mechanism of lipid transport across cellular compartments but will also open up new ways for rational drug design.
Original languageEnglish
Awarding Institution
  • Utrecht University
Supervisors/Advisors
  • Killian, Antoinette, Primary supervisor
  • Dessen, A., Co-supervisor, External person
  • Breukink, Eefjan, Co-supervisor
Award date30 Nov 2015
Publisher
Print ISBNs978-94-6299-223-8
Publication statusPublished - 30 Nov 2015

Keywords

  • peptidoglycan biosynthesis
  • bicelles
  • membrane proteins
  • SEDS proteins
  • X-ray crystallography
  • Lipid II

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