Biophysical and enzymological exploration of Mycobacterium tuberculosis phosphoserine phosphatase SerB2 self-assembly and regulation by L-serine

Candidate

Elise PIERSON

Promoter

Prof. Johan WOUTERS, UNamur, Department of chemistry, Laboratory of Structural Biological Chemistry (CBS)

Jury

• Prof. Catherine MICHAUX (département de chimie, UNamur), présidente
• Prof. Johan WOUTERS (département de chimie, UNamur), promoteur et secrétaire
• Prof. René WINTJENS (faculté de pharmacie, ULB)
• Prof. Marianne FILLET (département de pharmacie, ULiège)
• Prof. Xavier DE BOLLE (département de biologie, UNamur)

Summary

Homo-oligomerization, or protein self-assembly, is a phenomenon governing a wide variety of cellular functions. The homo-oligomeric state of an enzyme can, for instance, influence its function and/or activity, and consequently all the biochemical mechanisms arising therefrom. An emerging therapeutic strategy therefore consists in regulating the activity of an enzyme by interfering with its quaternary structure(s) using small molecules. The implementation of such an approach requires the thorough characterization of homo-oligomerization in the targeted system.

In the context of research against tuberculosis and the ever-growing threat to public health caused by the emergence of multidrug-resistant strains, this thesis focuses on the phosphoserine phosphatase SerB2 of M. tuberculosis (MtSerB2). Not only essential to the survival of the pathogen through its metabolic function in L-serine biosynthesis, the enzyme is also an interesting therapeutic target in light of its suspected role in host invasion.

This work consists of the exploration of MtSerB2 homo-oligomerization in the presence and absence of L-serine. The nature, structure, and activity of the oligomeric species formed by the enzyme and its homologs (natural or engineered) are studied by electrophoresis, size exclusion chromatography, light scattering in solution, X-ray crystallography, and enzyme kinetics. Combined, the results suggest that MtSerB2 is a morpheein. Multiple alternative quaternary assemblies of the enzyme are evidenced, including an active domain-swapped dimer, an inactive tetramer, and a low-activity trimer formed in the presence of L-serine. Their structural characteristics and formation mechanisms indicate that the interconversion between these species can take place through a conformationally flexible monomeric state. In addition to establishing the basis for the rational design of selective allosteric inhibitors, this equilibrium may provide answers to the moonlighting properties of MtSerB2.