The Molecular Basis of Substrate Recognition, Catalysis, and Regulation in Sortase Enzymes
- Author(s): Jacobitz, Alex William
- Advisor(s): Clubb, Robert T
- et al.
The Molecular Basis of Substrate Recognition, Catalysis, and Regulation in
Alex William Jacobitz
Doctor of Philosophy in Biochemistry and Molecular Biology
University of California, Los Angeles, 2015
Professor Robert T. Clubb, Chair
Gram-positive bacteria utilize sortase enzymes to catalyze a transpeptidation reaction that covalently links protein substrates to the cell wall or polymerizes proteinaceous pili. Sortase enzymes are membrane anchored cysteine transpeptidases that recognize a cell wall sorting signal motif at the C-terminus of their primary protein substrate and covalently attach it to an amino-nucleophile located in their secondary substrate. For cell wall anchoring sortases, this secondary substrate is the cell wall precursor lipid II, while for pilin polymerizing sortases it is a lysine sidechain within their pilin protein substrate. A number of major questions have remained unanswered concerning the binding interactions that govern the substrate specificity of these enzymes, their catalytic mechanism, and the mechanism through which appendages that contact the active site regulate their activity. This dissertation advances the understanding of the sortase mechanism through the elucidation of new sortase structures, and the characterization of their dynamic behavior.
Chapter three of this thesis describes the solution structure of the class D sortase from Bacillus anthracis by NMR. Class D enzymes anchor proteins involved in bacterial sporulation to the cell wall, and this is the first structure of a class D sortase to ever be determined. NMR analysis of the enzyme uncovered a rigid substrate binding pocket and a novel dimerization interface. Chapter four describes the crystal structure of the class B sortase from Staphylococcus aureus bound to a substrate analog. This work provided new insight into the biophysical basis of substrate recognition. The structure combined with computational modeling and molecular dynamics simulations led to the discovery of a substrate-stabilized oxyanion hole that is used to stabilize tetrahedral reaction intermediates. Molecular dynamics simulations and the high degree of sequence conservation inherent in these enzymes suggest that all members of the sortase superfamily will stabilize high energy reaction intermediates in a similar manner. Chapter five investigates the additional structural features some sortases use to regulate access to their active site, focusing on sortase C-1 from Streptococcus pneumoniae as a model. Using NMR, mutagenesis, and biochemical experiments this work demonstrated that the enzyme utilizes a rigid N-terminal appendage, termed the “lid,” to maintain the enzyme in an inactive state. The results of in vitro biochemical assays indicate that the lid prevents cleavage of the primary substrate by preventing access to the catalytic cysteine residue. Both in vitro activity and access to the active site cysteine could be increased by the incorporation of mutations which were shown by NMR to destabilize the lid and increase its flexibility. Based on these results we propose that on the cell surface, lid containing class C enzymes exist in a dormant state and are only activated during pilus biogenesis by interactions with either their substrate or other factors on the cell surface. The results of these experiments have provided new insight into substrate binding, catalysis, and regulation in sortase enzymes.