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Engineering Novel Substrate Specificity into the Low Molecular Weight Protein Tyrosine Phosphatase (LMWPTP) by Computational and Structure-guided Rational design
[Thesis]. Manchester, UK: The University of Manchester; 2019.
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Abstract
Protein phosphatases are regulatory enzymes with unique substrate specificity for serine/threonine/tyrosine/histidine phosphorylated proteins and phosphoinositides. They mediate critical cell processes such as cell metabolism, cell cycle, growth and differentiation. For example, phosphoinositide phosphatases act as regulatory enzymes that mediate critical cellular processes such as cell signalling, membrane trafficking, cell cycle and growth. Loss-of-function mutations in these enzymes are linked to a plethora of disease conditions. Tumour suppressor PTEN is the most frequently mutated gene in various forms of human cancer; Myotubularins are mutated in X-linked myotubular myopathy (XLMTM) and Charcot-Marie-Tooth disease type 4B (CMT4B); Synaptojanin I is mutated in early-onset progressive Parkinsonism. Engineered PTPs are therefore seen as useful tools to enhance our understanding of signalling pathways involving phosphorylation events. Here, we used a combination of structure-guided rational and computational design to alter the substrate specificity of Low Molecular Weight Protein Tyrosine Phosphatase (LMW-PTP), from tyrosine-specific to PI(3,5)P2-, PI(3)P-, PI(4)P-, and PI(5)P-specific phosphatase (Low Molecular Weight Phosphoinositide Phosphatase – LMWPIP). Four LMW-PIP mutants dephosphorylate phosphoinositides with single or broad substrate specificity. We combined Rosetta Enzyme designs containing frequently introduced mutations with molecular docking studies to assess binding affinity to the ligand substrate. This approach facilitates the design selection process by reducing the number of candidate mutants to be validated experimentally. We also designed mutants to be tested for specificity towards serine/threonine phospho-peptide. Finally, we set up a system in yeast that can be used for in vivo characterisation of LMWPTPB mutants, by cloning and confirming the expression of LMWPTP in yeast using western blot and RT-PCR. Engineered LMW-PIP mutants can be a useful tool in investigating lipid-regulated cell signalling pathways, and in the long term, could serve as biotherapeutics in enzyme replacement therapy.
Keyword(s)
LMWPTP; protein engineering; rational design; substrate specificity