Understanding PTPLP specificity: from atomic resolution to rational mutagenesis

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dc.contributor.authorCleland, Colyn P.
dc.contributor.authorUniversity of Lethbridge. Faculty of Arts and Science
dc.contributor.supervisorMosimann, Steven C.
dc.contributor.supervisorGirodat, Dylan
dc.date.accessioned2025-11-14T20:53:20Z
dc.date.available2025-11-14T20:53:20Z
dc.date.issued2025
dc.degree.levelPh.D
dc.description.abstractTo understand substrate specificity of protein tyrosine phosphatase-like myo-inositol phosphatases (PTPLPs), I determined the X-ray crystallographic structures of two divergent PTPLP’s (30% sequence identity), PhyAdm from Solidesulfovibrio magneticus and PhyAlp from Legionella pneumophila str. Paris, in complex with InsP6. Both enzymes feature a CRGG phosphate-binding loop (P-loop) sequence, a shared InsP6 dephosphorylation pathway and a novel InsP6 binding mode. Comparison with the previously solved PhyAsr structure in complex with InsP6, which features a CEAG P-loop sequence, revealed that conformational differences in InsP6 binding were linked to the identity of the x2 residue in the P-loop. This finding prompted the investigation of additional novel PTPLPs containing similar and divergent naturally occurring x1x2 P-loop variants. The results established a relationship between InsP6 dephosphorylation pathways and P-loop x1x2 residues, identifying the P-loop as a primary determinant of PTPLP specificity. To demonstrate an understanding of PTPLP specificity, I rationally engineered P-loop mutants of PhyAdm and PhyAsr that swapped their x1x2 residues. Each mutant exhibited the InsP6 dephosphorylation pathway characteristic of the other enzyme. Structural analysis of the PhyAsr P-loop mutant in complex with its new dephosphorylation products confirmed that the x1x2 residue pairing influences substrate binding modes within the active site. These findings significantly advance our understanding of PTPLP substrate specificity and suggest a variety of novel research directions to further expand upon the structure-function relationship of these enzymes.
dc.embargoNo
dc.identifier.urihttps://hdl.handle.net/10133/7224
dc.language.isoen
dc.publisherLethbridge, Alta. : University of Lethbridge, Dept. of Chemistry and Biochemistry
dc.publisher.departmentDepartment of Chemistry and Biochemistry
dc.publisher.facultyArts and Science
dc.relation.ispartofseriesThesis (University of Lethbridge. Faculty of Arts and Science)
dc.subjectPTPLPs
dc.subjectphosphate-binding loop sequences
dc.subjectPTPLP specificity
dc.subjectsubstrate binding modes
dc.subjectx1x2 residues
dc.subjectmio-inositol phosphatases
dc.subject.lcshDissertations, Academic
dc.subject.lcshInositol phosphates--Research
dc.subject.lcshProtein-tyrosine phosphatase--Research
dc.titleUnderstanding PTPLP specificity: from atomic resolution to rational mutagenesis
dc.typeThesis
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