Structure and substrate specificity of myo-inositol phosphatases at atomic resolution

Abstract

Protein tyrosine phosphatase-like myo-inositol phosphatases (PTPLPs) follow an ordered, sequential dephosphorylation pathway that utilizes the abundant myo-inositol- 1,2,3,4,5,6-hexakisphosphate (InsP6) to produce less-phosphorylated myo-inositol phosphates (IPs) containing between one and five phosphoryl groups. To understand PTPLP substrate specificity, I present multiple complex structures of Phytase A from Selenomonas ruminantium (PhyAsr) and Mitsuokella multacida (PhyAmm; a tandem repeat) with various IPs. From these structures I demonstrated that binding of IPs by these enzymes is consistent with a 'lock-and-key' binding mechanism, determined binding differences between InsP6 and less-phosphorylated IPs, and revised the existing PTPLP substrate specificity model. As part of this work, I have produced the first PhyAmm complex structures and demonstrated that the PhyAmm C-terminal repeat binds substrates using identical phosphoryl binding sites as PhyAsr. Further, I have provided evidence that differential substrate binding in the PhyAmm N- and C-terminal repeats is due to electrostatic differences and a loop insertion causing steric clashes.