Structure and function of PhyA from Desulfovibrio magneticus and use of its product in affinity pull-down experiments

The X-ray crystallographic structure of a divergent PTPLP from Desulfovibrio magneticus has been determined in the presence and absence of InsP6 substrate in order to identify the structural features that give rise to its 3-4-5 substrate specificity. These include a novel Phy loop conformation and R242 of the HCRGG.GR P-loop signature sequence. Further, PTPLPs containing an arginine following the cysteine nucleophile are expected to preferentially target the C4-phosphoryl of Ins(1,2,4,5,6)P5 and have a 3-4 hydrolytic pathway. Subsequently, both InsP6 and Ins(1,2,6)P3 were covalently attached to chromatographic resin and utilized as ’bait’ molecules in novel affinity pull-down experiments utilizing a phosphate control column and unfractionated whole cell lysates. The resulting interactomes contain a greater fraction of known IP interacting proteins than current literature methods and identify similar or larger number of total proteins. Despite these improvements, more work needs to be done to address non-specific binding to our columns.

Authors

• Cleland, Colyn P.
• University of Lethbridge. Faculty of Arts and Science

• protein complex
• substrate specificity
• PTP-like
• affinity pull-down
• Cysteine proteinases
• Inositol phosphates
• Phosphatases
• X-ray crystallography

Identifier

hdl:10133/5478

OAI identifier oai:identifier

oai:opus.uleth.ca:10133/5478