Inositol Phosphate Kinase Architecture: Practical Approaches and Lessons Learned.

Inositol phosphate and pyrophosphate kinases (InsPKs) regulate cellular signaling by phosphorylating inositol phosphates, which play key roles in diverse physiological processes from insulin signaling to cell proliferation. Here, we review the structural biology of these enzymes and our understanding of their distinctive architectures. InsPKs can be classified into two structural families, which consist of InsPKs and other enzymes: the protein kinase-like and ATP-Grasp, each with distinct catalytic features. While crystal structures have been determined for representative catalytic domains through the InsPK synthetic pathways, structural information remains limited to these domains, with little information to guide our understanding of regulatory mechanisms or architectures of multidomain InsPKs. Here, we review the evolution of InsPK classification from early substrate-based naming to modern structural family groupings. We highlight how structural insights have revealed relationships between seemingly unrelated enzymes and what can be understood about InsPKs from features they share with other enzymes. We also provide an overview of the decision points in InsPK structural biology, from protein engineering and expression to crystallization, emphasizing how choices at each stage impact both the likelihood of success and the biological relevance of the results. We discuss the tools and recent advances, such as AlphaFold structure predictions, which have provided new insights into previously intractable regions of InsPKs and offer guidance for future exploration of these kinases.