ABSTRACT
Many novel signal transduction domains are being identified in the wake of genome sequencing projects and improved sensitivity in homology-detection techniques. The functions of these domains are being discovered by hypothesis-driven experiments and structural genomics approaches. This article reviews the recent highlights of research on modular signaling domains, and the relative contributions and limitations of the various approaches being used.
Subject(s)
Genomics/methods , Protein Conformation , Protein Structure, Tertiary/physiology , Proteins/chemistry , Animals , Computational Biology , Humans , Models, Molecular , Protein Folding , Proteins/genetics , Proteins/physiology , Signal TransductionABSTRACT
Signaling domains have been identified by the analysis of data derived from biochemical studies, molecular cloning, or genetic studies. With the availability of genomic information from many organisms and the improved sensitivity in homology detection techniques, many new domains are being identified. In an attempt to understand biochemical and biological function of these domains, we have started a small-scale structural genomics, or structural biology with genomic approach. Two examples from our recent work are steroidogenic acute regulatory protein (StAR)-related lipid-transfer (START) domain and inositol polyphosphate 5-phosphatase catalytic (IPP5C) domain. Crystal structure of human MLN64-START domain revealed a hollowed-out protein containing a hydrophobic tunnel just large enough to bind one molecule of cholesterol and completely exclude it from solvent. This structure suggests that the START domain is a classical type of lipid transporter. On the contrary, the function of IPP5C domain has been extensively studied for a long time, but its catalytic mechanism, positional selectivity, and diverse substrate specificity remained mysterious due to the unavailability of three-dimensional structure. With the structural genomic approach, the first structure of IPP5C domain was solved from a S. pombe protein that is now known as SPsynaptojanin and the structure gave us answers to some of these questions.
Subject(s)
Carrier Proteins/chemistry , Genomics/methods , Membrane Proteins/chemistry , Phosphoproteins/chemistry , Phosphoric Monoester Hydrolases/chemistry , Animals , Carrier Proteins/genetics , Crystallography, X-Ray , Databases, Protein , Humans , Inositol Polyphosphate 5-Phosphatases , Mice , Models, Molecular , Phosphoproteins/genetics , Phosphoric Monoester Hydrolases/genetics , Protein Binding , Protein Structure, Tertiary , Signal Transduction/physiology , Structure-Activity RelationshipABSTRACT
Inositol-5-phosphatases are important enzymes involved in the regulation of diverse cellular processes from synaptic vesicle recycling to insulin signaling. We describe a comparative study of two representative inositol-5-phosphatases, Schizosaccharomyces pombe synaptojanin (SPsynaptojanin) and human SH2 domain-containing inositol-5-phosphatase SHIP2. We show that in addition to Mg2+, transition metals such as Mn2+, Co2+, and Ni2+ are also effective activators of SPsynaptojanin. In contrast, Ca2+ and Cu2+ are inhibitory. We provide evidence that Mg2+ binds the same site occupied by Ca2+ observed in the crystal structure of SPsynaptojanin complexed with inositol 1,4-bisphosphate (Ins(1,4)P2). Ionizations important for substrate binding and catalysis are defined for the SPsynaptojanin-catalyzed Ins(1,4,5)P3 reaction. Kinetic analysis with four phosphatidylinositol lipids bearing a 5-phosphate and 54 water-soluble inositol phosphates reveals that SP-synaptojanin and SHIP2 possess much broader substrate specificity than previously appreciated. The rank order for SPsynaptojanin is Ins(2,4,5)P3 > phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) approximately Ins(4,5)P2 approximately Ins(1,4,5)P3 approximately Ins(4,5,6)P3 > PtdIns(3,5)P2 approximately PtdIns(3,4,5)P3 approximately Ins(1,2,4,5)P4 approximately Ins(1,3,4,5)P4 approximately Ins-(2,4,5,6)P4 approximately Ins(1,2,4,5,6)P5. The rank order for SHIP2 is Ins(1,2,3,4,5)P5 > Ins(1,3,4,5)P4 > PtdIns(3,4,5)P4 approximately PtdIns(3,5)P2 approximately Ins(1,4,5,6)P4 approximately Ins(2,4,5,6)P4. Because inositol phosphate isomers elicit different biological activities, the extended substrate specificity for SPsynaptojanin and SHIP2 suggest that these enzymes likely have multiple roles in cell signaling and may regulate distinct pathways. The unique substrate specificity profiles and the importance of 2-position phosphate in binding also have important implications for the design of potent and selective SPsynaptojanin and SHIP2 inhibitors for pharmacological investigation.