ABSTRACT
Pumilio/fem-3 mRNA-binding factor (PUF) proteins possess a recognition code for bases A, U, and G, allowing designed RNA sequence specificity of their modular Pumilio (PUM) repeats. However, recognition side chains in a PUM repeat for cytosine are unknown. Here we report identification of a cytosine-recognition code by screening random amino acid combinations at conserved RNA recognition positions using a yeast three-hybrid system. This C-recognition code is specific and modular as specificity can be transferred to different positions in the RNA recognition sequence. A crystal structure of a modified PUF domain reveals specific contacts between an arginine side chain and the cytosine base. We applied the C-recognition code to design PUF domains that recognize targets with multiple cytosines and to generate engineered splicing factors that modulate alternative splicing. Finally, we identified a divergent yeast PUF protein, Nop9p, that may recognize natural target RNAs with cytosine. This work deepens our understanding of natural PUF protein target recognition and expands the ability to engineer PUF domains to recognize any RNA sequence.
Subject(s)
Caenorhabditis elegans Proteins/metabolism , Cytosine/chemistry , Cytosine/metabolism , Animals , Caenorhabditis elegans Proteins/chemistry , Caenorhabditis elegans Proteins/genetics , Crystallography, X-Ray , Humans , Protein Structure, Tertiary , RNA/chemistry , RNA/genetics , RNA/metabolism , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
Phospholipase C (PLC) isozymes are directly activated by heterotrimeric G proteins and Ras-like GTPases to hydrolyze phosphatidylinositol 4,5-bisphosphate into the second messengers diacylglycerol and inositol 1,4,5-trisphosphate. Although PLCs play central roles in myriad signaling cascades, the molecular details of their activation remain poorly understood. As described here, the crystal structure of PLC-beta2 illustrates occlusion of the active site by a loop separating the two halves of the catalytic TIM barrel. Removal of this insertion constitutively activates PLC-beta2 without ablating its capacity to be further stimulated by classical G protein modulators. Similar regulation occurs in other PLC members, and a general mechanism of interfacial activation at membranes is presented that provides a unifying framework for PLC activation by diverse stimuli.
Subject(s)
Type C Phospholipases/antagonists & inhibitors , Amino Acid Sequence , Animals , Binding Sites , COS Cells , Chlorocebus aethiops , Crystallography, X-Ray , Enzyme Activation , GTP-Binding Proteins/metabolism , Humans , Isoenzymes/antagonists & inhibitors , Isoenzymes/chemistry , Models, Molecular , Molecular Sequence Data , Phosphoinositide Phospholipase C/antagonists & inhibitors , Phosphoinositide Phospholipase C/chemistry , Phosphoinositide Phospholipase C/metabolism , Phospholipase C beta/antagonists & inhibitors , Phospholipase C beta/chemistry , Phospholipase C beta/isolation & purification , Phospholipase C beta/metabolism , Phospholipase C delta/antagonists & inhibitors , Phospholipase C delta/chemistry , Phospholipase C delta/metabolism , Protein Structure, Secondary , Sequence Deletion , Type C Phospholipases/chemistryABSTRACT
Although diverse signaling cascades require the coordinated regulation of heterotrimeric G proteins and small GTPases, these connections remain poorly understood. We present the crystal structure of the GTPase Rac1 bound to phospholipase C-beta2 (PLC-beta2), a classic effector of heterotrimeric G proteins. Rac1 engages the pleckstrin-homology (PH) domain of PLC-beta2 to optimize its orientation for substrate membranes. Gbetagamma also engages the PH domain to activate PLC-beta2, and these two activation events are compatible, leading to additive stimulation of phospholipase activity. In contrast to PLC-delta, the PH domain of PLC-beta2 cannot bind phosphoinositides, eliminating this mode of regulation. The structure of the Rac1-PLC-beta2 complex reveals determinants that dictate selectivity of PLC-beta isozymes for Rac GTPases over other Rho-family GTPases, and substitutions within PLC-beta2 abrogate its stimulation by Rac1 but not by Gbetagamma, allowing for functional dissection of this integral signaling node.
Subject(s)
Isoenzymes/chemistry , Isoenzymes/metabolism , Type C Phospholipases/chemistry , Type C Phospholipases/metabolism , rac1 GTP-Binding Protein/chemistry , rac1 GTP-Binding Protein/metabolism , Crystallography, X-Ray , Humans , Isoenzymes/genetics , Models, Molecular , Mutation/genetics , Phospholipase C beta , Protein Binding , Protein Structure, Quaternary , Static Electricity , Type C Phospholipases/genetics , rac1 GTP-Binding Protein/geneticsABSTRACT
Small GTPases function as molecular switches, which transduce cellular signals from upstream regulators to downstream effectors in a guanine nucleotide-dependent manner. Direct binding partners of small GTPases fall into four classes of both regulators and effectors that can be differentiated on the basis of the state of nucleotide required for binding. Here we describe a procedure for the rapid screening and quantitative assessment of direct interactions of the Rho family of small GTPases with effector molecules of the phospholipase Cbeta class of enzymes using surface plasmon resonance technology. The experimental format described is also readily adaptable toward characterizing guanine nucleotide-dependent binding events of both small and heterotrimeric G proteins with various classes of GTPase regulatory proteins.
Subject(s)
Isoenzymes/metabolism , Type C Phospholipases/metabolism , rac GTP-Binding Proteins/physiology , Animals , Humans , Phospholipase C beta , Spodoptera , Surface Plasmon Resonance/methodsABSTRACT
Heterotrimeric G proteins are molecular switches that regulate numerous signaling pathways involved in cellular physiology. This characteristic is achieved by the adoption of two principal states: an inactive, GDP bound state and an active, GTP bound state. Under basal conditions, G proteins exist in the inactive, GDP bound state; thus, nucleotide exchange is crucial to the onset of signaling. Despite our understanding of G protein signaling pathways, the mechanism of nucleotide exchange remains elusive. We employed phage display technology to identify nucleotide state-dependent Galpha binding peptides. Herein, we report a GDP-selective Galpha binding peptide, KB-752, that enhances spontaneous nucleotide exchange of Galpha(i) subunits. Structural determination of the Galpha(i1)/peptide complex reveals unique changes in the Galpha switch regions predicted to enhance nucleotide exchange by creating a GDP dissociation route. Our results cast light onto a potential mechanism by which Galpha subunits adopt a conformation suitable for nucleotide exchange.
