Structure of a signal transduction regulator, RACK1, from Arabidopsis thaliana

Abstract

The receptor for activated C-kinase 1 (RACK1) is a highly conserved WD40 repeat scaffold protein found in a wide range of eukaryotic species from Chlamydymonas to plants and humans. In tissues of higher mammals, RACK1 is ubiquitously expressed and has been implicated in diverse signaling pathways involving neuropathology, cellular stress, protein translation, and developmental processes. RACK1 has established itself as a scaffold protein through physical interaction with a myriad of signaling proteins ranging from kinases, phosphatases, ion channels, membrane receptors, G proteins, IP3 receptor, and with widely conserved structural proteins associated with the ribosome. In the plant Arabidopsis thaliana, RACK1A is implicated in diverse developmental and environmental stress pathways. Despite the functional conservation of RACK1-mediated protein-protein interactionregulated signaling modes, the structural basis of such interactions is largely unknown. Here we present the first crystal structure of a RACK1 protein, RACK1 isoform A from Arabidobsis thaliana, at 2.4 Å resolution, as a C-terminal fusion of the maltose binding protein. The structure implicates highly conserved surface residues that could play critical roles in protein-protein interactions and reveals the surface location of proposed post-transcriptionally modified residues. The availability of this structure provides a structural basis for dissecting RACK1-mediated cellular signaling mechanisms in both plants and animals.Keywords: RACK1; protein signaling; scaffolding protein; WD40; propeller; protein-protein interaction; drought Scaffold proteins are uniquely poised to integrate signals from multiple pathways. Such proteins generate considerable functional diversity by mediating concomitant and/ or promiscuous interactions with a vast array of protein partners. The receptor for activated C-kinase 1 (RACK1) is a member of the WD40 repeat family of b-propeller proteins. It was discovered through its ability to function as a scaffold protein, stabilizing signaling complexes involving protein kinase C (Ron and Mochly-Rosen 1994; . In metazoans, RACK1 is now postulated to coordinate many different signal transduction pathways, ranging from cell division to ion channel regulation, by interacting with more than 80 different proteins (McCahill et al. 2002;Alfarano et al. 2005; 4 Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi

A synergistic approach to protein crystallization: Combination of a fixed‐arm carrier with surface entropy reduction

“…We have successfully solved the structures of three of these proteins-2OST (Table I), 42 RACK1A, 43 and Der p 7 (Mueller et al (doi:10.1016)). We have also obtained crystals for two (TargetA and TargetB) of the remaining four proteins (Table III).…”

“…Gly4 was chosen as the starting residue based on secondary structure prediction, and by manually docking the N-terminus of related b-propeller proteins to the C-terminus of the MBP. 43 The structure of the MBP(C)-RACK1A is shown in Figure 3(A). A detailed analysis of the structure within the crystal lattice identifies several key features.…”

“…In this crystal form, there are three MBP(E)-Der p 7 molecules within the asymmetric unit. Following positioning of the MBP molecules using molecular replacement with an MBP model (PDB ID code 3DM0 43 ), discontinuous electron density was visible for the Der p 7 molecule. Although molecular replacement models using the structurally related proteins BPI (PDB ID code 1EWF 96 ) and Takeout1 (PDB ID code 3E8T 97 ) were unsuccessful, these structures provided a guide for chain tracing and manual model building.…”

Engineered Derivatives of Maltose-Binding Protein

“…In most cases, conformational flexibility between the MBP and the target protein works against crystallization, so fusion proteins expressed in the most common commercial MBP vectors are often modified to shorten the spacer between MBP and the target. This approach has been used to solve the structures of a number of proteins, among them human T cell leukemia virus type 1 gp21 (Kobe et al, 1999), the SarR protein from Staphylococcus aureus (Liu et al, 2001), yeast MATa1 (Ke & Wolberger, 2003), and RACK1 from Arabidopsis thaliana (Ullah et al, 2008).…”

Phosphorylation of RACK1 in plants

“…4 The protein sequences and the structure of RACK1 are conserved in plants. 8,9 Similar to mammalian RACK1, plant RACK1 interacts with a large group of proteins 10,11 and is involved in diverse biological processes, ranging from seed germination, leaf and root development to flowering, 8,12 and is also involved in responses to plant hormones, 8,13 and biotic and abiotic stresses. [13][14][15] At the molecular level, RACK1 protein is associated with ribosomes [16][17][18] and is involved in the regulation of protein translation 18 and miRNA abundance.…”