Structural and functional studies of MESD, a specialized chaperone protein of LRP5/6 in Wnt signaling
Abstract
<?Pub Inc> My Ph.D. thesis focuses on structural and functional studies of MESD, a newly discovered, novel specialized chaperone protein for proper folding of the BP domain in LDLR family, particularly for LRP5/6. LRP5 and LRP6 are critical co-receptors in the Wnt signaling pathway. Objective 1. Structure determination of MESD and its central core structure domain MESD(12-155). I have determined NMR structure of full length mouse MESD and MESD(12-155), the central core structural region of MESD which represents Boca, the homolog of MESD in the Drosophila. These high-resolution NMR structures indicated that MESD is a typical two domain protein, containing a well defined N-terminal core domain and a flexible C-terminal domain. The N-terminal domain is a tightly packed that is centered at a four stranded, anti-parallel β-sheet, surrounded by two rigid helices on the top and one helix in the bottom of the sheet. In addition, a flexible short helix is also located on the top of the sheet and this short helix contains several critical lysine residues that are crucial for binding to the receptors. The C-terminal domain is a lysine rich flexible domain containting two short helix. Using functional assays of different MESD domains, our data demonstrated that this domain is necessary and sufficient for mature LRP6 binding. Objective 2. Identification of the essential domains and critical residues in MESD for MESD's chaperone and escort functions. Combining structural and functional tools, I identified that MESD contains two independent structural/functional domains: The chaperone domain and the escort domain. The N-terminal core structural domain is the chaperone domain that promotes the proper folding and maturation of the BP domain, whereas the C-terminal flexible helical domain is the escort domain which safely-guards the mature LRP5/6 trafficking from the ER to Golgi and prevents pre-mature binding of the physiological ligands of the receptor inside ER. I further mapped critical residues in each domain that are critical for MESD's dual functions. I demonstrated that the mutations in one functional domain do not affect the other functions, suggesting that the MESD's dual biological functions are independent. However, extensive domain-domain interactions are observed in the MESD NMR structure, suggesting the complexity of MESD's structural/functional relationship which raises more fundamental questions of interplays of the dual functions of MESD during the Wnt signaling. Objective 3. Define the binding modes and switch mechanism of MESD and BP domain of LRP6. Using these critical residues as restraints, I built two complexes: the chaperone complex and the escort complex. The chaperone complex is between the chaperone domain of MESD and the BP domain of LRP6, whereas the escort domain is between the escort domain of MESD and the BP domain of LRP6. The chaperone domain of MESD covers the entire surface of the YWTD-end of the BP domain of LRP6, serving as a template for the proper folding of BP domain and preventing the newly translated EGF-repeat to bind to the YWTD-end. As a consequence, the newly translated EGF-repeat can only bind to the opposite end of the BP domain from the YWTD-end. Importantly, this opposite end of BP domain contains a large hydrophobic surface which may cause self-aggregation of the BP domain. The EGF-repeat binds to this large hydrophobic surface to eliminate self aggregation of the BP domain, ensuring proper folding of the receptor. Based on these two complexes, I derived detailed interaction modes of both the chaperone domain and the escort domain of MESD to the BP domain of LRP6. In contrast to the chaperone domain, the escort domain of MESD only covers half of the YWTD-end surface of the BP domain, leaving the other half surface open for interactions. The latter contains three exposed histidine residues which face an array of positively charged lysines of the chaperone domain of MESD. Based on my study, I proposed two switching mechanisms to explain how the chaperone complex switch to the escort complex after the BP domain become properly folded and mature receptor and how the escort complex releases MESD inside the Golgi to allow MESD recycling back to the ER. The hydrophobic interaction between EGF and YWTD β-propeller domain causes proper folding and maturation of the BP domain, triggering the switch between the chaperone complex to the escort complex to safely-guard the mature receptor trafficking from the ER to Golgi. Inside Golgi, the acidic pH caused the explosed histidines on the YWTD-end surface to become positively charged, which serves as a potential "histidine switch" to cause dissociation of MESD from the receptor through electrostatic interaction. In addition, the low pH environment of the Golgi may also cause MESD to adopt a molten globular structure, further causing its releasing from the receptor. (Abstract shortened by UMI.)
Subject Area
Molecular biology,Cellular biology,Biophysics
Recommended Citation
Jianglei Chen,
"Structural and functional studies of MESD, a specialized chaperone protein of LRP5/6 in Wnt signaling"
(January 1, 2009).
ETD Collection for Wayne State University.
Paper AAI3369660.
http://digitalcommons.wayne.edu/dissertations/AAI3369660
View More
