The long-term objective of this project is to provide a thorough understanding of how the low-density lipoprotein receptor (LDLR) folds into its native structure and recognizes its lipoprotein ligands. The LDLR is the primary mechanism for uptake of plasma cholesterol into cells. When the LDLR is unable to clear cholesterol-containing lipoproteins sufficiently from the blood, an elevated plasma cholesterol level results. A high plasma cholesterol level is a major risk for heart disease, the leading cause of death in the United States. Over 150 different mutations of the LDLR give rise to familial hypercholesterolemia (FH), which is characterized clinically by an elevated concentration of plasma LDL and cholesterol. Detailed structural and biochemical studies of LDLR-lipoprotein interactions have been elusive, because the receptor protein is large and membrane bound. However, the ligand-binding domain of the LDLR is composed of a series of autonomously structured, non-identical tandem repeats that can be studied in isolation from the rest of the receptor. In previous work, the PI has shown that a critical repeat (repeat 5) within the ligand-binding domain of the receptor can be folded to its native structure after expression in bacteria, and that calcium is required for proper folding of this domain. During the period of grant support, he plans (1) to determine the principles that govern proper folding of this prototypic repeat of the LDLR ligand-binding domain into its native structure, and (2) to elucidate the detailed molecular basis for ligand-binding by the LDLR, relying on the folding studies to identify potential sites of receptor-ligand interaction. This work will have broad implications for the mechanism of ligand interactions. This work will have broad implications for the mechanism of ligand recognition by the wide variety of proteins that contain structural motifs homologous to those found in the ligand-binding domain of the LDLR, including proteins implicated in G-protein couple signaling, brain development, and the immune response. Understanding the basis for ligand recognition by LDL-A repeats may ultimately allow the alteration of the ligand-binding properties of LDL-A repeats to create novel receptors for arbitrary target ligands. Eventually, small molecules may be identified which suppress the folding defects in some of the FH mutations, and which might serve as therapeutics for patients with FH.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL061001-03
Application #
6151376
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1998-02-01
Project End
2002-01-31
Budget Start
2000-02-01
Budget End
2001-01-31
Support Year
3
Fiscal Year
2000
Total Cost
$216,256
Indirect Cost
Name
Brigham and Women's Hospital
Department
Type
DUNS #
071723621
City
Boston
State
MA
Country
United States
Zip Code
02115
Estrada, Kristine; Fisher, Carl; Blacklow, Stephen C (2008) Unfolding of the RAP-D3 helical bundle facilitates dissociation of RAP-receptor complexes. Biochemistry 47:1532-9
Blacklow, Stephen C (2007) Versatility in ligand recognition by LDL receptor family proteins: advances and frontiers. Curr Opin Struct Biol 17:419-26
Koduri, Vidyasagar; Blacklow, Stephen C (2007) Requirement for natively unstructured regions of mesoderm development candidate 2 in promoting low-density lipoprotein receptor-related protein 6 maturation. Biochemistry 46:6570-7
Lee, Donghan; Walsh, Joseph D; Mikhailenko, Irina et al. (2006) RAP uses a histidine switch to regulate its interaction with LRP in the ER and Golgi. Mol Cell 22:423-30
Fisher, Carl; Beglova, Natalia; Blacklow, Stephen C (2006) Structure of an LDLR-RAP complex reveals a general mode for ligand recognition by lipoprotein receptors. Mol Cell 22:277-83
Abdul-Aziz, Dunia; Fisher, Carl; Beglova, Natalia et al. (2005) Folding and binding integrity of variants of a prototype ligand-binding module from the LDL receptor possessing multiple alanine substitutions. Biochemistry 44:5075-85
Jeon, Hyesung; Blacklow, Stephen C (2005) Structure and physiologic function of the low-density lipoprotein receptor. Annu Rev Biochem 74:535-62
Stolt, Peggy C; Chen, Ying; Liu, Pingsheng et al. (2005) Phosphoinositide binding by the disabled-1 PTB domain is necessary for membrane localization and Reelin signal transduction. J Biol Chem 280:9671-7
Beglova, Natalia; Jeon, Hyesung; Fisher, Carl et al. (2004) Cooperation between fixed and low pH-inducible interfaces controls lipoprotein release by the LDL receptor. Mol Cell 16:281-92
Boswell, Emma J; Jeon, Hyesung; Blacklow, Stephen C et al. (2004) Global defects in the expression and function of the low density lipoprotein receptor (LDLR) associated with two familial hypercholesterolemia mutations resulting in misfolding of the LDLR epidermal growth factor-AB pair. J Biol Chem 279:30611-21

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