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Current research is designed to understand molecular details of the interaction between human low-density lipoprotein receptor (LDLR) and ligand/regulatory proteins, including apolipoprotein E (apoE) and proprotein convertase subtilisin kexin 9 (PCSK9). To study this, we are employing discriminating receptor/ligand binding assays, including a fluorescence resonance energy transfer (FRET) assay and surface plasmon resonance (SPR) spectroscopy. Ongoing projects include the following:

Characterization of pH dependent conformational change in LDLR:
In 2002, the X-ray crystal structure of a large extra-cellular portion of human LDLR at endosomal pH (pH = 5.3) was reported (Figure 1).

Figure 1. LDLR extra-cellular domain structure. LDL-A repeats are labeled LA2-LA7. EGF repeats are labeled A, B and C. Note the interaction between the ß-propeller domain (cyan) and LDL-A repeats, LA4 and LA5. PDB code 1N7D.

A striking finding of this structure is that the β-propeller motif present in the EGF precursor homology domain makes close contact with LA4 and LA5. Since the structure was determined at pH 5.3 and LA4 and LA5 are critical for ligand interaction, these data provide a structural rationale for how LDLR releases bound ligand within the endosome. By acting as a pH sensitive, intra-molecular alternate ligand, the β-propeller motif is postulated to displace bound ligand as the pH drops from 7.4 in the cytosol to 5.3 in the endosomal compartment. In our project, the postulated pH dependent conformational change of LDLR will be characterized using intra-molecular FRET to report on the conformational status of the N- and C-termini of sLDLR in solution.

Studies of the ability of PCSK9 to modulate the interaction between LDLR and apoE: It is well known that PCSK9 regulates plasma cholesterol levels. Studies have demonstrated that PCSK9 binding to LDLR on the cell surface induces LDLR protein degradation in lysosomes rather than recycling to the cell surface. Although numerous studies have been conducted on the interaction between these two proteins, the molecular mechanism of PCSK9 mediated cholesterol lowering effect is still not clear. We hypothesize that PCSK9 has multiple functions in terms of lowering cholesterol levels in plasma. We are examining whether PCSK9 will interfere with apoE binding to LDLR. The PCSK9 domain that is responsible for the inhibition on the apoE binding to LDLR will be determined.

Studies of the molecular interaction between LDLR and PCSK9: The ability of PCSK9 to interact with the LDLR at endosomal pH prevents the recycling of the LDLR, and the PCSK9/LDLR complex shuttles to the lysozome, where degradation of the LDLR occurs. However the molecular mechanism of pH dependent binding affinities of PCSK9 toward LDLR remains to be determined. The crystal structures of the PCSK9 indicated no large conformational change occurs in this protein across a wide pH range (pH 4.6 ~ 10.5). There are 14 histidine residues in the C-terminal domain of PCSK9. The crystal structure of the C-terminal domain reveals an overall large number of histidine residues (Figure 2).

Figure 2. Overall of the PCSK9 structure. Prodomain, catalytic domain or C-terminal domain are colored in cyan, yellow or green, respectively. The cluster of histidine amino acid residues on the C-terminal domain are emphasized.

Histidine residues have been shown to act as switches in pH-dependent protein-protein interactions. So far, natural missense mutations in the C-terminal domain of PCSK9 induce either hypercholesterolemia (E482G, F515L, and H553R) or hypocholesterolemia (Q554E and Q619P). Moreover, it was also demonstrated that the C-terminal domain of PCSK9 is critical for co-localization of PCSK9 and LDLR in cell culture We hypothesize that stabilization of the binding interaction between PCSK9 and LDLR is related to low pH induced protonation of key histidine residues in the C-terminal domain of PCSK9.

Projects are conducted in collaboration with Robert O. Ryan (CHORI).

Research Assistants

Heidi Choi
Jennifer Matsuda


revised: Wednesday, March 25, 2009 5:05 PM


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