Abstract
The LDL receptor (LDL-R) mediates cholesterol metabolism in humans by binding and internalizing cholesterol transported by LDL. Several different molecular mechanisms have been proposed for the binding of LDL to LDL-R at neutral plasma pH and for its release at acidic endosomal pH. The crystal structure of LDL-R at acidic pH shows that the receptor folds back on itself in a closed form, obscuring parts of the ligand binding domain with the epidermal growth factor (EGF)-precursor homology domain. We have used a structure-based site-directed mutagenesis approach to examine 12 residues in the extracellular domain of LDL-R for their effect on LDL binding and release. Our studies show that the interface between the ligand binding domain and the EGF-precursor homology domain seen at acidic pH buries residues mediating both LDL binding and release. Our results are consistent with an alternative model of LDL-R whereby multiple modules of the extracellular domain interact with LDL at neutral pH, concurrently positioning key residues so that at acidic pH the LDL-R:LDL interactions become unfavorable, triggering release. After LDL release, the closed form of LDL-R may target its return to the cell surface.
Highlights
The LDL receptor (LDL-R) mediates cholesterol metabolism in humans by binding and internalizing cholesterol transported by LDL
We focused on residues that appear important to maintain the closed conformation of low density lipoprotein receptor (LDL-R) seen in the crystal structure at acidic pH and examined their effect on LDL binding and release
DNA mutagenesis Mutants of the human LDL receptor extracellular domain in pFastBac1 were generated with the Quickchange kit (Stratagene)
Summary
The LDL receptor (LDL-R) mediates cholesterol metabolism in humans by binding and internalizing cholesterol transported by LDL. The crystal structure of LDL-R at acidic pH shows that the receptor folds back on itself in a closed form, obscuring parts of the ligand binding domain with the epidermal growth factor (EGF)-precursor homology domain. Our studies show that the interface between the ligand binding domain and the EGF-precursor homology domain seen at acidic pH buries residues mediating both LDL binding and release. The extracellular domain of LDL-R contains seven cysteine-rich repeats (R1–R7, called LA1–LA7), two epidermal growth factor (EGF)-like repeats (EGF-A and EGF-B), a -propeller domain, and a third EGF-like repeat (EGF-C) tethered to the cell surface by a single transmembrane segment [8,9,10]. The side chains of Asp196, Asp200, Asp206, and Glu207 and the carbonyl oxygens of Trp193 and Gly198 coordinate the Ca2+ ion within R5, a cysteine-rich repeat essential for all lipoprotein binding [11]. The analogous Ca2+-binding residues in R4, Asp147, Asp151, Asp157, and Glu158 provide side chains, and Trp144 and Asp149 provide backbone carbonyl oxygens
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
