Abstract: TH-PO544
Structures of Megalin/LRP2 at Extracellular and Endosomal pH
Session Information
- Pathology and Lab Medicine
November 03, 2022 | Location: Exhibit Hall, Orange County Convention Center‚ West Building
Abstract Time: 10:00 AM - 12:00 PM
Category: Pathology and Lab Medicine
- 1700 Pathology and Lab Medicine
Authors
- Beenken, Andrew, Columbia University Irving Medical Center, New York, New York, United States
- Barasch, Jonathan M., Columbia University Irving Medical Center, New York, New York, United States
- Shapiro, Lawrence, Columbia University, New York, New York, United States
Background
The low-density lipoprotein receptor-related protein 2 (LRP2 or megalin) mediates endocytosis in species ranging from worms to humans and is implicated in diseases of the kidney and brain. The structural basis for its function remains one of the great unknowns of kidney biology. To understand the mechanisms of its ligand-binding and trafficking, we have solved structures of LRP2 at extracellular and endosomal pH.
Methods
We purified endogenous LRP2 from mouse kidney by isolating proximal tubule apical membrane, solubilizing in detergent, and purifying LRP2 from solubilized membrane using ion exchange chromatography and gel filtration. Protein was frozen in vitreous ice and then imaged with a Titan Krios microscope. Data was processed to yield cryo electron microscopy (cryo-EM) maps with resolutions of 2.8Å and 3.0Å for LRP2 at extracellular and endosomal pH, respectively.
Results
At each pH, LRP2 adopts a homodimeric assembly stabilized by pH-dependent interfaces (Figure). A significant change in tertiary structure between the two structures is mediated by pH-senstive Ca2+-coordinating sites. At endosomal pH, Ca2+ dissociates from multiple sites including both homodimer and intramolecular interfaces, enabling large-scale domain rearrangements that bury ligand-binding sites. A subset of human Donnai-Barrow loss of function missense mutations appear to impair homodimer assembly of LRP2 by perturbing pH-sensitive interfaces.
Conclusion
This work lays the foundation for further understanding the function and mechanism of the critical proximal tubule endocytic receptor, LRP2. In particular, our structural data highlights the central importance of pH-sensitive Ca2+ switches in LRP2’s structural transitions during cellular trafficking. Based on analysis of our structures and published genetic data, we unexpectedly find that LRP2 homodimerization appears conserved, necessitating a re-evaluation of the structural biology of related receptors, including LRP1 and LDLR.
Structures of the LRP2 homodimer at endosomal and extracellular pH
Funding
- Other NIH Support