Cooperation between Fixed and Low pH-Inducible Interfaces Controls Lipoprotein Release by the LDL Receptor  Natalia Beglova, Hyesung Jeon, Carl Fisher, Stephen C. Blacklow  Molecular Cell  Volume 16, Issue 2, Pages 281-292 (October 2004) DOI: 10.1016/j.molcel.2004.09.038

Figure 1 Ribbon Trace of the LDL Receptor Structure Determined at Endosomal pH The boxes enclose two interdomain interfaces that may be regulated by the change in pH that occurs upon moving from the cell surface to the endosome. The coordinates used for the figure are from PDB accession code 1N7D (Rudenko et al., 2002). Molecular Cell 2004 16, 281-292DOI: (10.1016/j.molcel.2004.09.038)

Figure 2 The Propeller Domain Is Required for Low pH-Mediated Ligand Release (A) Schematic diagram of the native LDL receptor and three deletion variants. (B) Flow cytometry plots. The amount of fluorescence from diI-LDL bound to cells expressing different receptor variants was detected before and after treatment with low pH buffer. The native receptor releases bound LDL (blue, pH 7.4) upon exposure to endosomal pH (red, pH 5.3), whereas receptors lacking the propeller domain do not. (C) Quantification of the fraction of bound LDL released after exposure to low pH. Molecular Cell 2004 16, 281-292DOI: (10.1016/j.molcel.2004.09.038)

Figure 3 Propeller Domain-Swap Experiments (A) Schematic of the native LDL receptor highlighting the propeller-EGF_C domain pair in gray. (B) Alignment of residues 501-638 of the LDLR propeller domain with the corresponding regions of LRP-6 propeller 2 and LRP6 propeller 4. LDLR residues in the long-range interface with LA4 and LA5 are indicated with asterisks and colored by residue type. (C) Flow cytometry plots, showing the amount of fluorescence from diI-LDL bound to cells before and after treatment with low pH buffer. (D) Quantification of the fraction of bound LDL released after exposure to low pH. Molecular Cell 2004 16, 281-292DOI: (10.1016/j.molcel.2004.09.038)

Figure 4 Histidine Residues in Interface I Are Required for Release of LDL at Low pH (A) Close-up view of interface I. The three interface histidines lie within the box at the center of the interface (B) Flow cytometry plots. Mutation of each individual histidine residue modestly compromises the ability of the receptor to release bound LDL at low pH, whereas simultaneous substitution of all three histidine residues prevents release of bound LDL. (C) Quantification of the fraction of bound LDL released after exposure to low pH. Molecular Cell 2004 16, 281-292DOI: (10.1016/j.molcel.2004.09.038)

Figure 5 Structure and dynamics of the LA7-EGF_A domain pair at neutral pH (A) Plot of hNOE as a function of residue number for the LA7-EGF_A pair. The hNOE profile of the LA7-EGF_A pair is the same at pH 5.2, 6.5, and 7.0, indicating the existence of an interface that does not vary with pH. (B) Best fit superposition of the 15 lowest energy neutral pH NMR structures of the LA7-EGF_A domain pair. The Cα trace and bound calcium ions are shown. (C) Ribbon trace of the LA7-EGF_A structure. The LA7 ribbon is blue, and the EGF_A ribbon is red. Disulfide bonds and calcium-coordinating side chains are illustrated in CPK colors. Bound calcium ions are yellow. (D) Close-up stereo view of the interface seen in the neutral pH NMR structure. Residues in a hydrophobic cluster that comprises the interface are labeled. Labels of residues harboring FH mutations are boxed. (E) Best fit superposition (stereo) of the neutral pH NMR structure (blue) onto the corresponding region of the crystal structure determined at endosomal pH (yellow). Molecular Cell 2004 16, 281-292DOI: (10.1016/j.molcel.2004.09.038)

Figure 6 Effect of Glycine Substitutions in the Linker Connecting LA7 to EGF_A (A) Two Gly mutations in the linker decouple the domains. Comparison of the HSQC spectra of native LA7-EGF_A (blue peaks) and the LA7-EGF_A mutant (red peaks) at neutral pH. Perturbed residues are labeled. (B) Chemical shift perturbations (detected by the HSQC spectra in A) mapped onto the structure of LA7-EGF_A. Mutated residues are colored red; perturbed residues are colored blue. (C) Flow cytometry plots, showing the amount of fluorescence from diI-LDL bound to cells before and after treatment with low pH buffer. Creation of the flexible linker interferes with release of bound LDL. (D) Specific release activity of the mutant receptor, normalized to the amount released by the native LDLR. Molecular Cell 2004 16, 281-292DOI: (10.1016/j.molcel.2004.09.038)

Figure 7 Schematic Proposing How Fixed and Flexible Connections among Domains Cooperate to Permit Interconversion between Open and Closed Conformations in Response to pH LA7, EGF_A, and EGF_B constitute a rigid scaffold that is invariant with pH. Wavy lines identify modules linked by connections likely to be flexible at the indicated pH, with freedom of movement for the ligand binding modules at neutral pH increasing as a function of distance from the rigid scaffold. LA modules are green, EGF-like modules are yellow, and the β propeller domain is pink. Molecular Cell 2004 16, 281-292DOI: (10.1016/j.molcel.2004.09.038)