Molecular studies of pH-dependent ligand interactions with the low-density lipoprotein receptor.
The release of ligand from the low-density lipoprotein receptor (LDLR) has been postulated to involve a "histidine switch"-induced intramolecular rearrangement that discharges bound ligand. A recombinant soluble low-density lipoprotein receptor (sLDLR) was employed in ligand binding experiments with a fluorescently tagged variant apolipoprotein E N-terminal domain (apoE-NT). Binding was monitored as a function of fluorescence resonance energy transfer (FRET) from excited Trp residues in sLDLR to an extrinsic fluorophore covalently attached to Trp-null apoE3-NT. In binding experiments with wild-type (WT) sLDLR, FRET-dependent AEDANS fluorescence decreased as the pH was lowered. To investigate the role of His190, His562, and His586 in sLDLR in pH-dependent ligand binding and discharge, site-directed mutagenesis studies were performed. Compared to WT sLDLR, triple His --> Ala mutant sLDLR displayed attenuated pH-dependent ligand binding and a decreased level of ligand release as a function of low pH. When these His residues were substituted for Lys, the positively charged side chain of which does not ionize over this pH range, ligand binding was nearly abolished at all pH values. When sequential His to Lys mutants were examined, the evidence suggested that His562 and His586 function cooperatively. Whereas the sedimentation coefficient for WT sLDLR increased when the pH was reduced from 7 to 5, no such change occurred in the case of the triple Lys mutant receptor or a His562Lys/His586Lys double mutant receptor. The data support the existence of a cryptic, histidine side chain ionization-dependent alternative ligand that modulates ligand discharge via conformational reorganization.
Center for Prevention of Obesity, Diabetes and Cardiovascular Disease, Children's Hospital Oakland Research Institute, 5700 Martin Luther King Jr. Way, Oakland, California 94609, USA.
Taichi
T
Yamamoto
Hsuan-Chih
HC
Chen
Emmanuel
E
Guigard
Cyril M
CM
Kay
Robert O
RO
Ryan
D019943:Amino Acid Substitution; D001057:Apolipoproteins E / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D006639:Histidine / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011973:Receptors, LDL / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D014364:Tryptophan / Q000737:chemistry
D001057:Apolipoproteins E; D008024:Ligands; D010446:Peptide Fragments; D011973:Receptors, LDL; D011994:Recombinant Proteins; D006639:Histidine; D014364:Tryptophan