Yamamoto Taichi, Chen Hsuan-Chih, Guigard Emmanuel, Kay Cyril M, Ryan Robert O
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.
Biochemistry. 2008 Nov 4;47(44):11647-52. doi: 10.1021/bi801117t. Epub 2008 Oct 11.
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.
低密度脂蛋白受体（LDLR）释放配体的过程被假定涉及一种“组氨酸开关”诱导的分子内重排，从而释放结合的配体。在与荧光标记的变体载脂蛋白E N端结构域（apoE-NT）进行的配体结合实验中，使用了重组可溶性低密度脂蛋白受体（sLDLR）。结合情况通过荧光共振能量转移（FRET）进行监测，FRET是从sLDLR中激发的色氨酸残基转移到共价连接到无色氨酸apoE3-NT的外部荧光团。在与野生型（WT）sLDLR的结合实验中，随着pH值降低，FRET依赖的AEDANS荧光减弱。为了研究sLDLR中His190、His562和His586在pH依赖的配体结合和释放中的作用，进行了定点诱变研究。与WT sLDLR相比，三重His→Ala突变体sLDLR表现出减弱的pH依赖的配体结合以及作为低pH函数的配体释放水平降低。当这些组氨酸残基被赖氨酸取代时，其带正电荷的侧链在此pH范围内不会电离，在所有pH值下配体结合几乎都被消除。当检查连续His到Lys突变体时，证据表明His562和His586协同发挥作用。当pH从7降低到5时，WT sLDLR的沉降系数增加，而三重赖氨酸突变体受体或His562Lys/His586Lys双突变体受体则没有这种变化。这些数据支持存在一种隐蔽的、依赖组氨酸侧链电离的替代配体，其通过构象重组调节配体释放。
