Department of Chemistry and Biochemistry, Freie Universität Berlin , 14195 Berlin, Germany.
Biochemistry. 2014 Oct 21;53(41):6603-14. doi: 10.1021/bi500548f. Epub 2014 Oct 10.
Niemann-Pick Type C disease is characterized by disrupted lipid trafficking within the late endosomal (LE)/lysosomal (Lys) cellular compartments. Cholesterol transport within the LE/Lys is believed to take place via a concerted hand-off mechanism in which a small (131aa) soluble cholesterol binding protein, NPC2, transfers cholesterol to the N-terminal domain (NTD) of a larger (1278aa) membrane-bound protein, NPC1(NTD). The transfer is thought to occur through the formation of a stable intermediate complex NPC1(NTD)-NPC2, in which the sterol apertures of the two proteins align to allow passage of the cholesterol molecule. In the working model of the NPC1(NTD)-NPC2 complex, the sterol apertures are aligned, but the binding pockets are bent with respect to one another. In order for cholesterol to slide from one binding pocket to the other, a conformational change must occur in the proteins, in the ligand, or in both. Here, we investigate the possibility that the ligand undergoes a conformational change, or isomerization, to accommodate the bent transfer pathway. To understand what structural factors influence the isomerization rate, we calculate the energy barrier to cholesterol isomerization in both the NPC1(NTD) and NPC2 binding pockets. Here, we use a combined quantum mechanical/molecular mechanical (QM/MM) energy function to calculate the isomerization barrier within the native NPC1(NTD) and NPC2 binding pockets before protein-protein docking as well as in the binding pockets of the NPC1(NTD)-NPC2 complex after docking has occurred. The results indicate that cholesterol isomerization in the NPC2 binding pocket is energetically favorable, both before and after formation of the NPC1(NTD)-NPC2 complex. The NPC1(NTD) binding pocket is energetically unfavorable to conformational rearrangement of the hydrophobic ligand because it contains more water molecules near the ligand tail and amino acids with polar side chains. For three NPC1(NTD) mutants investigated, L175Q/L176Q, L175A/L176A, and E191A/Y192A, the isomerization barriers were all found to be higher than the barrier calculated in the NPC2 binding pocket. Our results indicate that cholesterol isomerization in the NPC2 binding pocket, either before or after docking, may ensure an efficient transfer of cholesterol to NPC1(NTD).
尼曼-匹克 C 型疾病的特征是晚期内体（LE）/溶酶体（Lys）细胞区室中的脂质运输紊乱。据信胆固醇在 LE/Lys 中的运输是通过一种协同的手递机制进行的，在该机制中，一种小的（131aa）可溶性胆固醇结合蛋白 NPC2 将胆固醇转移到较大的（1278aa）膜结合蛋白 NPC1（NTD）的 N 端结构域（NTD）。据认为，这种转移是通过形成一个稳定的中间复合物 NPC1（NTD）-NPC2 来实现的，其中两个蛋白质的固醇孔道对齐，以允许胆固醇分子通过。在 NPC1（NTD）-NPC2 复合物的工作模型中，固醇孔道对齐，但相对于彼此，结合口袋弯曲。为了使胆固醇从一个结合口袋滑到另一个结合口袋，蛋白质、配体或两者都必须发生构象变化。在这里，我们研究了配体是否发生构象变化或异构化以适应弯曲的转移途径的可能性。为了了解哪些结构因素会影响异构化速率，我们计算了 NPC1（NTD）和 NPC2 结合口袋中胆固醇异构化的能垒。在这里，我们使用量子力学/分子力学（QM/MM）能量函数来计算 NPC1（NTD）和 NPC2 结合口袋中原位 NPC1（NTD）和 NPC2 结合口袋的异构化势垒，以及在 NPC1（NTD）-NPC2 复合物形成后结合口袋的异构化势垒。结果表明，NPC2 结合口袋中的胆固醇异构化在形成 NPC1（NTD）-NPC2 复合物之前和之后都是能量有利的。NPC1（NTD）结合口袋不利于疏水配体的构象重排，因为它在配体尾部附近含有更多的水分子和带有极性侧链的氨基酸。对于三种研究的 NPC1（NTD）突变体 L175Q/L176Q、L175A/L176A 和 E191A/Y192A，发现异构化势垒均高于 NPC2 结合口袋中计算出的势垒。我们的结果表明，NPC2 结合口袋中的胆固醇异构化，无论是在对接之前还是之后，都可能确保胆固醇有效地转移到 NPC1（NTD）。
