Natarajan S K, Stern L J, Sadegh-Nasseri S
Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
J Immunol. 1999 Mar 15;162(6):3463-70.
Certain class II MHC-peptide complexes are resistant to SDS-induced dissociation. This property, which has been used as an in vivo as well as an in vitro peptide binding assay, is not understood at the molecular level. Here we have investigated the mechanistic basis of SDS stability of HLA-DR1 complexes by using a biosensor-based assay and SDS-PAGE with a combination of wild-type and mutant HLA-DR1 and variants of hemagglutinin peptide HA306-318. Experiments with wild-type DR1 along with previously published results establish that the SDS-stable complexes are formed only when the hydrophobic pocket 1 (P1) is occupied by a bulky aromatic (Trp, Phe, Tyr) or an aliphatic residue (Met, Ile, Val, Leu). To further explore whether the SDS sensitivity is primarily due to the exposed hydrophobic regions, we mutated residue beta Gly86 at the bottom of P1 to tyrosine, presumably reducing the depth of the pocket and the exposure of hydrophobic residues and increasing the contacts between subunits. In direct contrast to wild-type DR1, the peptide-free mutant DR1 exists as an alpha/beta heterodimer in SDS. Moreover, the presence of a smaller hydrophobic residue, such as alanine, as P1 anchor with no contribution from any other anchor is sufficient to enhance the SDS stability of the mutant complexes, demonstrating that the basis of SDS resistance may be localized to P1 interactions. The good correlation between SDS sensitivity and the exposure of hydrophobic residues provides a biochemical rationale for the use of this assay to investigate the maturation of class II molecules and the longevity of the complexes.
某些II类主要组织相容性复合体（MHC）-肽复合物对十二烷基硫酸钠（SDS）诱导的解离具有抗性。这一特性已被用作体内和体外肽结合试验，但在分子水平上尚未被理解。在此，我们通过使用基于生物传感器的分析方法以及结合野生型和突变型HLA-DR1以及血凝素肽HA306-318变体的SDS聚丙烯酰胺凝胶电泳（SDS-PAGE），研究了HLA-DR1复合物SDS稳定性的机制基础。野生型DR1的实验以及先前发表的结果表明，只有当疏水口袋1（P1）被一个大的芳香族（色氨酸、苯丙氨酸、酪氨酸）或脂肪族残基（甲硫氨酸、异亮氨酸、缬氨酸、亮氨酸）占据时，才会形成SDS稳定的复合物。为了进一步探究SDS敏感性是否主要归因于暴露的疏水区域，我们将P1底部的β Gly86残基突变为酪氨酸，推测这会减少口袋的深度和疏水残基的暴露，并增加亚基之间的接触。与野生型DR1形成直接对比的是，无肽突变型DR1在SDS中以α/β异二聚体形式存在。此外，作为P1锚定物的较小疏水残基（如丙氨酸）的存在，且没有任何其他锚定物的贡献，就足以增强突变复合物的SDS稳定性，这表明SDS抗性的基础可能定位于P1相互作用。SDS敏感性与疏水残基暴露之间的良好相关性为使用该试验来研究II类分子的成熟以及复合物的寿命提供了生化依据。
