Springer S, Doring K, Skipper J C, Townsend A R, Cerundolo V
Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, U.K.
Biochemistry. 1998 Mar 3;37(9):3001-12. doi: 10.1021/bi9717441.
Major histocompatibility complex (MHC) class I molecules bind peptides in the endoplasmic reticulum (ER). For this binding reaction, when performed in vitro, widely differing association rates have been reported. We have expressed empty soluble H-2Db class I molecules in Chinese hamster ovary (CHO) cells and generated complete sets of association, dissociation, and equilibrium constants of unmodified peptides using tritium-labeled peptides and stopped-flow fluorescence spectroscopy. We find that (i) the transition midpoint of temperature denaturation (Tm) of the protein is shifted from 30.5 to 56 degrees C upon the binding of a high-affinity peptide. (ii) With the peptide SV-324-332 (sequence FAPGNYPAL) at 4 degrees C, the dissociation rate constant of 1.02 x 10(-5) s-1 and an equilibrium constant of 8.5 x 10(7) M-1 predict an association rate constant of 870 M-1 s-1 for a simple one-step model of binding. (iii) In contrast, binding of this peptide proceeds much faster, with 1.4 x 10(6) M-1 s-1. These "mismatch kinetics" suggest that peptide binding occurs in several steps, most likely via a conformational rearrangement of the peptide binding groove. The structure of the peptide-class I complex at the time-point of peptide recognition may therefore be different from the equilibrium crystal structures. (iv) Association of modified peptides, in the presence of detergent, or above the Tm of the empty molecule is considerably slower. This might explain why fast on-rates have not been observed in previous studies.
主要组织相容性复合体（MHC）I类分子在内质网（ER）中结合肽段。对于这种体外进行的结合反应，已报道了差异很大的缔合速率。我们在中国仓鼠卵巢（CHO）细胞中表达了空的可溶性H-2Db I类分子，并使用氚标记的肽段和停流荧光光谱法生成了未修饰肽段的完整缔合、解离和平衡常数集。我们发现：（i）高亲和力肽段结合后，蛋白质的温度变性转变中点（Tm）从30.5℃移至56℃。（ii）在4℃下，对于肽段SV-324-332（序列FAPGNYPAL），解离速率常数为1.02×10⁻⁵ s⁻¹，平衡常数为8.5×10⁷ M⁻¹，对于简单的一步结合模型，预测缔合速率常数为870 M⁻¹ s⁻¹。（iii）相比之下，该肽段的结合速度要快得多，为1.4×10⁶ M⁻¹ s⁻¹。这些“错配动力学”表明肽段结合分几步进行，很可能是通过肽段结合槽的构象重排。因此，在肽段识别时间点的肽段-I类复合体结构可能与平衡晶体结构不同。（iv）在存在去污剂的情况下或高于空分子的Tm时，修饰肽段的缔合要慢得多。这可能解释了为什么在先前的研究中未观察到快速的结合速率。
