Rupert P B, Mollah A K, Mossing M C, Matthews B W
Institute of Molecular Biology Howard Hughes Medical Institute and Department of Physics 1229, University of Oregon, Eugene, OR 97403, USA.
J Mol Biol. 2000 Mar 3;296(4):1079-90. doi: 10.1006/jmbi.1999.3498.
It was previously shown that the Cro repressor from phage lambda, which is a dimer, can be converted into a stable monomer by a five-amino acid insertion. Phe58 is the key residue involved in this transition, switching from interactions which stabilize the dimer to those which stabilize the monomer. Structural studies, however, suggested that Phe58 did not penetrate into the core of the monomer as well as it did into the native dimer. This was strongly supported by the finding that certain core-repacking mutations, including in particular, Phe58-->Trp, increased the stability of the monomer. Unexpectedly, the same substitution also increased the stability of the native dimer. At the same time it decreased the affinity of the dimer for operator DNA. Here we describe the crystal structures of the Cro F58W mutant, both as the monomer and as the dimer. The F58W monomer crystallized in a form different from that of the original monomer. In contrast to that structure, which resembled the DNA-bound form of Cro, the F58W monomer is closer in structure to wild-type (i.e. non-bound) Cro. The F58W dimer also crystallizes in a form different from the native dimer but has a remarkably similar overall structure which tends to confirm the large changes in conformation of Cro on binding DNA. Introduction of Trp58 perturbs the position occupied by the side-chain of Arg38, a DNA-contact residue, providing a structural explanation for the reduction in DNA-binding affinity. The improved thermal stability is seen to be due to the enhanced solvent transfer free energy of Trp58 relative to Phe58, supplemented in the dimer structure, although not the monomer, by a reduction in volume of internal cavities.
先前的研究表明，来自噬菌体λ的Cro阻遏蛋白是一种二聚体，通过插入五个氨基酸可转化为稳定的单体。苯丙氨酸58（Phe58）是这一转变过程中的关键残基，它从稳定二聚体的相互作用转变为稳定单体的相互作用。然而，结构研究表明，Phe58在单体核心中的嵌入程度不如在天然二聚体中。这一观点得到了以下发现的有力支持：某些核心重排突变，特别是Phe58→Trp，增加了单体的稳定性。出乎意料的是，相同的取代也增加了天然二聚体的稳定性。同时，它降低了二聚体与操纵基因DNA的亲和力。在此，我们描述了Cro F58W突变体作为单体和二聚体的晶体结构。F58W单体以与原始单体不同的形式结晶。与类似Cro与DNA结合形式的结构不同，F58W单体在结构上更接近野生型（即未结合）Cro。F58W二聚体也以与天然二聚体不同的形式结晶，但具有非常相似的整体结构，这倾向于证实Cro在结合DNA时构象的巨大变化。引入色氨酸58（Trp58）扰乱了作为DNA接触残基的精氨酸38（Arg38）侧链所占据的位置，为DNA结合亲和力的降低提供了结构上的解释。热稳定性的提高被认为是由于Trp58相对于Phe58的溶剂转移自由能增强，在二聚体结构中，尽管单体结构中没有，但内部空腔体积的减小也起到了补充作用。
