SGX Pharmaceuticals, San Diego, CA 92121, USA.
J Mol Biol. 2010 Feb 19;396(2):406-30. doi: 10.1016/j.jmb.2009.11.051. Epub 2009 Nov 26.
The DeltaF508 mutation in nucleotide-binding domain 1 (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) is the predominant cause of cystic fibrosis. Previous biophysical studies on human F508 and DeltaF508 domains showed only local structural changes restricted to residues 509-511 and only minor differences in folding rate and stability. These results were remarkable because DeltaF508 was widely assumed to perturb domain folding based on the fact that it prevents trafficking of CFTR out of the endoplasmic reticulum. However, the previously reported crystal structures did not come from matched F508 and DeltaF508 constructs, and the DeltaF508 structure contained additional mutations that were required to obtain sufficient protein solubility. In this article, we present additional biophysical studies of NBD1 designed to address these ambiguities. Mass spectral measurements of backbone amide (1)H/(2)H exchange rates in matched F508 and DeltaF508 constructs reveal that DeltaF508 increases backbone dynamics at residues 509-511 and the adjacent protein segments but not elsewhere in NBD1. These measurements also confirm a high level of flexibility in the protein segments exhibiting variable conformations in the crystal structures. We additionally present crystal structures of a broader set of human NBD1 constructs, including one harboring the native F508 residue and others harboring the DeltaF508 mutation in the presence of fewer and different solubilizing mutations. The only consistent conformational difference is observed at residues 509-511. The side chain of residue V510 in this loop is mostly buried in all non-DeltaF508 structures but completely solvent exposed in all DeltaF508 structures. These results reinforce the importance of the perturbation DeltaF508 causes in the surface topography of NBD1 in a region likely to mediate contact with the transmembrane domains of CFTR. However, they also suggest that increased exposure of the 509-511 loop and increased dynamics in its vicinity could promote aggregation in vitro and aberrant intermolecular interactions that impede trafficking in vivo.
核苷酸结合域 1（NBD1）中的 DeltaF508 突变是囊性纤维化跨膜电导调节因子（CFTR）的主要病因。先前对人类 F508 和 DeltaF508 结构域的生物物理研究表明，仅存在局限于残基 509-511 的局部结构变化，且折叠速率和稳定性仅存在微小差异。这些结果令人瞩目，因为 DeltaF508 被广泛认为会破坏结构域折叠，其依据是它阻止 CFTR 从内质网中转运出来。然而，先前报道的晶体结构并非来自匹配的 F508 和 DeltaF508 结构，且 DeltaF508 结构包含了获得足够蛋白可溶性所需的额外突变。在本文中，我们提出了针对这些歧义的进一步的 NBD1 生物物理研究。在匹配的 F508 和 DeltaF508 结构中，对 NBD1 中骨干酰胺（1）H/（2）H 交换率的质谱测量表明，DeltaF508 增加了残基 509-511 及其相邻蛋白片段的骨架动力学，但在 NBD1 的其他部位则没有。这些测量还证实了在晶体结构中表现出可变构象的蛋白片段具有较高的灵活性。我们还呈现了更广泛的人类 NBD1 结构的晶体结构，其中包括一个含有天然 F508 残基的结构，以及在存在更少和不同的可溶性突变的情况下含有 DeltaF508 突变的其他结构。唯一一致的构象差异发生在残基 509-511。该环中的残基 V510 的侧链在所有非 DeltaF508 结构中大多被掩埋，但在所有 DeltaF508 结构中完全暴露在溶剂中。这些结果强化了 DeltaF508 对 NBD1 表面形貌的干扰在可能与 CFTR 跨膜域相互作用的区域中的重要性。然而，它们还表明，509-511 环的暴露增加及其附近的动力学增加可能促进体外聚集和妨碍体内转运的异常分子间相互作用。
