Solution Structure of the LIM-Homeodomain Transcription Factor Complex Lhx3/Ldb1 and the Effects of a Pituitary Mutation on Key Lhx3 Interactions

Lhx3 is a LIM-homeodomain (LIM-HD) transcription factor that regulates neural cell subtype specification and pituitary development in vertebrates, and mutations in this protein cause combined pituitary hormone deficiency syndrome (CPHDS). The recently published structures of Lhx3 in complex with each of two key protein partners, Isl1 and Ldb1, provide an opportunity to understand the effect of mutations and posttranslational modifications on key protein-protein interactions. Here, we use small-angle X-ray scattering of an Ldb1-Lhx3 complex to confirm that in solution the protein is well represented by our previously determined NMR structure as an ensemble of conformers each comprising two well-defined halves (each made up of LIM domain from Lhx3 and the corresponding binding motif in Ldb1) with some flexibility between the two halves. NMR analysis of an Lhx3 mutant that causes CPHDS, Lhx3(Y114C), shows that the mutation does not alter the zinc-ligation properties of Lhx3, but appears to cause a structural rearrangement of the hydrophobic core of the LIM2 domain of Lhx3 that destabilises the domain and/or reduces the affinity of Lhx3 for both Ldb1 and Isl1. Thus the mutation would affect the formation of Lhx3-containing transcription factor complexes, particularly in the pituitary gland where these complexes are required for the production of multiple pituitary cell types and hormones.Lhx3是一类LIM同源结构域（LIM-homeodomain, LIM-HD）转录因子，在脊椎动物中调控神经细胞亚型特化与垂体发育；该蛋白的突变会引发联合垂体激素缺乏综合征（combined pituitary hormone deficiency syndrome, CPHDS）。近期发表的Lhx3分别与两个关键蛋白伴侣Isl1和Ldb1结合的复合物结构，为理解突变与翻译后修饰如何影响关键蛋白-蛋白相互作用提供了研究契机。本研究通过对Ldb1-Lhx3复合物进行小角X射线散射（small-angle X-ray scattering）分析，证实溶液状态下该蛋白的构象可由我们此前解析的核磁共振（nuclear magnetic resonance, NMR）结构很好地表征：该结构以构象异构体集合的形式呈现，包含两个结构明晰的结构模块，分别由Lhx3的LIM结构域与Ldb1中对应的结合基序构成，且两个模块之间存在一定柔性。针对引发CPHDS的Lhx3突变体Lhx3(Y114C)的核磁共振分析结果显示，该突变并未改变Lhx3的锌配位特性，但似乎引发了Lhx3的LIM2结构域疏水核心的结构重排，进而使该结构域不稳定，或降低Lhx3与Ldb1、Isl1的结合亲和力。综上，该突变会影响含Lhx3的转录因子复合物的形成，尤其在垂体组织中，这类复合物是多种垂体细胞类型与激素生成所必需的。