Bernat Bryan, Pal Gabor, Sun Miao, Kossiakoff Anthony A
Department of Biochemistry and Molecular Biology and Institute for Biophysical Dynamics, Cummings Life Sciences Center, University of Chicago, 920 East 58th Street, Chicago, IL 60637, USA.
Proc Natl Acad Sci U S A. 2003 Feb 4;100(3):952-7. doi: 10.1073/pnas.0235023100. Epub 2003 Jan 27.
Signaling in the human growth hormone (hGH)-human GH receptor system is initiated by a controlled sequential two-step hormone-induced dimerization of two hGH receptors via their extracellular domains (ECDs). Little is currently known about the energetics governing the important regulatory step in receptor signaling (step 2) because of previously existing experimental barriers in characterizing the binding of the second receptor (ECD2). A further complication is that ECD2 binds through contacts from two spatially distinct sites: through its N-terminal domain to hGH, and to ECD1 through its C-terminal domain, which forms a pseudo-2-fold symmetrical interaction between the stems of the two receptors. We report here a detailed evaluation of the energetics of step 2 binding using a modified surface plasmon resonance method that is able to measure accurately the kinetics of the trimolecular binding process and separate the effects of the two binding sites. The binding kinetics of 23 single and 126 ECD1-ECD2 pair-wise alanine mutations was measured. Although both of the ECD2 binding interfaces were found to be important, the ECD1-ECD2 stem-stem contact is the stronger of the two. It was determined that most residues in the binding interfaces act in additive fashion, and that the six residues common in both ECDs contribute very differently to homodimerization depending on which ECD they reside in. This interface is characterized by a binding "hot-spot" consisting of a core of three residues in ECD1 and two in ECD2. There is no similar hot-spot in the N-terminal domain of ECD2 binding to Site2 of hGH. This study suggests ways to engineer ECD molecules that will bind specifically to either Site1 or Site2 of hGH, providing novel reagents for biophysical and biological studies.
人类生长激素（hGH）-人类生长激素受体系统中的信号传导是由两个hGH受体通过其细胞外结构域（ECD）进行的受控顺序两步激素诱导二聚化启动的。由于先前在表征第二个受体（ECD2）的结合方面存在实验障碍，目前对受体信号传导中重要调节步骤（步骤2）的能量学知之甚少。另一个复杂之处在于，ECD2通过两个空间上不同的位点进行结合：通过其N端结构域与hGH结合，并通过其C端结构域与ECD1结合，这在两个受体的茎之间形成了一种伪二倍体对称相互作用。我们在此报告使用改进的表面等离子体共振方法对步骤2结合的能量学进行的详细评估，该方法能够准确测量三分子结合过程的动力学，并分离两个结合位点的影响。测量了23个单突变和126个ECD1-ECD2成对丙氨酸突变的结合动力学。虽然发现ECD2的两个结合界面都很重要，但ECD1-ECD2茎-茎接触在两者中更强。已确定结合界面中的大多数残基以加性方式起作用，并且两个ECD中共同的六个残基根据它们所在的ECD对同源二聚化的贡献非常不同。该界面的特征是一个结合“热点”，由ECD1中的三个残基核心和ECD2中的两个残基组成。ECD2与hGH的位点2结合的N端结构域中没有类似的热点。这项研究提出了设计能够特异性结合hGH的位点1或位点2的ECD分子的方法，为生物物理和生物学研究提供了新的试剂。
