Jossart Christian, Coupal Martin, McNicoll Normand, Fournier Alain, Wilkes Brian C, De Léan André
Département de Pharmacologie, Faculté de Médecine, Université de Montréal, Montréal, Québec, Canada.
Biochemistry. 2005 Feb 22;44(7):2397-408. doi: 10.1021/bi048251y.
Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) are loop-shaped peptidic hormones that have multiple actions on body fluid homeostasis. Their physiological effects are mediated through the activation of their receptor, natriuretic peptide receptor A (NPRA). This receptor is a member of the membrane guanylyl cyclase family and catalyzes cyclic guanosine monophosphate (cGMP) production following its activation. To map the binding site of human NPRA, we applied the methionine proximity assay method to this receptor. We photolabeled NPRA mutants, presenting a single methionine in the binding domain of the receptor, and used benzoylphenylalanine- (Bpa-) substituted peptides at positions 0, 3, 18, 26, and 28 of the ligand. We identified that the N-terminus of the peptide is interacting with the region between Asp(177) and Val(183) of the receptor. Arg(3) is interacting in the vicinity of Phe(172). Leu(18) binds close to Val(116). Phe(26) binds in the vicinity of His(195), and the C-terminal Tyr(28) is located close to Met(173). We next proceeded with photolabeling of a dual Bpa-substituted peptide and showed that the N-terminus and Leu(18) interact with opposite receptor subunits. On the basis of our results, a molecular model of peptide-bound NPRA was developed by homology modeling with the C-type natriuretic peptide- (CNP-) bound natriuretic peptide receptor C (NPRC) crystal structure. The model has been validated by molecular dynamics simulations. Our work provides a rational basis for interpreting and predicting natriuretic peptide binding to the human NPRA.
心房利钠肽（ANP）和脑利钠肽（BNP）是环状肽类激素，对体液平衡具有多种作用。它们的生理效应是通过其受体利钠肽受体A（NPRA）的激活来介导的。该受体是膜鸟苷酸环化酶家族的成员，激活后催化环磷酸鸟苷（cGMP）的产生。为了绘制人NPRA的结合位点，我们将甲硫氨酸邻近分析法应用于该受体。我们对在受体结合域中呈现单个甲硫氨酸的NPRA突变体进行了光标记，并在配体的第0、3、18、26和28位使用苯甲酰苯丙氨酸（Bpa）取代的肽。我们确定肽的N末端与受体的Asp(177)和Val(183)之间的区域相互作用。Arg(3)在Phe(172)附近相互作用。Leu(18)靠近Val(116)结合。Phe(26)在His(195)附近结合，C末端的Tyr(28)靠近Met(173)定位。接下来，我们对双Bpa取代的肽进行了光标记，并表明N末端和Leu(18)与相反的受体亚基相互作用。基于我们的结果，通过与C型利钠肽（CNP）结合的利钠肽受体C（NPRC）晶体结构进行同源建模，建立了肽结合的NPRA的分子模型。该模型已通过分子动力学模拟验证。我们的工作为解释和预测利钠肽与人NPRA的结合提供了合理的基础。
