Flanagan C A, Rodic V, Konvicka K, Yuen T, Chi L, Rivier J E, Millar R P, Weinstein H, Sealfon S C
Department of Neurology, Fishberg Research Center in Neurobiology, Mount Sinai School of Medicine, New York, New York 10029, USA.
Biochemistry. 2000 Jul 18;39(28):8133-41. doi: 10.1021/bi000085g.
Mutation of Asp(2.61(98)) at the extracellular boundary of transmembrane helix 2 of the gonadotropin-releasing hormone (GnRH) receptor decreased the affinity for GnRH. Using site-directed mutagenesis, ligand modification, and computational modeling, different side chain interactions of Asp(2.61(98)) that contribute to high-affinity binding were investigated. The conservative Asp(2. 61(98))Glu mutation markedly decreased the affinity for a series of GnRH analogues containing the native His(2) residue. This mutant showed smaller decreases in affinity for His(2)-substituted ligands. The loss of preference for His(2)-containing ligands in the mutant receptor shows that Asp(2.61(98)) determines the specificity for His(2). Analysis of the affinities of a series of position 2-substituted ligands suggests that a hydrogen bond forms between Asp(2.61(98)) and the delta NH group of His(2) and that Asp(2. 61(98)) forms a second hydrogen bond with the ligand. Substitution of Asp(2.61(98)) with an uncharged residue further decreased the affinity for all ligands and also decreased receptor expression. Computational modeling indicates an intramolecular ionic interaction of Asp(2.61(98)) with Lys(3.32(121)) in transmembrane helix 3. The uncharged, Lys(3.32(121))Gln mutation also markedly decreased agonist affinity. The modeling and the similar phenotypes of mutants with uncharged substitutions for Asp(2.61(98)) or Lys(3.32(121)) are consistent with the presence of this helix 2-helix 3 interaction. These studies support a dual role for Asp(2.61(98)): formation of an interhelical interaction with Lys(3.32(121)) that contributes to the structure of the agonist binding pocket and an interaction with His(2) of GnRH that helps stabilize agonist complexing.
促性腺激素释放激素（GnRH）受体跨膜螺旋2细胞外边界处的天冬氨酸（Asp(2.61(98))）发生突变会降低对GnRH的亲和力。利用定点诱变、配体修饰和计算建模，研究了有助于高亲和力结合的Asp(2.61(98))的不同侧链相互作用。保守的Asp(2. 61(98))Glu突变显著降低了对一系列含有天然His(2)残基的GnRH类似物的亲和力。该突变体对His(2)取代配体的亲和力下降较小。突变受体中对含His(2)配体偏好性的丧失表明Asp(2.61(98))决定了对His(2)的特异性。对一系列2位取代配体亲和力的分析表明，Asp(2.61(98))与His(2)的δNH基团之间形成了氢键，并且Asp(2. 61(98))与配体形成了第二个氢键。用不带电荷的残基取代Asp(2.61(98))会进一步降低对所有配体的亲和力，同时也会降低受体表达。计算建模表明，跨膜螺旋3中的Asp(2.61(98))与Lys(3.32(121))存在分子内离子相互作用。不带电荷的Lys(3.32(121))Gln突变也显著降低了激动剂亲和力。对Asp(2.61(98))或Lys(3.32(121))用不带电荷的残基取代的突变体进行的建模和相似表型与这种螺旋2-螺旋3相互作用的存在一致。这些研究支持了Asp(2.61(98))的双重作用：与Lys(3.32(121))形成螺旋间相互作用，这有助于激动剂结合口袋的结构形成；以及与GnRH的His(2)相互作用，这有助于稳定激动剂复合物。
