A computationally designed water-soluble variant of a G-protein-coupled receptor: the human mu opioid receptor.
A computationally designed water-soluble variant of a G-protein-coupled receptor: the human mu opioid receptor.
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DOI：
10.1371/journal.pone.0066009
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Liu R
中科院分区：
文献类型：
--
作者：
Perez-Aguilar JM;Xi J;Matsunaga F;Cui X;Selling B;Saven JG;Liu R
g蛋白偶联受体（gpcr）在多种生理过程中发挥着重要作用，被广泛应用于药物治疗。尽管它们很重要，但研究gpcr一直存在问题，因为很难以保留其配体结合能力的形式分离大量这些膜蛋白。通过突变外部，跨膜残基来创建水溶性gpcr变体，为克服这些困难提供了一种潜在的方法。在这里，我们提出了第一个涉及计算设计、表达和表征人类GPCR的水溶性变体的研究，人类mu阿片受体（MUR），它与疼痛和成瘾有关。利用比较（同源）模型和已知的GPCR结构建立了跨膜结构域的原子结构。该结构与随后确定的小鼠受体结构高度相似，并用于计算设计跨膜区域外部残基的53个突变，产生可溶于水介质的变体。该突变体在大肠杆菌中高产表达，且具有水溶性。该变体与天然人类MUR具有相同的结构和功能相关特征，包括螺旋二级结构和对拮抗剂纳曲酮的相似亲和力（K d = 65 nM）。还研究了胆固醇和二硫键对受体变异稳定性的作用。这项研究举例说明了计算方法在生产水溶性gpcr变体方面的潜力，这些变体可以在水溶液中进行结构和功能相关的表征。
G-protein-coupled receptors (GPCRs) play essential roles in various physiological processes, and are widely targeted by pharmaceutical drugs. Despite their importance, studying GPCRs has been problematic due to difficulties in isolating large quantities of these membrane proteins in forms that retain their ligand binding capabilities. Creating water-soluble variants of GPCRs by mutating the exterior, transmembrane residues provides a potential method to overcome these difficulties. Here we present the first study involving the computational design, expression and characterization of water-soluble variant of a human GPCR, the human mu opioid receptor (MUR), which is involved in pain and addiction. An atomistic structure of the transmembrane domain was built using comparative (homology) modeling and known GPCR structures. This structure was highly similar to the subsequently determined structure of the murine receptor and was used to computationally design 53 mutations of exterior residues in the transmembrane region, yielding a variant intended to be soluble in aqueous media. The designed variant expressed in high yield in Escherichia coli and was water soluble. The variant shared structural and functionally related features with the native human MUR, including helical secondary structure and comparable affinity for the antagonist naltrexone (K d = 65 nM). The roles of cholesterol and disulfide bonds on the stability of the receptor variant were also investigated. This study exemplifies the potential of the computational approach to produce water-soluble variants of GPCRs amenable for structural and functionally related characterization in aqueous solution.
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DOI：
10.1073/pnas.89.22.10915
发表时间：
1992-11-15
影响因子：
11.1
作者：
HENIKOFF, S;HENIKOFF, JG
通讯作者：
HENIKOFF, JG
DOI：
10.1002/prot.340230412
发表时间：
1995-12-01
期刊：
PROTEINS-STRUCTURE FUNCTION AND GENETICS
影响因子：
--
作者：
Frishman, D;Argos, P
通讯作者：
Argos, P
影响因子：
2.4
作者：
Muller, Isabelle;Sarramegna, Valerie;Talmont, Franck
通讯作者：
Talmont, Franck
影响因子：
3
作者：
Jain E;Bairoch A;Duvaud S;Phan I;Redaschi N;Suzek BE;Martin MJ;McGarvey P;Gasteiger E
通讯作者：
Gasteiger E
影响因子：
14.9
作者：
Davis IW;Leaver-Fay A;Chen VB;Block JN;Kapral GJ;Wang X;Murray LW;Arendall WB 3rd;Snoeyink J;Richardson JS;Richardson DC
通讯作者：
Richardson DC