Heteronuclear (H-1, C-13, N-15) NMR assignments and solution structure of the monocyte chemoattractant protein-1 (MCP-1) dimer
Heteronuclear (H-1, C-13, N-15) NMR assignments and solution structure of the monocyte chemoattractant protein-1 (MCP-1) dimer
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DOI：
10.1021/bi9602270
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发表时间：
1996-05-28
期刊：
影响因子：
2.9
通讯作者：
Domaille, PJ
中科院分区：
文献类型：
--
作者：
Handel, TM;Domaille, PJ
用异核多维核磁共振确定了单核细胞趋化蛋白-1(MCP-1或MCAF)均二聚体的全高分辨三维溶液结构。MCP-1是一个小蛋白家族的成员，通过协调特定的白细胞向免疫挑战区域募集，在免疫监视中发挥关键作用。通过在大肠杆菌中的表达，该蛋白被C-13和N-15等同位素富集，并通过结合异核双共振和三重共振实验获得了完整的序列特异性共振指认。二级结构是根据NOE的特征模式、C-13(α/β)化学位移、(3)J(HNHα)标量耦合的测量和酰胺质子缓慢交换的模式来推断的。由于MCP-1形成对称的同源二聚体，因此进行了额外的实验，以明确地建立四元联系。来自这些新颖实验的NOE与更传统的异核分离NOE测量在迭代策略中合并，以划分显式亚基间/亚基内接触和其中两者都被保留为模棱两可的类别之间的限制。每个残基有30多个限制，三维结构定义良好，主干RMSD为0.37埃，平均为5-69个二聚体残基。我们将其结构与最近报道的相关趋化因子MIP-1β和RANTES进行了比较，强调了它们在受体特异性和功能方面的差异，并解释了MCP-1突变体的已知生物活性数据。
A full high-resolution three-dimensional solution structure of the monocyte chemoattractant protein-1 (MCP-1 or MCAF) homodimer has been determined by heteronuclear multidimensional NMR. MCP-1 is a member of a family of small proteins which play a crucial role in immune surveillance by orchestrating the recruitment of specific leukocytes to areas of immune challenge. The protein was uniformly isotopically enriched with C-13 and N-15 by expression in Escherichia coli, and complete sequence-specific resonance assignments were obtained by a combination of heteronuclear double- and triple-resonance experiments. The secondary structure was deduced from characteristic patterns of NOEs, C-13(alpha/beta) chemical shifts, measurements of (3)J(HNH alpha) scalar couplings, and patterns of slowly exchanging amide protons. Because MCP-1 forms symmetrical homodimers, additional experiments were carried out to unambiguously establish the quaternary contacts. NOEs from these novel experiments were merged with more traditional heteronuclear separated NOE measurements in an iterative strategy to partition the restraints between explicit inter/intrasubunit contacts and a class wherein both were retained as ambiguous. With more than 30 restraints per residue, the three-dimensional structure is well-defined with a backbone rmsd of 0.37 Angstrom to the mean over residues 5-69 of the dimer. We compare the structure with those recently reported for the related chemokines MIP-1 beta and RANTES and highlight the differences in terms of receptor specificity and function as well as interpret the known biological activity data of MCP-1 mutants.