Inhibition mechanism of cytokine activity of human autocrine motility factor examined by crystal structure analyses and site-directed mutagenesis studies.
Inhibition mechanism of cytokine activity of human autocrine motility factor examined by crystal structure analyses and site-directed mutagenesis studies.
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通过晶体结构分析和定点诱变研究检查人自分泌运动因子细胞因子活性的抑制机制。
DOI：
10.1016/s0022-2836(02)00186-9
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发表时间：
2002
期刊：
影响因子：
--
通讯作者：
Nakamura,KazuoT
中科院分区：
文献类型：
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作者：
Tanaka,Nobutada;Haga,Arayo;Uemura,Hiroshi;Akiyama,Hiroko;Funasaka,Tatsuyoshi;Nagase,Hisamitsu;Raz,Avraham;Nakamura,KazuoT
自分泌运动因子(AMF)是一种肿瘤分泌的细胞因子，在体外刺激细胞迁移，在体内促进转移。AMF在基因上与细胞外细胞因子神经白细胞(NLK)和成熟因子(MF)以及细胞内的磷酸己糖异构酶(PHI)相同。人AMF的无抑制物开放型和结合抑制物(4-磷酸红细胞，E4P，一种强烈抑制AMF细胞因子活性的抑制剂)封闭形式的晶体结构分别在1.9？和2.4？分辨率下测定。当E4P结合时，抑制剂结合部位周围发生局部构象变化(从开放到关闭)。E4P结合结构表明，人AMF的抑制物(细胞因子活性)结合部位与PHIs的(酶活性抑制物)结合部位非常相似。本研究清楚地表明，AMF和PHI的酶和细胞因子功能区域存在结构重叠，并提出了碳水化合物磷酸基团抑制AMF细胞因子活性的两种可能机制。一种可能的情况是，该化合物可能会竞争AMF与AMF受体(AMFR)的碳水化合物部分结合，AMF受体是一种糖基化的七跨膜螺旋蛋白。另一种情况是，抑制剂结合时局部构象的变化可能会影响AMF-AMFR相互作用。为了研究这些残基在抑制剂结合部位的作用，我们制备了两个突变型AMF。His389被Phe氢键连接到E4P的羟基，Thr215被Asp氢键连接到E4P的磷酸基团，导致突变的AMF细胞因子活性受损。这些结果表明，这些氨基酸在识别AMFR的碳水化合物部分中起到了作用。由于E4P是具有AMF抑制剂活性的最小化合物之一，了解目前的晶体结构将为更有效的AMF抑制剂的先导化合物设计提供见解。
Autocrine motility factor (AMF), a tumor-secreted cytokine, stimulates cell migration in vitro and metastasis in vivo. AMF is genetically identical with the extracellular cytokines neuroleukin (NLK) and maturation factor (MF) and, interestingly, the intracellular enzyme phosphohexose isomerase (PHI). The crystal structures of the inhibitor-free open form and the inhibitor (erythrose 4-phosphate, E4P, a strong inhibitor of AMF's cytokine activity)-bound closed form of human AMF have been determined at 1.9Å and 2.4Å resolution, respectively. Upon E4P binding, local conformation changes (open to closed) occur around the inhibitor-binding site. The E4P-bound structure shows that the location of the inhibitor (of cytokine activity) binding site of human AMF is very similar to those of the inhibitor (of enzymatic activity) binding sites of PHIs. The present study shows clearly that there is structural overlap of the regions responsible for the enzymatic and cytokine functions of AMF and PHI and suggests two scenarios for the inhibition mechanism of cytokine activity of AMF by the carbohydrate phosphate group. One likely scenario is that the compound could compete for AMF binding with the carbohydrate moiety of the AMF receptor (AMFR), which is a glycosylated seven-transmembrane helix protein. The other scenario is that the local conformation changes upon inhibitor binding may affect the AMF–AMFR interactions. To examine roles of the residues in the inhibitor-binding site, two mutant AMFs were prepared. Replacements of His389, which is hydrogen-bonded to the hydroxyl group of E4P by Phe, and Thr215, which is hydrogen-bonded to the phosphate group of E4P by Asp, result in mutant AMFs that are impaired in cytokine activity. These results suggest a role for these amino acids in recognition of a carbohydrate moiety of the AMFR. Since the E4P is one of the smallest compounds having AMF inhibitor activity, knowledge of the present crystal structure would provide an insight into the lead compound design of more effective AMF inhibitors.