Crystal structure of a mini-intein reveals a conserved catalytic module involved in side chain cyclization of asparagine during protein splicing
Crystal structure of a mini-intein reveals a conserved catalytic module involved in side chain cyclization of asparagine during protein splicing
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DOI：
10.1074/jbc.m306197200
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发表时间：
2003-10-03
影响因子：
4.8
通讯作者：
Rao, ZH
中科院分区：
文献类型：
--
作者：
Ding, Y;Xu, MQ;Rao, ZH
我们已经确定了一个154个残基内含肽的晶体结构，来自集胞藻属菌株PCC 6803的dnaB基因，并将其细化到2.0埃的分辨率。X-射线结构表明，该内含肽具有两个催化位点，似乎分别负责N和C-末端易裂键的剪接和裂解。保守的内含肽区段F残基是最后一个内含肽残基Asn-154的侧链环化的催化位点的重要组成部分。这些数据表明，His-143的咪唑环参与了Asn-154侧链Ndelta原子的活化，导致Asn-154羰基碳的亲核攻击。用Ala或Gln取代His-143导致C-末端切割的抑制。His-153、Asp-136和水分子似乎通过接触Asn-154的羰基氧来稳定过渡态，从而构成氧阴离子结合位点。结构和诱变数据也支持Thr-138和Ser-155的羟基之间的紧密接触，其侧链参与S -> O酰基移位，在亲核试剂取向中起重要作用。我们的结构模型表明，这种催化模块是保守的C-末端亚结构域的内含肽从不同的生物体。
We have determined the crystal structure of a 154-residue intein derived from the dnaB gene of Synechocystis sp. strain PCC6803 and refined it to a 2.0-Angstrom resolution. The x-ray structure suggests that this intein possesses two catalytic sites that appear to be separately responsible for splicing and cleavage of the Nand C-terminal scissile bonds. The conserved intein block F residues are the important components of a catalytic site for side chain cyclization of the last intein residue, Asn-154. The data suggest that the imidazole ring of His-143 is involved in the activation of the side chain Ndelta atom of Asn-154, leading to a nucleophilic attack on the carbonyl carbon of Asn-154. Substitution of His-143 with Ala or Gln resulted in the inhibition of C-terminal cleavage. His-153, Asp-136, and a water molecule appear to constitute an oxyanion binding site by contacting the carbonyl oxygen of Asn-154 to stabilize the transition state. The structure and mutagenesis data also support that the close contact between the hydroxyl groups of Thr-138 and Ser-155, whose side chain participates in an S --> O acyl shift, plays an important role in the nucleophile orientation. Our structural modeling suggests that this catalytic module is conserved in the C-terminal subdomains of inteins from diverse organisms.