Structure of the Cladosporium fulvum Avr4 effector in complex with (GlcNAc)6 reveals the ligand-binding mechanism and uncouples its intrinsic function from recognition by the Cf-4 resistance protein.
Structure of the Cladosporium fulvum Avr4 effector in complex with (GlcNAc)6 reveals the ligand-binding mechanism and uncouples its intrinsic function from recognition by the Cf-4 resistance protein.
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DOI：
10.1371/journal.ppat.1007263
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发表时间：
2018-08
期刊：
影响因子：
6.7
通讯作者：
Fisher AJ
中科院分区：
文献类型：
--
作者：
Hurlburt NK;Chen LH;Stergiopoulos I;Fisher AJ
效应器是微生物衍生的分泌蛋白，在感染期间调节宿主免疫具有重要功能。CfAvr4是一种来自番茄病原菌黄枝孢子菌的效应蛋白，也是真菌效应蛋白家族的创始成员，它通过结合真菌几丁质并保护真菌免受几丁质酶的侵害来促进寄主。Avr4与几丁质的结合是由家族14的碳水化合物结合模块（CBM14）介导的，这是一种广泛存在于所有生命领域的CBM。迄今为止，人们对Avr4效应蛋白结合几丁质和同源的Cf-4植物免疫受体识别几丁质的结构基础仍然知之甚少。利用x射线晶体学，我们以1.95Å的分辨率解析了CfAvr4与壳聚糖[(GlcNAc)6]配合物的晶体结构。这是CBM14蛋白及其配体的第一个共晶结构，进一步揭示了Avr4效应蛋白与CBM14家族成员结合（GlcNAc）6的分子机制。结构表明，CfAvr4的两个分子通过配体相互作用，形成一个三维分子三明治，将两个（GlcNAc）6分子封装在二聚体组装体中。与以往对CBM14其他成员的假设相反，壳聚糖结合域（ChBD）延伸到CfAvr4的整个长度，（GlcNAc）6的还原端位于n端附近，非还原端位于c端。对与（GlcNAc）6相互作用的残基进行定点诱变，阐明了Avr4 ChBD的精确地形和氨基酸组成，并进一步表明这些残基不单独介导Cf-4免疫受体对CfAvr4的识别。相反，这些研究强调了cf -4介导的识别依赖于CfAvr4在叶外体中的稳定性和抗蛋白水解能力，并为该效应家族在结构上将内在功能与免疫受体识别分离提供了证据。微生物在感染过程中调动一系列分泌的效应物来操纵宿主，而宿主则利用同源免疫受体来感知效应物并进行防御。迄今为止，效应器功能的结构基础和免疫受体的识别仍然知之甚少。在这里，我们展示了CfAvr4与壳聚糖酶复合物的晶体结构，CfAvr4是一种CBM14凝集素，也是真菌效应家族的创始成员，它结合并保护真菌细胞壁上的几丁质免受几丁质酶的侵害。这是第一个CBM14蛋白与其配体共结晶的结构，进一步揭示了Avr4效应物的功能。具体来说，通过利用结构和功能数据，我们阐明了CfAvr4与配体结合的分子基础，并表明两个效应分子通过配体结合在一起形成三明治结构，在二聚体组装中层压两个壳聚糖分子。我们进一步发现同源的Cf-4免疫受体对CfAvr4的识别不是通过与壳聚糖酶直接相互作用的残基介导的，从而在结构上使Avr4的配体结合功能与Cf-4的识别解耦，并挑战了Cf-4对Avr4效应物的广泛识别源于感知与配体结合相关的残基的早期假设。
Effectors are microbial-derived secreted proteins with an essential function in modulating host immunity during infections. CfAvr4, an effector protein from the tomato pathogen Cladosporium fulvum and the founding member of a fungal effector family, promotes parasitism through binding fungal chitin and protecting it from chitinases. Binding of Avr4 to chitin is mediated by a carbohydrate-binding module of family 14 (CBM14), an abundant CBM across all domains of life. To date, the structural basis of chitin-binding by Avr4 effector proteins and of recognition by the cognate Cf-4 plant immune receptor are still poorly understood. Using X-ray crystallography, we solved the crystal structure of CfAvr4 in complex with chitohexaose [(GlcNAc)6] at 1.95Å resolution. This is the first co-crystal structure of a CBM14 protein together with its ligand that further reveals the molecular mechanism of (GlcNAc)6 binding by Avr4 effector proteins and CBM14 family members in general. The structure showed that two molecules of CfAvr4 interact through the ligand and form a three-dimensional molecular sandwich that encapsulates two (GlcNAc)6 molecules within the dimeric assembly. Contrary to previous assumptions made with other CBM14 members, the chitohexaose-binding domain (ChBD) extends to the entire length of CfAvr4 with the reducing end of (GlcNAc)6 positioned near the N-terminus and the non-reducing end at the C-terminus. Site-directed mutagenesis of residues interacting with (GlcNAc)6 enabled the elucidation of the precise topography and amino acid composition of Avr4’s ChBD and further showed that these residues do not individually mediate the recognition of CfAvr4 by the Cf-4 immune receptor. Instead, the studies highlighted the dependency of Cf-4-mediated recognition on CfAvr4’s stability and resistance against proteolysis in the leaf apoplast, and provided the evidence for structurally separating intrinsic function from immune receptor recognition in this effector family. Microbes mobilize an array of secreted effectors to manipulate their hosts during infections, whereas in response, hosts utilize cognate immune receptors to perceive effectors and mount a defense. To date, the structural basis of effector function and recognition by immune receptors are still poorly understood. Here we present the crystal structure in complex with chitohexaose of CfAvr4, a CBM14 lectin and the founding member of a fungal effector family that binds and protects chitin in fungal cell-walls from chitinases. This is the first structure of a CBM14 protein to be co-crystalized with its ligand that further reveals how Avr4 effectors function. Specifically, by leveraging structural and functional data, we elucidate the molecular basis for ligand-binding by CfAvr4 and show that two effector molecules are brought together through the ligand to form a sandwich structure that laminates two chitohexaose molecules within the dimeric assembly. We further show that recognition of CfAvr4 by the cognate Cf-4 immune receptor is not mediated through residues directly interacting with chitohexaose, thereby structurally uncoupling the ligand-binding function of Avr4 from recognition by Cf-4 and challenging early postulations that the broad recognition of Avr4 effectors by Cf-4 stems from perceiving residues implicated in binding their ligand.
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