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Structures of a dimodular nonribosomal peptide synthetase reveal conformational flexibility
Science ( IF 44.7 ) Pub Date : 2019-11-07 , DOI: 10.1126/science.aaw4388
Janice M. Reimer 1 , Maximilian Eivaskhani 1 , Ingrid Harb 1 , Alba Guarné 1 , Martin Weigt 2 , T. Martin Schmeing 1
Affiliation  

Moving modules drive biosynthesis Modular biosynthesis of small molecules—where enzyme units can be swapped in and out of assembly line complexes to produce desired products—is a distant goal in the lab despite a huge diversity of modular systems in nature. Part of the challenge is in understanding how modules interact and hand off intermediates. Reimer et al. determined crystal structures of portions of a nonribosomal peptide synthetase, including a full dimodule. Module positioning differed between these structures even when the same intermediate was attached to the enzyme. The authors used small-angle x-ray scattering to confirm that large conformational changes are possible during biosynthesis and handoff between modules. Science, this issue p. eaaw4388 Flexibility in modular biosynthetic complexes is explored. INTRODUCTION Nonribosomal peptide synthetases (NRPSs) are microbial megaenzymes that make a wide variety of small-molecule products, including many that are clinically used as antitumors, antibiotics, or immunosuppressants. Nonribosomal peptide synthesis proceeds with assembly-line logic, where each station on the NRPS assembly line is a multidomain unit called a module. An excellent understanding of the structures and activities of isolated modules has been established, but much less is known about how modules work with each other in the context of the larger NRPS. Structural investigation of multimodular NRPSs is needed to understand NRPS architecture, organization, and intramodular function during the synthetic cycle of an NRPS and to facilitate the longstanding goal of bioengineering for production of new-to-nature bioactive small molecules. RATIONALE To gain insight into outstanding trans- and supermodular questions in NRPS function, we performed x-ray crystallography with a series of constructs of the dimodular NRPS protein linear gramicidin synthetase subunit A (LgrA). We performed complementary small-angle x-ray scattering experiments to analyze the behavior of the NRPS in solution. We also performed direct coupling analysis to confirm the biological relevance and evolutionary conservation of observed interdomain interfaces. Both the structures and direct coupling analyses were used to guide mutagenesis studies designed to enhance the activity of a chimeric NRPS. RESULTS We have determined five independent crystal structures of constructs of LgrA, bound with a series of ligands and intermediate analogs, to resolutions between 2.2 and 6 Å. The crystallized constructs include the complete initiation module and from one to all three canonical domains from the elongation module. Some structures are in markedly different conformations, inferring large movements, and each structure seems to be in a catalytically relevant state. Small-angle x-ray scattering indicates that LgrA is also very flexible in solution, confirming that markedly different conformations are a bona fide feature of NRPS biology. The structures reveal previously unobserved states, including a full condensation conformation, where the thiolation (T) domains from both the initiation and elongation modules are simultaneously bound at the condensation (C) domain. Similar conformations in high-resolution structures allow analyses of the productive T:C domain-domain interface, which mediates the only known functional link between modules. Direct coupling analysis applied to large collections of NRPS sequences provides strong support for the biological relevance and evolutionary conservation of observed interdomain interfaces. Furthermore, both the structures and coupling scores for mutational effects were used to guide bioengineering, and we were able to double the activity of a module-swapped chimeric NRPS by introducing two point mutations at the unnatural T:C domain-domain interface. CONCLUSION The structures and small-angle x-ray scattering show NRPSs undergo very large conformational changes and challenge the general assumption that NRPSs have regular higher-order architecture. They demonstrate that there is no strict coupling between the catalytic state of a particular module and the overall conformation of the multimodular NRPS and suggest that the T:C interaction for condensation is the only point where adjacent modules must coordinate. This feature can be exploited in module-swapping bioengineering to produce new useful nonribosomal peptides. Structures of a dimodular NRPS protein reveal the central condensation state and infer very large conformational changes. A series of crystal structures of the dimodular nonribosomal peptide synthetase protein LgrA includes a structure of the condensation state (left). Condensation is the central event in synthesis, elongating the peptide intermediate and passing it to the downstream module. Additional structures in condensation and thiolation states show large conformational differences (indicated by arrows), which are supported by solution small-angle x-ray scattering data. These structures show decoupling of the catalytic state and overall conformation and imply that coordination of adjacent modules’ catalytic states is only required at condensation. The structures and coevolution analyses enable improvement of activity of a module-swapped chimeric enzyme (bottom left). Nonribosomal peptide synthetases (NRPSs) are biosynthetic enzymes that synthesize natural product therapeutics using a modular synthetic logic, whereby each module adds one aminoacyl substrate to the nascent peptide. We have determined five x-ray crystal structures of large constructs of the NRPS linear gramicidin synthetase, including a structure of a full core dimodule in conformations organized for the condensation reaction and intermodular peptidyl substrate delivery. The structures reveal differences in the relative positions of adjacent modules, which are not strictly coupled to the catalytic cycle and are consistent with small-angle x-ray scattering data. The structures and covariation analysis of homologs allowed us to create mutants that improve the yield of a peptide from a module-swapped dimodular NRPS.

中文翻译:

双模块非核糖体肽合成酶的结构揭示了构象灵活性

移动模块驱动生物合成 小分子的模块化生物合成——其中酶单元可以交换进出装配线复合体以生产所需的产品——尽管自然界中有大量的模块化系统,但在实验室中仍是一个遥远的目标。部分挑战在于理解模块如何交互和传递中间体。雷默等人。确定了非核糖体肽合成酶部分的晶体结构,包括完整的双模块。即使将相同的中间体连接到酶上,这些结构之间的模块定位也不同。作者使用小角度 X 射线散射来确认在生物合成和模块之间的切换过程中可能发生大的构象变化。科学,这个问题 p。eaaw4388 探索了模块化生物合成复合物的灵活性。引言 非核糖体肽合成酶 (NRPS) 是微生物巨酶,可制造多种小分子产品,包括许多临床上用作抗肿瘤剂、抗生素或免疫抑制剂的产品。非核糖体肽合成以装配线逻辑进行,其中 NRPS 装配线上的每个站都是一个称为模块的多域单元。对孤立模块的结构和活动有了很好的理解,但在更大的 NRPS 背景下,模块如何相互工作却知之甚少。需要对多模块 NRPS 进行结构研究,以了解 NRPS 合成周期中的 NRPS 结构、组织和模块内功能,并促进生物工程生产新自然生物活性小分子的长期目标。基本原理为了深入了解 NRPS 功能中突出的跨模块和超模块问题,我们使用一系列双模块 NRPS 蛋白线性短杆菌肽合成酶亚基 A (LgrA) 的构建体进行了 X 射线晶体学。我们进行了互补的小角度 X 射线散射实验来分析 NRPS 在溶液中的行为。我们还进行了直接耦合分析,以确认观察到的域间界面的生物学相关性和进化保守性。结构和直接耦合分析都用于指导旨在增强嵌合 NRPS 活性的诱变研究。结果我们已经确定了 LgrA 构建体的五种独立晶体结构,与一系列配体和中间体类似物结合,分辨率在 2.2 和 6 Å 之间。结晶构建体包括完整的起始模块和来自延伸模块的从一个到所有三个规范域。一些结构具有明显不同的构象,推断出较大的运动,并且每个结构似乎都处于催化相关状态。小角度 X 射线散射表明 LgrA 在溶液中也非常灵活,证实显着不同的构象是 NRPS 生物学的真正特征。这些结构揭示了以前未观察到的状态,包括完整的缩合构象,其中来自起始和延伸模块的硫醇化 (T) 域同时结合在缩合 (C) 域上。高分辨率结构中的类似构象允许分析高效的 T:C 域-域界面,它介导了模块之间唯一已知的功能链接。应用于大量 NRPS 序列集合的直接耦合分析为观察到的域间界面的生物学相关性和进化保守性提供了强有力的支持。此外,突变效应的结构和耦合评分都用于指导生物工程,我们能够通过在非自然 T:C 域-域界面引入两个点突变来使模块交换嵌合 NRPS 的活性加倍。结论 结构和小角度 X 射线散射表明 NRPS 经历了非常大的构象变化,并挑战了 NRPS 具有规则高阶结构的一般假设。他们证明特定模块的催化状态与多模块 NRPS 的整体构象之间没有严格的耦合,并表明缩合的 T:C 相互作用是相邻模块必须协调的唯一点。可以在模块交换生物工程中利用此功能来生产新的有用的非核糖体肽。二模块 NRPS 蛋白的结构揭示了中央缩合状态并推断出非常大的构象变化。双模块非核糖体肽合成酶蛋白 LgrA 的一系列晶体结构包括缩合状态的结构(左)。缩合是合成中的核心事件,它拉长了肽中间体并将其传递到下游模块。缩合和硫醇化状态的其他结构显示出很大的构象差异(用箭头表示),这得到了溶液小角度 X 射线散射数据的支持。这些结构显示出催化状态和整体构象的解耦,并暗示仅在缩合时需要相邻模块催化状态的协调。结构和共同进化分析能够提高模块交换嵌合酶的活性(左下)。非核糖体肽合成酶 (NRPS) 是使用模块化合成逻辑合成天然产物治疗剂的生物合成酶,其中每个模块向新生肽添加一种氨酰基底物。我们已经确定了 NRPS 线性短杆菌肽合成酶的大型构建体的五种 X 射线晶体结构,包括一个完整的核心双模块结构,其构象是为缩合反应和肽基底物传递而组织的。结构揭示了相邻模块相对位置的差异,这些差异与催化循环没有严格耦合,并且与小角度 X 射线散射数据一致。同源物的结构和协变分析使我们能够创建突变体,以提高来自模块交换双模块 NRPS 的肽产量。
更新日期:2019-11-07
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