Communication Breakdown: Dissecting the COM Interfaces between the Subunits of Nonribosomal Peptide Synthetases
Communication Breakdown: Dissecting the COM Interfaces between the Subunits of Nonribosomal Peptide Synthetases
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DOI：
10.1021/acscatal.1c02113
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发表时间：
2021-08-16
期刊：
影响因子：
12.9
通讯作者：
Lewandowski, Jozef R.
中科院分区：
文献类型：
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作者：
Fage, Christopher D.;Kosol, Simone;Lewandowski, Jozef R.
非核糖体肽是一种结构多样且具有生物活性的天然产物，是由多结构域酶组装线非核糖体肽合成酶（NRPSs）合成的。虽然NRPSs的核心催化结构域甚至整个蛋白质亚基已经在结构上被阐明，但很少有关于促进亚基之间相互作用（从而转移生物合成中间体）的对接结构域的生物物理研究报道。在本研究中，我们仔细研究了在NRPS亚基末端发现的介导供体外映(E)和受体缩合(C)结构域之间通信的COM结构域。通过x射线晶体学、圆二色光谱、溶液和固态核磁共振光谱以及分子动力学（MD）模拟的结合，我们提供了直接的证据，证明一个内在无序的供体COM区域在与合适的受体结合时折叠成一个动态的螺旋基序。此外，我们的核磁共振滴定和碳足迹实验阐明了COM相互作用界面所涉及的残基，我们的MD模拟显示了与实验数据一致的折叠。尽管我们的研究结果证实了先前提出的螺旋手相互作用模式，但它们也强调了将COM界面视为动态整体而不是单一刚性结构的重要性，并建议工程实验除了稳定最终复合物的相互作用外，还应考虑瞬时引导折叠的相互作用。通过活性测定和亲和力测量，我们进一步证实了供体COM区域在结合受体C结构域中的作用，并暗示这一短基序很容易转座非同源结构域串扰。最后，我们的生物信息学分析表明，COM结构域在天然产物途径中广泛存在，并且在上述规范类型之外的接口上起作用，这为彻底表征这些对接结构域设定了高度优先级。我们的发现为未来合理设计NRPS域-域相互作用的尝试奠定了基础，最终目标是产生生物活性分子。
Nonribosomal peptides are a structurally diverse and bioactive class of natural products constructed by multidomain enzymatic assembly lines known as nonribosomal peptide synthetases (NRPSs). While the core catalytic domains and even entire protein subunits of NRPSs have been structurally elucidated, little biophysical work has been reported on the docking domains that promote interactions-and thus transfer of biosynthetic intermediates-between subunits. In the present study, we closely examine the COM domains that mediate COMmunication between donor epimerization (E) and acceptor condensation (C) domains found at the termini of NRPS subunits. Through a combination of X-ray crystallography, circular dichroism spectroscopy, solution- and solid-state NMR spectroscopy, and molecular dynamics (MD) simulations, we provide direct evidence for an intrinsically disordered donor COM region that folds into a dynamic helical motif upon binding to a suitable acceptor. Furthermore, our NMR titration and carbene footprinting experiments illuminate the residues involved at the COM interaction interface, and our MD simulations demonstrate folding consistent with experimental data. Although our results lend credence to the previously proposed helix-hand mode of interaction, they also underscore the importance of viewing COM interfaces as dynamic ensembles rather than single rigid structures and suggest that engineering experiments should account for the interactions which transiently guide folding in addition to those which stabilize the final complex. Through activity assays and affinity measurements, we further substantiate the role of the donor COM region in binding the acceptor C domain and implicate this short motif as readily transposable for noncognate domain crosstalk. Finally, our bioinformatics analyses show that COM domains are widespread in natural product pathways and function at interfaces beyond the canonical type described above, setting a high priority for thorough characterization of these docking domains. Our findings lay the groundwork for future attempts to rationally engineer NRPS domain-domain interactions with the ultimate goal of generating bioactive molecules.