Regulatory and Catalytic Domains of Poly(ADP-ribose) Polymerases Cross-Complement for DNA-Break-Dependent Allosteric Stimulation of Catalytic Activity.

IF 3.5 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY
ACS Chemical Biology Pub Date : 2025-03-21 Epub Date: 2025-02-12 DOI:10.1021/acschembio.4c00582
Makwana Milee, Shanavas P Greeshma, Waghela Deeksha, Eerappa Rajakumara
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引用次数: 0

Abstract

Allosteric regulation is achieved by regulatory domains that sense stimuli and induce conformational changes in the functional domain that performs the catalytic activity of the enzyme. Poly-ADP-ribose polymerases (PARPs) are modular enzymes present across all domains of life including Archaea, Bacteria, and Eukarya. A typical domain architecture of PARPs consists of a conserved C-terminal catalytic domain (CAT) associated with multiple distinct N-terminal sensory and/or regulatory domains which together serve as regulatory region (REG). In this study, we investigated whether REG of different orthologs and paralogs of PARPs from mammals (hPARP1 and hPARP2), plants (atPARP2), and bacteria (haPARP) can assemble with CAT of each other to generate functional chimeric assemblies. We have employed qualitative and quantitative enzyme activity assays along with binding studies to examine these in vitro chimeric assemblies. The cis-complemented REG and CAT of hPARP2 exhibited micromolar binding affinity, suggesting that these domains can interact independent of allosteric ligands. Also, our results show that REG and CAT of PARP proteins can assemble in a functionally active conformation in the presence of DNA implying that REG and CAT are not required to be present on a single polypeptide for catalytic activity stimulation. Interestingly, only CAT of atPARP2 displayed functional complementation with REG of the other studied PARPs. Conversely, REG of hPARP1 and atPARP2 failed to cross-complement CAT of other PARPs while REG of hPARP2 showed robust cross-complementation. Our novel studies on chimeric PARP assemblies can be developed as a powerful synthetic biology tool to interrogate and control their activities in living cells. In addition, by co-engineering non-complementing REG and CAT domains of different PARPs, new functional chimeric PARPs can be developed for selective allosteric ligand-dependent regulation of PARP systems. Furthermore, our study can facilitate the understanding of the coevolution of REG and CAT domains in PARP enzymes.

聚(adp -核糖)聚合酶交叉补体对dna断裂依赖性变构刺激催化活性的调控和催化结构域。
变构调节是通过感知刺激和诱导功能域构象变化的调节域来实现的,功能域执行酶的催化活性。聚adp核糖聚合酶(PARPs)是存在于所有生命领域的模块化酶,包括古生菌、细菌和真核生物。PARPs的典型结构域由一个保守的c端催化结构域(CAT)和多个不同的n端感觉和/或调节结构域组成,这些结构域共同构成调控区(REG)。在这项研究中,我们研究了来自哺乳动物(hPARP1和hPARP2)、植物(atPARP2)和细菌(haPARP)的parp的不同同源物和类似物的REG是否可以与彼此的CAT组装产生功能嵌合组装。我们采用定性和定量酶活性测定以及结合研究来检查这些体外嵌合组装。hPARP2的顺式互补的REG和CAT具有微摩尔结合亲和力,表明这些结构域可以独立于变构配体相互作用。此外,我们的研究结果表明,PARP蛋白的REG和CAT可以在DNA存在的情况下以功能活跃的构象组装,这意味着REG和CAT不需要存在于单个多肽上以刺激催化活性。有趣的是,只有atPARP2的CAT与其他研究parp的REG表现出功能性互补。相反,hPARP1和atPARP2的REG不能交叉互补其他PARPs的CAT,而hPARP2的REG则表现出强大的交叉互补。我们对嵌合PARP组装体的新研究可以作为一种强大的合成生物学工具来询问和控制它们在活细胞中的活性。此外,通过共同工程不同PARP的非互补的REG和CAT结构域,可以开发出新的功能嵌合PARP,用于PARP系统的选择性变构配体依赖性调节。此外,我们的研究有助于理解PARP酶中REG和CAT结构域的共同进化。
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来源期刊
ACS Chemical Biology
ACS Chemical Biology 生物-生化与分子生物学
CiteScore
7.50
自引率
5.00%
发文量
353
审稿时长
3.3 months
期刊介绍: ACS Chemical Biology provides an international forum for the rapid communication of research that broadly embraces the interface between chemistry and biology. The journal also serves as a forum to facilitate the communication between biologists and chemists that will translate into new research opportunities and discoveries. Results will be published in which molecular reasoning has been used to probe questions through in vitro investigations, cell biological methods, or organismic studies. We welcome mechanistic studies on proteins, nucleic acids, sugars, lipids, and nonbiological polymers. The journal serves a large scientific community, exploring cellular function from both chemical and biological perspectives. It is understood that submitted work is based upon original results and has not been published previously.
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