In spore forming microbes, germination protease (GPR) plays a key role in the initiation of the germination process. A critical step during germination is the degradation of small acid-soluble proteins (SASPs), which protect spore DNA from external stresses (UV, heat, low temperature, etc.). Inactive zymogen GPR can be activated by autoprocessing of the N-terminal pro-sequence domain. Activated GPR initiates the degradation of SASPs; however, the detailed mechanisms underlying the activation, catalysis, regulation, and substrate recognition of GPR remain elusive. In this study, we determined the crystal structure of GPR from Paenisporosarcina sp. TG-20 (PaGPR) in its inactive form at a resolution of 2.5 A. Structural analysis showed that the active site of PaGPR is sterically occluded by an inhibitory loop region (residues 202–216). The N-terminal region interacts directly with the self-inhibitory loop region, suggesting that the removal of the N-terminal pro-sequence induces conformational changes, which lead to the release of the self-inhibitory loop region from the active site. In addition, comparative sequence and structural analyses revealed that PaGPR contains two highly conserved Asp residues (D123 and D182) in the active site, similar to the putative aspartic acid protease GPR from Bacillus megaterium. The catalytic domain structure of PaGPR also shares similarities with the sequentially non-homologous proteins HycI and HybD. HycI and HybD are metal-loproteases that also contain two Asp (or Glu) residues in their active site, playing a role in metal binding. In summary, our results provide useful insights into the activation process of PaGPR and its active conformation.

中文翻译：

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对嗜冷生发蛋白酶PaGPR及其自动抑制环的结构见解。

在形成孢子的微生物中，发芽蛋白酶（GPR）在发芽过程的启动中起关键作用。萌发过程中的关键步骤是降解小酸溶性蛋白质（SASP），从而保护孢子DNA免受外部压力（紫外线，热，低温等）。可以通过自动加工N端前序列域来激活无活性的酶原GPR。激活的GPR会引发SASP的降解；然而，GPR的激活，催化，调节和底物识别背后的详细机制仍然难以捉摸。在这项研究中，我们确定了Paenisporosarcina中GPR的晶体结构sp。TG-20（PaGPR）处于非活性状态，分辨率为2.5A。结构分析表明，PaGPR的活性位点在空间上被抑制性环区域（残基202-216）封闭。N-末端区域直接与自抑制环区域相互作用，这表明N-末端前序序列的去除诱导构象变化，这导致自抑制位点区域从活性位点释放。此外，比较序列和结构分析表明，Pa GPR在活性位点包含两个高度保守的Asp残基（D123和D182），类似于来自巨大芽孢杆菌的假定的天冬氨酸蛋白酶GPR 。PaGPR的催化结构域结构也与顺序非同源蛋白HycI和HybD具有相似性。HycI和HybD是金属蛋白酶，在它们的活性位点也包含两个Asp（或Glu）残基，在金属结合中起作用。总之，我们的结果为Pa GPR的激活过程及其活性构象提供了有用的见解。