Institute of Molecular Biophysics, Florida State University, Tallahassee, FL, 32306, USA.
Department of Medical Biochemistry and Cell Biology, University of Gothenburg, Gothenburg, 40530, Sweden.
Commun Biol. 2022 Mar 29;5(1):279. doi: 10.1038/s42003-022-03187-1.
The small RNA-mediated immunity in bacteria depends on foreign RNA-activated and self RNA-inhibited enzymatic activities. The multi-subunit Type III-A CRISPR-Cas effector complex (Csm) exemplifies this principle and is in addition regulated by cellular metabolites such as divalent metals and ATP. Recognition of the foreign or cognate target RNA (CTR) triggers its single-stranded deoxyribonuclease (DNase) and cyclic oligoadenylate (cOA) synthesis activities. The same activities remain dormant in the presence of the self or non-cognate target RNA (NTR) that differs from CTR only in its 3'-protospacer flanking sequence (3'-PFS). Here we employ electron cryomicroscopy (cryoEM), functional assays, and comparative cross-linking to study in vivo assembled mesophilic Lactococcus lactis Csm (LlCsm) at the three functional states: apo, the CTR- and the NTR-bound. Unlike previously studied Csm complexes, we observed binding of 3'-PFS to Csm in absence of bound ATP and analyzed the structures of the four RNA cleavage sites. Interestingly, comparative crosslinking results indicate a tightening of the Csm3-Csm4 interface as a result of CTR but not NTR binding, reflecting a possible role of protein dynamics change during activation.
细菌中的小 RNA 介导的免疫依赖于外源 RNA 激活和自身 RNA 抑制的酶活性。多亚基 III-A 型 CRISPR-Cas 效应复合物 (Csm) 就是这一原理的典范，此外还受到细胞代谢物如二价金属离子和 ATP 的调节。外源或同源靶 RNA (CTR) 的识别触发其单链脱氧核糖核酸酶 (DNase) 和环状寡聚腺苷酸 (cOA) 合成活性。在存在与 CTR 仅在其 3'-原间隔侧翼序列 (3'-PFS) 不同的自身或非同源靶 RNA (NTR) 的情况下，相同的活性处于休眠状态。在这里，我们使用电子 cryoEM（冷冻电镜）、功能测定和比较交联来研究在三种功能状态下体内组装的嗜温乳球菌 Csm（LlCsm）：apo、CTR 和 NTR 结合。与以前研究过的 Csm 复合物不同，我们观察到 3'-PFS 在没有结合 ATP 的情况下与 Csm 结合，并分析了四个 RNA 切割位点的结构。有趣的是，比较交联结果表明，由于 CTR 但不是 NTR 结合，Csm3-Csm4 界面的紧密度增加，反映了激活过程中蛋白质动力学变化的可能作用。
