![]() There are numerous outstanding questions pertaining to SgrAI structure, function, and the intriguing ways in which its activity and specificity are modulated through filamentation. Specifically, our model proposes that the conformation of DNA in the low-activity nonfilamentous form is more preferred when secondary site sequences are bound than that of the high-activity filamentous form, which as a result decreases the propensity of SgrAI to filament when bound to secondary site sequences substituted at the second bp ( From this observation, we proposed a mechanism for secondary site activity resulting from changes in the energy landscape of low-activity and high-activity conformations of SgrAI, which stem from changes in base stacking energies. When compared with the structure of SgrAI bound to either primary or secondary site DNA in the nonfilamentous form, the earlier 3.5 Å resolution structure of filamentous SgrAI indicated a change in the base stacking of the bound DNA at the base pair substituted in a subset of secondary sequences ( i.e., those substituted in the second position, CCCCGGYG). KeywordsĪbbreviations: CTF ( contrast transfer function), DBD ( DNA-bound SgrAI dimer), FSC ( Fourier shell correlation), PDB ( Protein Data Bank), ROO ( run-on oligomeric), SP ( scissile phosphate or scissile phosphodiester group), UCSF ( University of California San Francisco) Collectively, these multiple new observations clarify the mechanism of expansion of DNA sequence specificity of SgrAI, including the indirect readout of sequence-dependent DNA structure, changes in protein–DNA interactions, and the disorder-to-order transition of a crucial DNA recognition element. Second, we present an X-ray crystal structure of DNA-free (apo) SgrAI resolved to 2.0 Å resolution, which reveals a disordered loop involved in DNA recognition. This structure reveals important conformational changes that contribute to the catalytic mechanism and the binding of a second divalent cation in the enzyme active site, which is expected to contribute to increased DNA cleavage activity of SgrAI in the filamentous state. First, we present the cryo-EM structure of filamentous SgrAI bound to intact primary site DNA and Ca 2+ resolved to ∼2.5 Å within the catalytic center, which represents the trapped enzyme–DNA complex prior to the DNA cleavage reaction. Herein, we describe two new structures of the SgrAI enzyme that shed light on its catalytic function. ![]() However, the mechanistic bases underlying these events remain unclear. Both outcomes-the acceleration of DNA cleavage and the expansion of sequence specificity-are proposed to regulate critical processes in bacterial innate immunity. For the filament-forming sequence-specific DNA endonuclease SgrAI, the process of filamentation both accelerates its DNA cleavage activity and expands its DNA sequence specificity, thus allowing for many additional DNA sequences to be rapidly cleaved. Glycobiology and Extracellular MatricesĮnzyme filamentation is a widespread phenomenon that mediates enzyme regulation and function. ![]()
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