TY - JOUR
T1 - Pretransition state and apo structures of the filamentforming enzyme SgrAI elucidate mechanisms of activation and substrate specificity
AU - Shan, Zelin
AU - Ghadirian, Niloofar
AU - Lyumkis, Dmitry
AU - Horton, Nancy C.
N1 - Funding Information:
Funding and additional information—Research reported in this publication was supported by the National Science Foundation under grant no. MCB-1934291 (to N. C. H. and D. L.) and by the Hearst Foundations (to D. L.). The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of NIGMS or NIH.
Funding Information:
Acknowledgments—We thank Bill Anderson and Jean-Christophe Ducom at The Scripps Research Institute for help with EM data collection and network infrastructure. Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, is supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research and by the National Institutes of Health, National Institute of General Medical Sciences (P30GM133894).
Publisher Copyright:
© 2022 THE AUTHORS.
PY - 2022/4/1
Y1 - 2022/4/1
N2 - Enzyme filamentation is a widespread phenomenon that mediates enzyme regulation and function. For the filamentforming 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. Both outcomes-the acceleration of DNA cleavage and the expansion of sequence specificity-are proposed to regulate critical processes in bacterial innate immunity. However, the mechanistic bases underlying these events remain unclear. Herein, we describe two new structures of the SgrAI enzyme that shed light on its catalytic function. First, we present the cryo-EM structure of filamentous SgrAI bound to intact primary site DNA and Ca2+ resolved to _2.5 Å within the catalytic center, which represents the trapped enzyme-DNA complex prior to the DNA cleavage reaction. 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. 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. 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.
AB - Enzyme filamentation is a widespread phenomenon that mediates enzyme regulation and function. For the filamentforming 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. Both outcomes-the acceleration of DNA cleavage and the expansion of sequence specificity-are proposed to regulate critical processes in bacterial innate immunity. However, the mechanistic bases underlying these events remain unclear. Herein, we describe two new structures of the SgrAI enzyme that shed light on its catalytic function. First, we present the cryo-EM structure of filamentous SgrAI bound to intact primary site DNA and Ca2+ resolved to _2.5 Å within the catalytic center, which represents the trapped enzyme-DNA complex prior to the DNA cleavage reaction. 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. 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. 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.
UR - http://www.scopus.com/inward/record.url?scp=85127256323&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85127256323&partnerID=8YFLogxK
U2 - 10.1016/j.jbc.2022.101760
DO - 10.1016/j.jbc.2022.101760
M3 - Article
C2 - 35202658
AN - SCOPUS:85127256323
VL - 298
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 4
M1 - 101760
ER -