The novel interaction and catalytic mechanism of the new ubiquitin sidE and ubiquitin and its ligands.

On May 3, Cell magazine published online that the Institute of Biophysics of the Chinese Academy of Sciences' Institute of Biophysics' High School of Science and the New York University research paper, The R. Insights into Non-Canonical, Catalyzed by SidE.
the work analyzed the high-resolution crystal structure of the new ubiquitin synthase SidE and ubiquitin and ligands from the highly pathogenic Legionella pneumophila, and combined with a large number of biochemical experiments and mutant analysis, revealing the novel interaction and catalytic mechanism of SidE and ubiquitin and its ligands.
ubiquitin modification regulates almost all life activities of eukaryotic cells.
in the long-term game with the host, pathogens have evolved a variety of ways to interfere with the host's ubiquitin modification system, so as to enhance the efficiency of infection and avoid immune surveillance.
previously reported ubiquitization modifications (whether the host itself or the pathogen are involved) are catalyzed by the classic E1-E2-E3 trienzyme cascade reaction.
and recently identified sidE family derived from legions of pulmonary bacteria can perform atypical ubiquitinmodification of multiple host proteins.
the family has four members: SidE, SdeA, SdeB and SdeC, whose domain domains from the N-end to the C end are the DUB domain, the PDE domain, the mART domain and the coil-coil domain.
in the classic ubiquitin modification system, through the cascade reaction of E1-E2-E3, in the presence of ATP and Mg ions, the C-end covalent connection of ubiquitin to the end of the protein lysine.
and the catalytic process in which the SidE family protein is involved is completely different: first, the mART domain connects the ADP-ribose group of the NAD molecule to the 42nd-ranked arginine of the ubiquitin, generating ADP-ribosylated ubiquitin (ADPPru) -Ub); then, the PDE domain further cuts the phosphate dieste bonds in ADPr-Ub to generate phospho-ribosylated uquitbiin (Pr-Ub) and at the same time connect the Pr-Ub covalent to the serine of the substance.
However, the specific molecular mechanisms of mART domain binding and modification of ubiquitin, as well as PDE domain binding and hydrolyzed ADPr-Ub, remain unclear.
in this work, the researchers successfully obtained SidE's high-resolution crystal structure with ubiquitin and its ligands in its two-step catalytic reaction through numerous attempts.
SidE's apo structure shows that the catalytic pockets of the mART domain and the PDE domain are far away from each other and in different directions, suggesting that the catalytic processes of the two domains are independent of each other, and that the two domains are closely bound by a conservative base sequence, and when this interaction is lost, the activity of both domains is affected.
the composite structure of the mART domain and the ubiquitin and the cofactor NAD shows that the mART domain uses a highly conservative area of its surface to participate in the integration of ubiquitin, and this interface is very close to the catalytic center of mART; The 2-bit and 74-bit argine is tightly bound to the pocket sized by acidic amino acids in the mART domain, while NAD is bound to the classic R-S-E motif of the mART domain, and its niacinamine group is heading towards the 42-bit argine of ubiquitin, waiting for the catalytic formation of ADPr-Ub.
the composite structure of the PDE domain and the ubiquitin and ADP-ribose indicates that the ubiquitin is combined with the side of the catalytic pocket of the PDE domain, the Lys6-Thr9 region and His68 contribute the main binding point, the Arg42 is oriented towards the active center of the PDE domain;
all of these key amino acids involved in binding and catalyzing, the researchers used mutant experiments to perform enzyme live verification.
, the researchers also found that the identification of the SidE family protein on substrates does not depend on the specific three-dimensional structure of the substrate protein, which can be modified by ubiquition as long as the substrate protein contains serine that can enter the SidE catalytic pocket.
these results not only fully illustrate the novel ubiquitin modification mechanism of SidE family protein, but also provide the basis for the development of biological tools based on this new ubiquitin modification system.
the work was done by Gao's team in collaboration with New York University.
Gao Wei is the main communication author of the paper, and Wang Yong, assistant researcher of Gao Wei's research group, is the first author of the paper.
Shanghai light source to help in the crystal diffraction data collection process, Biophysics Institute platform Yang Fuquan and Xie Zhensheng in mass spectrometry analysis to give great help, Biophysics Institute researcher Yan Yang in cell experiments to give guidance.
the work has been funded by the National Natural Science Foundation of China, the Chinese Academy of Sciences pilot special program, the biophysics institute launched funds and the Central Group Department's Youth Thousand Program.
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