6fx7
Crystal structure of in vitro evolved Af1521
Structural highlights
FunctionY1521_ARCFU Removes ADP-ribose from glutamate residues in proteins bearing a single ADP-ribose moiety. Inactive towards proteins bearing poly-ADP-ribose. Catalyzes removal of a phosphate group from ADP-ribose 1-phosphate (Appr1p), but with low efficiency.[1] [2] Publication Abstract from PubMedProtein ADP-ribosylation is a reversible post-translational modification that regulates important cellular functions. The identification of modified proteins has proven challenging and has mainly been achieved via enrichment methodologies. Random mutagenesis was used here to develop an engineered Af1521 ADP-ribose binding macro domain protein with 1000-fold increased affinity towards ADP-ribose. The crystal structure reveals that two point mutations K35E and Y145R form a salt bridge within the ADP-ribose binding domain. This forces the proximal ribose to rotate within the binding pocket and, as a consequence, improves engineered Af1521 ADPr-binding affinity. Its use in our proteomic ADP-ribosylome workflow increases the ADP-ribosylated protein identification rates and yields greater ADP-ribosylome coverage. Furthermore, generation of an engineered Af1521 Fc fusion protein confirms the improved detection of cellular ADP-ribosylation by immunoblot and immunofluorescence. Thus, this engineered isoform of Af1521 can also serve as a valuable tool for the analysis of cellular ADP-ribosylation under in vivo conditions. Engineering Af1521 improves ADP-ribose binding and identification of ADP-ribosylated proteins.,Nowak K, Rosenthal F, Karlberg T, Butepage M, Thorsell AG, Dreier B, Grossmann J, Sobek J, Imhof R, Luscher B, Schuler H, Pluckthun A, Leslie Pedrioli DM, Hottiger MO Nat Commun. 2020 Oct 15;11(1):5199. doi: 10.1038/s41467-020-18981-w. PMID:33060572[3] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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