Maritza Vatta1, Bronwyn Lyons2, Kayla A Heney3, Taylor Lidster4, A Rod Merrill1. 1. Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON N1G 2W1, Canada. 2. Department of Biochemistry and Molecular Biology and Center for Blood Research, University of British Columbia, 2350 Health Sciences Mall, Vancouver, BC V6T 1Z3, Canada. 3. Department of Biochemistry, McGill University, Montreal, QC H3G 1Y6, Canada. 4. Department of Biological Sciences, Brock University, St. Catherines, ON L2S 3A1, Canada.
Abstract
Scabin is a mono-ADP-ribosyltransferase toxin/enzyme and possible virulence factor produced by the agriculture pathogen, Streptomyces scabies. Recently, molecular dynamic approaches and MD simulations revealed its interaction with both NAD+ and DNA substrates. An Essential Dynamics Analysis identified a crab-claw-like mechanism, including coupled changes in the exposed motifs, and the Rβ1-RLa-NLc-STTβ2-WPN-WARTT-(QxE)ARTT sequence motif was proposed as a catalytic signature of the Pierisin family of DNA-acting toxins. A new fluorescence assay was devised to measure the kinetics for both RNA and DNA substrates. Several protein variants were prepared to probe the Scabin-NAD-DNA molecular model and to reveal the reaction mechanism for the transfer of ADP-ribose to the guanine base in the DNA substrate. The results revealed that there are several lysine and arginine residues in Scabin that are important for binding the DNA substrate; also, key residues such as Asn110 in the mechanism of ADP-ribose transfer to the guanine base were identified. The DNA-binding residues are shared with ScARP from Streptomyces coelicolor but are not conserved with Pierisin-1, suggesting that the modification of guanine bases by ADP-ribosyltransferases is divergent even in the Pierisin family.
Scabin is a mono-ADP-ribosyltransferase toxin/enzyme an class="Disease">nd possible virulence factor produced by the agriculture pathogen, Streptomyces scabies. Recently, molecular dynamic approaches and MD simulations revealed its interaction with both NAD+ and DNA substrates. An Essential Dynamics Analysis identified a crab-claw-like mechanism, including coupled changes in the exposed motifs, and the Rβ1-RLa-NLc-STTβ2-WPN-WARTT-(QxE)ARTT sequence motif was proposed as a catalytic signature of the Pierisin family of DNA-acting toxins. A new fluorescence assay was devised to measure the kinetics for both RNA and DNA substrates. Several protein variants were prepared to probe the Scabin-NAD-DNA molecular model and to reveal the reaction mechanism for the transfer of ADP-ribose to the guanine base in the DNA substrate. The results revealed that there are several lysine andarginine residues in Scabin that are important for binding the DNA substrate; also, key residues such as Asn110 in the mechanism of ADP-ribose transfer to the guanine base were identified. The DNA-binding residues are shared with ScARP from Streptomyces coelicolor but are not conserved with Pierisin-1, suggesting that the modification of guanine bases by ADP-ribosyltransferases is divergent even in the Pierisin family.
Authors: Paul D Adams; Pavel V Afonine; Gábor Bunkóczi; Vincent B Chen; Ian W Davis; Nathaniel Echols; Jeffrey J Headd; Li-Wei Hung; Gary J Kapral; Ralf W Grosse-Kunstleve; Airlie J McCoy; Nigel W Moriarty; Robert Oeffner; Randy J Read; David C Richardson; Jane S Richardson; Thomas C Terwilliger; Peter H Zwart Journal: Acta Crystallogr D Biol Crystallogr Date: 2010-01-22
Authors: Emeline Lawarée; Gytis Jankevicius; Charles Cooper; Ivan Ahel; Stephan Uphoff; Christoph M Tang Journal: Cell Rep Date: 2020-02-04 Impact factor: 9.423