Deacylation Mechanism by SIRT2 Revealed in the 1′-SH-2′-O-Myristoyl Intermediate Structure

Abstract

Summary Sirtuins are NAD-dependent deacylases. Previous studies have established two important enzymatic intermediates in sirtuin-catalyzed deacylation, an alkylamidate intermediate I, which is then converted to a bicyclic intermediate II. However, how intermediate II is converted to products is unknown. Based on potent SIRT2-specific inhibitors we developed, here we report crystal structures of SIRT2 in complexes with a thiomyristoyl (TM) inhibitor and carba-NAD or NAD. Interestingly by soaking crystals with NAD, we capture a distinct covalent catalytic intermediate (III) that is different from the intermediates I and II. In this intermediate, the covalent bond between the S and the myristoyl carbonyl carbon is broken and we believe this intermediate III is the decomposition product of II in route to form the end products. MALDI-TOF data further support the intermediate III formation. This is the first time such an intermediate is captured by X-ray crystallography and provides more mechanistic insights into sirtuin-catalyzed reactions.

A Small‐Molecule SIRT2 Inhibitor That Promotes K‐Ras4a Lysine Fatty‐Acylation

“…ChemMedChem 2019, 14,744 -748 www.chemmedchem.org reported crystal structure of SIRT2 in complexw ithamyristoyl peptide( PDB ID:4 X3O), suggested that the introduction of a hydroxy group on TM allowed the compound to form hydrogen bonds with the amino acid backbone of SIRT2, specifically with Gln265, Val266, and Asn267 (Figure 3). [11] Similarh ydrogen bonds were also observed when am odel wasg enerated with JH-T4 and the established SIRT3 and H3K9 myristoyl peptide crystal structure,5 BWN ( Figure S1). [9a] The ability to form hydrogen bonds makes JH-T4 am ore efficient suicide substrate, which leads to the increased inhibition of SIRT1 andS IRT3,a s well as better inhibition of the defatty-acylation activity of SIRT2.…”

Evolved, Selective Erasers of Distinct Lysine Acylations

“…The exchange of positions of F60 and W67 induced by the crotonyl group in CobBac2 leads to the accumulation of Int. III (38), probably by preventing its hydrolysis due to increased shielding against water. This conclusion is supported by the observation that the equivalent of F60 (F33) in Thermotoga maritima Sir2 shields the reaction intermediates from premature hydrolysis and ADP-ribose formation (16), and that the equivalent of W67 (Y40) is predicted by MD simulations to control the access of water to the active site (42).…”

“…We obtained crystals of the complex by co-crystallization after 16 h ( Figure 4A) and by soaking for 36 h ( Figure 4B). We observed a continuous density that linked the ADP-ribose moiety to the crotonyl group via the 2'-OH of the ribose ( Figure 4B (38). We modelled the analogous crotonyl-Int.…”

A Designed Peptide Targets Two Types of Modifications of p53 with Anti-cancer Activity

“…Covalent modification of proteins is a powerful way to modulate macromolecular functions (Yang et al, 2016), representing a pivotal target for chemico-biological interventions (Jongkees et al, 2017;Mulder et al, 2016;Wang et al, 2017). Protein modifications may be regulated in a spatial-temporal manner by adaptor proteins, which function by modulating protein-protein interactions (Koch et al, 2017).…”