Resolving The Agonistic Mechanism Of Sirt6 Protein Based On Molecular Dynamics Simulations

Overview

SIRT6 is widely involved in deacetylation of histones and non-histone proteins, which is closely related to DNA damage repair, telomere integrity maintenance, transcriptional regulation and cellular energy metabolism. small molecule agonists of SIRT6 have good potential for drug formation, but the details of the agonism mechanism of SIRT6 protein are still not well understood. To address this issue, this study used μs-scale molecular dynamics simulations to firstly elucidate the structural basis for the recognition of different substrate proteins by the SIRT6 protein, and further found that small-molecule agonists can improve the catalytic efficiency of SIRT6 by stabilising the protein and the stability of the NAD+-substrate peptide complex. This study provides new insights for further development of efficient agonists for SIRT6.

Figure 1. (A) Protein structure of SIRT6; (B) small molecule agonist; (C) enzyme catalysis mechanism.

Methods and Discussion

The first step of the enzyme-catalysed reaction of SIRT6 is the modification of an acetyl group on the lysine residue of the substrate by transferring the acetyl group modification to NAD+. Therefore, the spatial position of NAD+ relative to the acetylated lysine substrate is important for catalysing the reaction. In this paper, molecular dynamics simulations of SIRT6 and acetylated substrate and long-chain fatty acylated substrate were firstly carried out respectively, as shown in Fig. 2, when SIRT6 binds the long-chain substrate, the long-chain fatty acyl group of the substrate sufficiently fills up the substrate-binding pocket of SIRT6, which can effectively maintain NAD+ in the appropriate catalytic conformation. In contrast, when SIRT6 binds acetylated substrates, the conformation of NAD+ is flipped because the catalytic pocket is not effectively occupied, and the catalytic reaction is difficult to occur efficiently.

Figure 2. Schematic representation of SIRT6 binding to (A) long-chain fatty acylated substrates and (B) acetylated substrates; (C) NAD+ conformation flip upon SIRT6 binding to acetylated substrates; (D-F) Distribution of key reaction site distances, calculated free energies, and reaction dihedral angles in the two catalytic systems.

Inspired by the mechanism of SIRT6 substrate selectivity, the researchers further analysed the mechanism by which endogenous long-chain fatty acids agonise SIRT6 deacetylation activity. Previous biochemical studies have suggested that endogenous long-chain fatty acids share the same catalytic pocket as the lipoyl groups of long-chain fatty acyl substrates. The researchers first docked the endogenous long-chain fatty acid MYA into the substrate pocket of SIRT6 using a molecular docking technique and selected two representative conformations as the starting conformations for the simulations (Figure 3). The results of the simulations showed that the starting conformation I was more effective in shortening the distance between the key catalytic sites and facilitating the deacetylation reaction. During the simulation of starting conformation I, the MYA small molecule undergoes a rotation of about 90°, moves out of the water transport channel of the bound substrate, stabilises near the α3 helix, and highly overlaps with the binding pockets of small-molecule agonists such as MDL-801, UBCS039, and 12q.

Figure 3. Modelling of (B) key reaction site distances, (C) estimated free energy distributions and (D) SIRT6-MYA stabilised interaction patterns after SIRT6 binds endogenous fatty acids in (A) two predicted conformations.

Inspired by this result, the researchers further explored whether there is a conserved agonist mechanism between different SIRT6 small molecule agonists. As shown in Figure 4, the researchers simulated the molecular dynamics trajectories of SIRT6 after binding the agonists UBCS039, MDL-801, and 12q. Compared with the system without bound agonists, SIRT6 binding to different agonists were effective in preventing NAD+ conformational flip and facilitating the interaction between substrate and cofactor NAD+.

Figure 4. Molecular dynamics simulations of SIRT6 binding to different agonists.

 Conclusions

This study further elucidated the activation mechanism of SIRT6 at the atomic level and proposed a research strategy to approximate the catalytic activity of the SIRT6 protein by the distance of the key catalytic site versus the dihedral angle distribution, which is expected to facilitate the design and optimisation of novel SIRT6 agonists.

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