Computational Structural Biology of S-nitrosylation of Cancer Targets

Abstract

Nitric oxide (NO) plays an essential role in redox signaling in normal and pathological cellular conditions. In particular, it is well known to react in vivo with cysteines by the so-called S-nitrosylation reaction. S-nitrosylation is a selective and reversible post-translational modification that exerts a myriad of different effects, such as the modulation of protein conformation, activity, stability, and biological interaction networks. We have appreciated, over the last years, the role of S-nitrosylation in normal and disease conditions. In this context, structural and computational studies can help to dissect the complex and multifaceted role of this redox post-translational modification. In this review article, we summarized the current state-of-the-art on the mechanism of S-nitrosylation, along with the structural and computational studies that have helped to unveil its effects and biological roles. We also discussed the need to move new steps forward especially in the direction of employing computational structural biology to address the molecular and atomistic details of S-nitrosylation. Indeed, this redox modification has been so far an underappreciated redox post-translational modification by the computational biochemistry community. In our review, we primarily focus on S-nitrosylated proteins that are attractive cancer targets due to the emerging relevance of this redox modification in a cancer setting.

Keywords: (de)nitrosylating enzymes; S-nitrosylation; cysteine; molecular dynamics simulations; redox cancer biology; redox modifications.

Figure 1

Most relevant cysteine oxidation states in the context of S-nitrosylation and possible pathways of interconversion. The figure is meant to be illustrative and does not depict the complete reaction schemes. R groups are part of the protein main chain.

Figure 2

Mechanisms of denitrosylation. Thioredoxin-system (top): The reduced Thioredoxin (Trx(SH)₂) denitrosylates the S-nitrosylated protein (Prot-SNO), releasing Prot-SH and the oxidized Trx (Trx(S₂)), which is further reduced back to (Trx(SH)₂) by Thioredoxin Reductase TrxR in a NADPH-dependent reaction. GSNOR-system (bottom): Prot-SNO is denitrosylated by S-transnitrosylation with glutathione (GSH), forming a reduced protein thiol (Prot-SH) and GSNO. The latter is then reduced by GSNOR to GSSG at the expenses of NADH and one GSH molecule. The resulting GSSG is further reduced by the Glutaredoxin enzyme (Grx) to give back the reduced GSH molecules—this step is not depicted here.

Figure 3

Effects of S-nitrosylation on protein function. For each class, we reported the examples of the target proteins that we discussed in the main text.

Figure 4

RSNO resonance structures, balanced between the neutral (center), the zwitterion (left) and the ion pair (right) forms. The trans conformer is depicted here, but the cis is also possible though less stable, as mentioned in section Physical Models for S-nitrosothiols (RSNOs). The neutral form is the most abundant one, the other ones being only minor conformations with dramatically opposite features—hence the dual reactivity of RSNOs with nucleophiles. The relative abundance of the three RSNO forms is highly depend on its microenvironment and the nature of the R group. For instance, the neutral/zwitterion/ion pair ratio is 79/11/10% vs. 75/15/10% for HSNO and CH₃SNO respectively.

Figure 5

SNO reactivity. We here illustrated the main reactions involving RSNOs. Top: homolytic cleavage of the S-N bond, consisting in the loss of nitric oxide with a remaining thyil radical species. Left: S-thiolation between RSNO and a R'SH thiol, leading to the formation of a disulfide bridge RSSR' and a nitroxyl moiety HNO. Right: S-transnitrosylation process inducing the transfer of NO from RSNO to a R'SH thiol. The detailed mechanisms of these reactions are still poorly understood and thus not depicted here.