Journal of Molecular Biology
Volume 395, Issue 4, 29 January 2010, Pages 844-859
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Structural Analysis of Cysteine S-Nitrosylation: A Modified Acid-Based Motif and the Emerging Role of Trans-Nitrosylation

https://doi.org/10.1016/j.jmb.2009.10.042Get rights and content

Abstract

S-Nitrosylation, the selective and reversible addition of nitric oxide (NO) moiety to cysteine (Cys) sulfur in proteins, regulates numerous cellular processes. In recent years, proteomic approaches that are capable of identifying nitrosylated Cys residues have been developed. However, the features underlying the specificity of Cys modification with NO remain poorly defined. Previous studies suggested that S-nitrosylated Cys may be flanked by an acid–base motif or hydrophobic areas and show high reactivity, low pKa, and high sulfur atom exposure. In the current study, we prepared an extensive, manually curated data set of proteins with S-nitrosothiols, accounting for a variety of biochemical functions, organisms of origin, and physiological responses to NO. Analysis of this generic NO-Cys data set revealed that proximal acid–base motif, Cys pKa, sulfur atom exposure, and Cys conservation or hydrophobicity in the vicinity of the modified Cys do not define the specificity of S-nitrosylation. Instead, this analysis revealed a revised acid–base motif, which is located more distantly to the Cys and has its charged groups exposed. We hypothesize that, rather than being strictly used for direct activation of Cys, the modified acid–base motif is engaged in protein–protein interactions thereby contributing to trans-nitrosylation as an important and widespread mechanism for reversible modification of Cys with NO moiety. For proteins lacking the revised motif, we discuss alternative mechanisms including a potential role of nitrosoglutathione as a trans-acting agent.

Introduction

S-Nitrosylation, the covalent addition of a nitric oxide (NO) moiety to the sulfur atom of cysteine (Cys) residues, is a reversible protein posttranslational modification; its function in signal transduction was initially established in smooth vascular muscle cells where, upon binding to the heme iron of guanylate cyclase1 and consequent cGMP (cyclic guanosine monophosphate)-dependent protein kinase-mediated activation of potassium channels,2 NO was found to be involved in a vasodilatative process. In the last decade, S-nitrosylation has drawn much attention from the biomedical community and led to a growing understanding of its role in a variety of signaling pathways under both physiological and pathological conditions.3, 4, 5, 6

Several methods, each with its own advantages and weaknesses, have been developed to characterize nitrosylated proteins, such as the Saville–Griess method, gas-phase chemiluminescence, mass spectrometry, and the biotin switch method.7, 8, 9, 10 However, some of these methods (e.g., Saville–Griess and gas-phase chemiluminescence) are unable to identify the modified Cys in S-nitrosylated proteins. Nevertheless, over the past decade, the number of identified S-nitrosylation sites has grown from a few, usually identified via laborious analyses, to hundreds, identified by sophisticated high-throughput proteomic approaches.11, 12, 13 In particular, the biotin switch method has recently been extensively used for identification of targets of S-nitrosylation in various model systems.14, 15, 16, 17

Taking advantage of a growing number of NO-Cys sites, bioinformatics analyses were then carried out based on several reported features of these sites initially observed by manual analyses. Although no linear sequence motifs were identified, these studies revealed a recurrence (even if not uniform) of an acid–base motif in the sequences flanking S-nitrosylated residues.18 A recent study19 analyzed common sequence and tertiary structure features in a set of 18 S-nitrosylated Cys, revealing that if the acid–base motif could be a general feature of S-nitrosylated Cys,20, 21 it is not at the sequence level. By analyzing available structures of proteins in the data set (i.e., 4 proteins out of 18 for which crystal structures were available), the authors found the motif within 7 Å of the modified Cys. However, it was also pointed that, given the different chemistries probably involved in S-nitrosylation in vivo, the motif is not a conserved feature of all Cys residues that are modified with NO.

Another feature often invoked for the structural environment of NO-Cys sites is hydrophobicity around the modified Cys. It was shown that hydrophobic protein surfaces can concentrate lipophilic NO and molecular oxygen, allowing formation of efficient nitrosating species (N2O3) precisely at the site of hydrophobic thiol, for example, Cys132 in argininosuccinate synthetase.22 Thus, selective targeting of Cys132 by S-nitrosylation could be achieved due to specific structural features of the environment around Cys. An overall hydrophobic content and a nearby positive charge (e.g., His116 in argininosuccinate synthetase) could facilitate thiol deprotonation, promoting the formation of thiolate, a target of NO. A further aspect often suggested to play a role in S-nitrosylation is low pKa of Cys, but no detailed analysis has been reported. Indeed, formation of the NO adduct to Cys also occurs at higher pH (around 9) in thioredoxin 1 (Trx1). All these features, as well as physiological aspects of S-nitrosylation, have been discussed in detail in several reviews.23, 24

Studies conducted thus far with regard to the general features of NO-Cys sites invariantly suggest a role of tertiary structure around the modifiable Cys residues, but no extensive studies have been reported that analyze various classes of S-nitrosylated proteins. Likewise, tools capable of predicting new NO-Cys sites are not available. To examine whether NO-Cys sites have common properties, we built a data set of 55 nonredundant proteins containing 70 NO-Cys sites; the analysis of this data set for general features of NO-Cys sites is described here.

Section snippets

Reference data sets

We first built a data set of proteins containing S-nitrosylated Cys residues based on literature reports (Supplementary Information, Table S1). We only considered proteins with established crystal or NMR structures and proteins that could be modeled by standard homology modeling approaches using the Swiss Model server and used nonredundant proteins with less than 50% sequence identity to any other protein in the data set. We also required the proteins in our data set to have established

Discussion

Bioinformatics approaches have been applied to examine different types of protein modifications (phosphorylation, acetylation, etc.), with variable success in terms of the ability to use common patterns for predictive purposes. These studies have helped in identifying some of the common features responsible for specificity of these modifications, as well as providing the basis for rules and patterns implemented in predictive algorithms (e.g., various Web-accessible services at Expasy‡

Sequence analysis

A set of NO-Cys-containing proteins was collected by manually curated literature searches and selecting proteins reported to be nitrosylated in more than one study. Sequence alignments were prepared with PSI-BLAST against the NCBI nonredundant protein database with the following search parameters: expectation value, 1 × 10 4; expectation value for multipass model, 1 × 10 3; and maximal number of output sequences, 1000. Cys conservation for proteins was determined with an in-house Python-script by

Acknowledgements

We thank Dr. Jonathan Stamler for discussion and helpful insights.

This study was supported by NIH GM065204 (to V.N.G.).

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