Inhibition of Mycobacterium tuberculosis PknG by non-catalytic rubredoxin domain specific modification: reaction of an electrophilic nitro-fatty acid with the Fe–S center

Gil, Magdalena; Graña, Martín; Schopfer, Francisco J.; Wagner, Tristan; Denicola, Ana; Freeman, Bruce A.; Alzari, Pedro M.; Batthyány, Carlos; Durán, Rosario

doi:10.1016/j.freeradbiomed.2013.06.021

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Inhibition of Mycobacterium tuberculosis PknG by non-catalytic rubredoxin domain specific modification: reaction of an electrophilic nitro-fatty acid with the Fe–S center

12 2013
By Gil, Magdalena ; Graña, Martín ; Schopfer, Francisco J. ; ...
http://dx.doi.org/10.1016/j.freeradbiomed.2013.06.021
Source: Free Radic Biol Med. 2013; 65:150-161

[PDF-1.41 MB]

English

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Details:

Alternative Title:

Free Radic Biol Med
Personal Author:

Gil, Magdalena ; Graña, Martín ; Schopfer, Francisco J. ; Wagner, Tristan ; Denicola, Ana ; ... More +

Gil, Magdalena ; Graña, Martín ; Schopfer, Francisco J. ; Wagner, Tristan ; Denicola, Ana ; Freeman, Bruce A. ; Alzari, Pedro M. ; Batthyány, Carlos ; Durán, Rosario Less -
Description:

PknG from Mycobacterium tuberculosis is a Ser/Thr protein kinase that regulates key metabolic processes within the bacterial cell as well as signaling pathways from the infected host cell. This multidomain protein has a conserved canonical kinase domain with N- and C-terminal flanking regions of unclear functional roles. The N-terminus harbors a rubredoxin-like domain (Rbx), a bacterial protein module characterized by an iron ion coordinated by four cysteine residues. Disruption of the Rbx-metal binding site by simultaneous mutations of all the key cysteine residues significantly impairs PknG activity. This encouraged us to evaluate the effect of a nitro-fatty acid (9- and 10-nitro-octadeca-9-cis-enoic acid; OA-NO2) on PknG activity. Fatty acid nitroalkenes are electrophilic species produced during inflammation and metabolism that react with nucleophilic residues of target proteins (i.e., Cys and His), modulating protein function and subcellular distribution in a reversible manner. Here, we show that OA-NO2 inhibits kinase activity by covalently adducting PknG remote from the catalytic domain. Mass spectrometry-based analysis established that cysteines located at Rbx are the specific targets of the nitroalkene. Cys-nitroalkylation is a Michael addition reaction typically reverted by thiols. However, the reversible OA-NO2-mediated nitroalkylation of the kinase results in an irreversible inhibition of PknG. Cys adduction by OA-NO2 induced iron release from the Rbx domain, revealing a new strategy for the specific inhibition of PknG. These results affirm the relevance of the Rbx domain as a target for PknG inhibition and support that electrophilic lipid reactions of Rbx-Cys may represent a new drug strategy for specific PknG inhibition.
Subjects:

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Alkenes Bacterial Proteins Catalytic Domain Circular Dichroism Fatty Acids Mutagenesis, Site-Directed Mycobacterium Tuberculosis Nitro Compounds Protein Serine-Threonine Kinases Rubredoxins Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Keywords:

[+]

Nitrated Fatty Acids Nitroalkene PknG Rubredoxin Ser/Thr Kinase
Source:

Free Radic Biol Med. 2013; 65:150-161
Pubmed ID:

23792274
Pubmed Central ID:

PMC4061738
Document Type:

Journal Article
Funding:

NIHR01-HL058115/HL/NHLBI NIH HHSUnited States/ ; R01AT-006822/AT/NCCIH NIH HHSUnited States/ ; R01-HL64937/HL/NHLBI NIH HHSUnited States/ ; P01-HL103455/HL/NHLBI NIH HHSUnited States/ ; U17 CE002009/CE/NCIPC CDC HHSUnited States/ ; ... More +

NIHR01-HL058115/HL/NHLBI NIH HHSUnited States/ ; R01AT-006822/AT/NCCIH NIH HHSUnited States/ ; R01-HL64937/HL/NHLBI NIH HHSUnited States/ ; P01-HL103455/HL/NHLBI NIH HHSUnited States/ ; U17 CE002009/CE/NCIPC CDC HHSUnited States/ ; P01 HL103455/HL/NHLBI NIH HHSUnited States/ ; R01 HL058115/HL/NHLBI NIH HHSUnited States/ ; R01 AT006822/AT/NCCIH NIH HHSUnited States/ ; R01 HL064937/HL/NHLBI NIH HHSUnited States/ Less -
Volume:

65
Collection(s):

CDC Public Access
Main Document Checksum:

[+]

urn:sha-512:36646e54e9f20dd4d9b20ccbfd87de02c83a9d31a7aea51d6d8d14ebae381bcf60299e69e6df26900ccee04690607064edfe3490c0884c617d6333f84959404c
Download URL:

https://stacks.cdc.gov/view/cdc/41966/cdc_41966_DS1.pdf
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