title: Inhibition of Mycobacterium tuberculosis PknG by non-catalytic rubredoxin domain specific modification: reaction of an electrophilic nitro-fatty acid with the Fe-S center.
abstract: 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.
authors.affiliation: Unidad de Bioquímica y Proteómica Analíticas, Institut Pasteur de Montevideo, Uruguay; Unidad de Bioquímica y Proteómica Analíticas, Instituto de Investigaciones Biológicas Clemente Estable, Ministerio de Educación y Cultura, Uruguay.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Gil
authors.affiliation: Unidad de Bioinformática, Institut Pasteur de Montevideo, Uruguay.
authors.forename: Martín
authors.initials: M
authors.lastname: Graña
authors.affiliation: Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA.
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Schopfer
authors.affiliation: Unité de Microbiologie Structurale & CNRS URA 2185, Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris Cedex 15, France.
authors.forename: Tristan
authors.initials: T
authors.lastname: Wagner
authors.affiliation: Laboratorio de Fisicoquímica Biológica, Facultad de Ciencias, Universidad de la República, Uruguay.
authors.forename: Ana
authors.initials: A
authors.lastname: Denicola
authors.affiliation: Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA.
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Freeman
authors.affiliation: Unité de Microbiologie Structurale & CNRS URA 2185, Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris Cedex 15, France.
authors.forename: Pedro M
authors.initials: PM
authors.lastname: Alzari
authors.affiliation: Unidad de Bioquímica y Proteómica Analíticas, Institut Pasteur de Montevideo, Uruguay; Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Uruguay. Electronic address: batthyany@pasteur.edu.uy.
authors.forename: Carlos
authors.initials: C
authors.lastname: Batthyány
authors.affiliation: Unidad de Bioquímica y Proteómica Analíticas, Institut Pasteur de Montevideo, Uruguay; Unidad de Bioquímica y Proteómica Analíticas, Instituto de Investigaciones Biológicas Clemente Estable, Ministerio de Educación y Cultura, Uruguay. Electronic address: duran@pasteur.edu.uy.
authors.forename: Rosario
authors.initials: R
authors.lastname: Durán
mesh_terms: D000475:Alkenes / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D020134:Catalytic Domain / Q000502:physiology; D002942:Circular Dichroism; D005227:Fatty Acids / Q000737:chemistry / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009169:Mycobacterium tuberculosis / Q000378:metabolism*; D009574:Nitro Compounds / Q000737:chemistry / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000378:metabolism*; D012416:Rubredoxins / Q000737:chemistry / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D000475:Alkenes; D001426:Bacterial Proteins; D005227:Fatty Acids; D009574:Nitro Compounds; D012416:Rubredoxins; C484411:PknH protein, Mycobacterium tuberculosis; D017346:Protein Serine-Threonine Kinases
keywords: 8-anilino-1-naphthalenesulfonic acid ammonium salt; 9- and 10-nitro-9-cis-octadecaenoic acids; 9-octadecenoic acid; ANS; BPS; ESI; IAM; IPTG; LC; Mycobacterium tuberculosis; Nitrated fatty acids; Nitroalkene; OA oleic acid; OA-NO(2) nitro-oleic acid; PknG; Rbx; Rubredoxin; Ser/Thr kinase; bathophenanthroline disulfonate; electrospray ionization; iodoacetamide; isopropyl β-D-1-thiogalactopyranoside; liquid chromatography; rubredoxin