title: Characterization of intermediates in the oxidation of zinc fingers in human immunodeficiency virus type 1 nucleocapsid protein P7.
abstract: Oxidants targeted toward inactivation of the nucleocapsid zinc finger protein are under development as antiviral agents, especially for use against human immunodeficiency virus. In the present study, electrospray ionization-mass spectrometry is used to follow in situ the progress of the reactions of 2,2'-dithiodipyridine and disulfiram with recombinant nucleocapsid protein p7 (Ncp7) from human immunodeficiency virus-1 at pH 7.4. Both reagents react with the two zinc fingers in the protein, resulting in the ejection of two zinc ions and the formation of oxidized apo-Ncp7 with three intramolecular disulfide bonds. The ejection of zinc by 2,2'-dithiodipyridine occurs in two steps. Alkylation of unreacted cysteine residues with N-ethylmaleimide after a 2-min reaction with 2,2'-dithiodipyridine reveals that the carboxyl-terminal zinc finger is disrupted first. Cys-49, Cys-36, and, to a lesser extent, Cys-39 are all shown to be target residues for initial electrophilic attack. In the reaction of disulfiram with Ncp7, ejection of the two zinc ions also occurs in two steps; however, the fully oxidized apo-Ncp7 is formed more rapidly. Thus, after a 40-min reaction, 45% of native Ncp7 is oxidized by 2,2'-dithiodipyridine, whereas 75% is oxidized by disulfiram.
authors.affiliation: Structural Biochemistry Center, University of Maryland Baltimore County, 21250, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Hathout
authors.forename: D
authors.initials: D
authors.lastname: Fabris
authors.forename: M S
authors.initials: MS
authors.lastname: Han
authors.forename: R C
authors.initials: RC
authors.lastname: Sowder
authors.forename: L E
authors.initials: LE
authors.lastname: Henderson
authors.forename: C
authors.initials: C
authors.lastname: Fenselau
mesh_terms: D015082:2,2'-Dipyridyl / Q000031:analogs & derivatives / Q000737:chemistry; D002213:Capsid / Q000737:chemistry*; D036022:Capsid Proteins; D004220:Disulfides / Q000737:chemistry; D004221:Disulfiram / Q000737:chemistry; D005033:Ethylmaleimide / Q000737:chemistry; D015683:Gene Products, gag / Q000737:chemistry*; D015497:HIV-1 / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D010084:Oxidation-Reduction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013439:Sulfhydryl Reagents / Q000737:chemistry; D014764:Viral Proteins; D016335:Zinc Fingers / Q000187:drug effects*; D054301:gag Gene Products, Human Immunodeficiency Virus
chemical_list: D036022:Capsid Proteins; D004220:Disulfides; D015683:Gene Products, gag; C071884:NCP7 protein, Human immunodeficiency virus 1; D013439:Sulfhydryl Reagents; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; C001105:2,2'-dipyridyl disulfide; D015082:2,2'-Dipyridyl; D005033:Ethylmaleimide; D004221:Disulfiram