Molecular basis of guanine nucleotide dissociation inhibitor activity of human neuroglobin by chemical cross-linking and mass spectrometry.
Oxidized human neuroglobin (Ngb), a heme protein expressed in the brain, has been proposed to act as a guanine nucleotide dissociation inhibitor (GDI) for the GDP-bound form of the heterotrimeric G protein alpha-subunit (Galpha(i)). Here, to elucidate the molecular mechanism underlying the GDI activity of Ngb, we used an glutathione-S-transferase pull-down assay to confirm that Ngb competes with G-protein betagamma-subunits (Gbetagamma) for binding to Galpha(i), and identified the Galpha(i)-binding site in Ngb by chemical cross-linking with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and sulfo-N-hydroxysuccinimide, coupled with mass spectrometry (MS). Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS analysis for tryptic peptides derived from the cross-linked Ngb-Galpha(i) complex revealed several binding regions in Ngb. Furthermore, MALDI-TOF/TOF MS analysis of the cross-linked Ngb and Galpha(i) peptides, together with the MS/MS scoring method, predicted cross-linking between Glu60 (Ngb) and Ser206 (Galpha(i)), and between Glu53 (Ngb) and Ser44 (Galpha(i)). Because Ser206 of Galpha(i) is located in the region that contacts Gbetagamma, binding of Ngb could facilitate the release of Gbetagamma from Galpha(i). Binding of Ngb to Galpha(i) would also inhibit the exchange of GDP for GTP, because Ser44 (Galpha(i)) is adjacent to the GDP-binding site and Glu53 (Ngb), which is cross-linked to Ser44 (Galpha(i)), could be located close to GDP. Thus, we have identified, for the first time, the sites of interaction between Ngb and Galpha(i), enabling us to discuss the functional significance of this binding on the GDI activity of Ngb.
Kitatsuji
Chihiro
C
Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan.
Kurogochi
Masaki
M
Nishimura
Shin-Ichiro
S
Ishimori
Koichiro
K
Wakasugi
Keisuke
K
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D044385:GTP-Binding Protein alpha Subunits / Q000378:metabolism; D044387:GTP-Binding Protein beta Subunits / Q000378:metabolism; D005914:Globins / Q000737:chemistry* / Q000378:metabolism*; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000378:metabolism*; D000078787:Neuroglobin; D011485:Protein Binding; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D003432:Cross-Linking Reagents; D044385:GTP-Binding Protein alpha Subunits; D044387:GTP-Binding Protein beta Subunits; D020730:Guanine Nucleotide Dissociation Inhibitors; D009419:Nerve Tissue Proteins; D000078787:Neuroglobin; D011993:Recombinant Fusion Proteins; D005914:Globins