期刊
JOURNAL OF MOLECULAR BIOLOGY
卷 368, 期 1, 页码 150-160出版社
ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2007.02.002
关键词
neuroglobin; guanine nucleotide dissociation inhibitor; cross-linking; MALDI-TOF 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 (G(xi). Here, to elucidate the molecular mechanism underlying the GD1 activity of Ngb, we used an glutathione-S-transferase pun-down assay to confirm that Ngb competes with G-protein beta gamma-subunits (G beta gamma) for binding to G alpha(i), and identified the G alpha(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-G alpha(i) complex revealed several binding regions in Ngb. Furthermore, MALDI-TOF/TOF MS analysis of the cross-linked Ngb and Gai peptides, together with the MS/MS scoring method, predicted cross-linking between Glu60 (Ngb) and Ser206 (G alpha(i)), and between Glu53 (Ngb) and Ser44 (G alpha(i)). Because Ser206 of G alpha(i) is located in the region that contacts G beta gamma, binding of Ngb could facilitate the release of G beta gamma from G alpha(i). Binding of Ngb to Gai would also inhibit the exchange of GDP for GTP, because Ser44 (G alpha(i)) is adjacent to the GDP-binding site and Glu53 (Ngb), which is cross-linked to Ser44 (G alpha(i)), could be located close to GDP. Thus, we have identified, for the first time, the sites of interaction between Ngb and Gai, enabling us to discuss the functional significance of this binding on the GDI activity of Ngb. (c) 2007 Elsevier Ltd. All rights reserved.
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