PMID- 17337004
OWN - NLM
STAT- MEDLINE
DCOM- 20070606
LR  - 20181201
IS  - 0022-2836 (Print)
IS  - 0022-2836 (Linking)
VI  - 368
IP  - 1
DP  - 2007 Apr 20
TI  - Molecular basis of guanine nucleotide dissociation inhibitor activity of human 
      neuroglobin by chemical cross-linking and mass spectrometry.
PG  - 150-60
AB  - 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.
FAU - Kitatsuji, Chihiro
AU  - Kitatsuji C
AD  - Department of Molecular Engineering, Graduate School of Engineering, Kyoto 
      University, Kyoto 615-8510, Japan.
FAU - Kurogochi, Masaki
AU  - Kurogochi M
FAU - Nishimura, Shin-Ichiro
AU  - Nishimura S
FAU - Ishimori, Koichiro
AU  - Ishimori K
FAU - Wakasugi, Keisuke
AU  - Wakasugi K
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20070207
PL  - Netherlands
TA  - J Mol Biol
JT  - Journal of molecular biology
JID - 2985088R
RN  - 0 (Cross-Linking Reagents)
RN  - 0 (GTP-Binding Protein alpha Subunits)
RN  - 0 (GTP-Binding Protein beta Subunits)
RN  - 0 (Guanine Nucleotide Dissociation Inhibitors)
RN  - 0 (Nerve Tissue Proteins)
RN  - 0 (Neuroglobin)
RN  - 0 (Recombinant Fusion Proteins)
RN  - 9004-22-2 (Globins)
SB  - IM
MH  - Amino Acid Sequence
MH  - Animals
MH  - Cattle
MH  - Cross-Linking Reagents/*pharmacology
MH  - GTP-Binding Protein alpha Subunits/metabolism
MH  - GTP-Binding Protein beta Subunits/metabolism
MH  - Globins/*chemistry/*metabolism
MH  - Guanine Nucleotide Dissociation Inhibitors/*chemistry/*metabolism
MH  - Humans
MH  - Models, Biological
MH  - Models, Molecular
MH  - Molecular Sequence Data
MH  - Nerve Tissue Proteins/*chemistry/*metabolism
MH  - Neuroglobin
MH  - Protein Binding
MH  - Rats
MH  - Recombinant Fusion Proteins/chemistry/metabolism
MH  - *Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
EDAT- 2007/03/06 09:00
MHDA- 2007/06/07 09:00
CRDT- 2007/03/06 09:00
PHST- 2006/11/09 00:00 [received]
PHST- 2007/02/01 00:00 [revised]
PHST- 2007/02/01 00:00 [accepted]
PHST- 2007/03/06 09:00 [pubmed]
PHST- 2007/06/07 09:00 [medline]
PHST- 2007/03/06 09:00 [entrez]
AID - S0022-2836(07)00165-9 [pii]
AID - 10.1016/j.jmb.2007.02.002 [doi]
PST - ppublish
SO  - J Mol Biol. 2007 Apr 20;368(1):150-60. doi: 10.1016/j.jmb.2007.02.002. Epub 2007 
      Feb 7.