A putative consensus sequence for the nucleotide-binding site of annexin A6.
Reaction-induced infrared difference spectroscopy (RIDS) has been used to investigate the nature of interactions of human annexin A6 (ANXA6) with nucleotides. RIDS results for ANXA6, obtained after the photorelease of GTP-gamma-S, ATP, or P(i) from the respective caged compounds, were identical, suggesting that the interactions between the nucleotide and ANXA6 were dominated by the phosphate groups. Phosphate-induced structural changes in ANXA6 were small and affected only seven or eight amino acid residues. The GTP fluorescent analogue, 2'(3')-O-(2,4,6-trinitrophenyl)guanosine 5'-triphosphate (TNP-GTP), quenched tryptophan fluorescence of ANXA6 when bound to the protein. A binding stoichiometry of 1 mol of nucleotide/mol ANXA6 was established with a K(D) value of 2.8 microM for TNP-GTP. The bands observed on RIDS of ANXA6 halves (e.g., N-terminal half, ANXA6a, and C-terminal half, ANXA6b) were similar to those of the whole molecule. However, their amplitudes were smaller by a factor of 2 compared to those of whole ANXA6. TNP-GTP bound to both fragments of ANXA6 with a stoichiometry of 0.5 mol/mol. However, the binding affinities of ANXA6a and ANXA6b differed from that of ANXA6. Simulated molecular modeling revealed a nucleotide-binding site which was distributed in two distinct domains. Residues K296, Y297, K598, and K644 of ANXA6 were less than 3 A from the bound phosphate groups of either GTP or ATP. The presence of two identical sequences in ANXA6 with the F-X-X-K-Y-D/E-K-S-L motif, located in the middle of ANXA6, at residues 293-301 (within ANXA6a) and at 641-649 (within ANXA6b), suggested that the F-X-X-K-Y-D/E-K-S-L motif was the putative sequence in ANXA6 for nucleotide binding.
Bandorowicz-Pikula
Joanna
J
Department of Cellular Biochemistry, Nencki Institute of Experimental Biology, 02-093 Warsaw, Poland. bandor@nencki.gov.pl
Kirilenko
Aneta
A
van Deursen
Ruud
R
Golczak
Marcin
M
Kühnel
Michael
M
Lancelin
Jean-Marc
JM
Pikula
Slawomir
S
Buchet
René
R
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D017317:Annexin A6 / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D003198:Computer Simulation; D016384:Consensus Sequence; D018360:Crystallography, X-Ray; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000737:chemistry / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010710:Phosphates / Q000737:chemistry / Q000378:metabolism; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D017550:Spectroscopy, Fourier Transform Infrared; D013873:Thionucleotides / Q000737:chemistry / Q000378:metabolism
D017317:Annexin A6; D010710:Phosphates; D020033:Protein Isoforms; D011994:Recombinant Proteins; D013873:Thionucleotides; C061507:P(3)-1-(2-nitro)phenylethylguanosine 5'-O-triphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate