Characterization of the zinc-binding site of the histidine-proline-rich glycoprotein associated with rabbit skeletal muscle AMP deaminase.
The AMP deaminase-associated variant of histidine-proline-rich glycoprotein (HPRG) is isolated from rabbit skeletal muscle by a modification of the protocol previously used for the purification of AMP deaminase. This procedure yields highly pure HPRG suitable for investigation by x-ray absorption spectroscopy of the zinc-binding behavior of the protein. X-ray absorption spectroscopy analysis of a 2:1 zinc-HPRG complex shows that zinc is bound to the protein, most probably in a dinuclear cluster where each Zn(2+) ion is coordinated, on average, by three histidine ligands and one heavier ligand, likely a sulfur from a cysteine. 11 cysteines of HPRG from different species are totally conserved, suggesting that five disulfide bridges are essential for the proper folding of the protein. At least another cysteine is present at different positions in the histidine-proline-rich domain of HPRG in all species, suggesting that this cysteine is the candidate for zinc ligation in the muscle variant of HPRG. The same conclusion is likely to be true for the six histidines used by the protein as zinc ligands. The presence in muscle HPRG of a specific zinc-binding site permits us to envisage the addition of HPRG into the family of metallochaperones. In this view, HPRG may enhance the in vivo stability of metalloenzymes such as AMP deaminase.
Mangani
Stefano
S
Dipartimento di Chimica, Università di Siena, Via Aldo Moro, 53100-Siena, Italy.
Meyer-Klaucke
Wolfram
W
Moir
Arthur J G
AJ
Ranieri-Raggi
Maria
M
Martini
Daniela
D
Raggi
Antonio
A
D000659:AMP Deaminase / Q000302:isolation & purification / Q000378:metabolism*; D000209:Acute-Phase Proteins / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008958:Models, Molecular; D018482:Muscle, Skeletal / Q000201:enzymology*; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D011817:Rabbits; D013056:Spectrophotometry, Ultraviolet; D015032:Zinc / Q000378:metabolism*; D000099053:Histidine-Rich Glycoprotein
D000209:Acute-Phase Proteins; D011506:Proteins; D000099053:Histidine-Rich Glycoprotein; D000659:AMP Deaminase; D015032:Zinc