Structure of hemocyanin subunit CaeSS2 of the crustacean Mediterranean crab Carcinus aestuarii.
Arthropodan hemocyanins are giant respiratory proteins responsible for oxygen transport. They exhibit unusual assemblies of up to 48 structural subunits. Hemocyanin from Carcinus aestuarii contains three major and two minor structural subunits. Here, we reveal the primary structure of the gamma-type 75 kDa subunit of Carcinus aestuarii hemocyanin, CaeSS2, and combine structure-based sequence alignments, tryptophan fluorescence, and glycosylation analyses to provide insights into the structural and functional organisation of CaeSS2. We identify three functional domains and three conserved histidine residues that most likely participate in the formation of the copper active site in domain 2. Oxygen-binding ability of Carcinus aestuarii Hc and its structural subunit 2 was studied using CD and fluorescence spectroscopy. Removing the copper dioxygen system from the active site led to a decrease of the melting temperature, which can be explained by a stabilizing effect of the binding metal ion. To study the quenching effect of the active site copper ions in hemocyanins, the copper complex Cu(II)(PuPhPy)2+ was used, which appears as a very strong quencher of the tryptophan emission. Furthermore, the structural localization was clarified and found to explain the observed fluorescence behavior of the protein. Sugar analysis reveals that CaeSS2 is glycosylated, and oligosaccharide chains connected to three O-glycosylated and one N-glycosylated sites were found.
Dolashka-Angelova
Pavlina
P
Institute of Organic Chemistry, Bulgarian Academy of Sciences, G. Bonchev 9, Sofia 1113, Bulgaria. pda54@yahoo.com
Dolashki
Alexandar
A
Savvides
Savvas N
SN
Hristova
Rumyana
R
Van Beeumen
Jozef
J
Voelter
Wolfgang
W
Devreese
Bart
B
Weser
Ulrich
U
Di Muro
Paolo
P
Salvato
Benedetto
B
Stevanovic
Stefan
S
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003300:Copper / Q000737:chemistry; D003445:Crustacea; D006433:Hemocyanins / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010455:Peptides / Q000737:chemistry / Q000235:genetics; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics; D016415:Sequence Alignment
D010455:Peptides; D021122:Protein Subunits; D003300:Copper; D006433:Hemocyanins