Determination of the solution structure of the N-domain plus linker of Antarctic eel pout antifreeze protein RD3.
RD3, a new antifreeze protein (AFP) extracted from antarctic eel pout is a single polypeptide divided into homologous N-terminal (residues Asn(1)-Glu(64)) and C-terminal (residues Ser(74)-Glu(134)) domains, each of which has a high sequence identity with Type III AFP. A 9-residue linker (-D(65)GTTSPGLK(73)-) connects these two domains in tandem and is thought to play a significant role in defining the nature of the intact molecule. The present paper shows for the first time the solution structure and preliminary (15)N-NMR backbone dynamics data of the N-domain plus the linker of recombinant RD3 protein (RD3-Nl: residues 1-73) by employing homo- and heteronuclear multidimensional NMR spectroscopy. Forty converged structures of RD3-Nl were successfully calculated by using a total of 958 NMR-derived structural restraints. It was found that the N-domain of RD3-Nl has a globular form comprising six beta-strands, three type III turns, and several loops, which stabilize a flat, ice-binding site formed on one side of this domain. Further, the linker portion appears to have a definitive structure, which is independent of the globular N-domain. This definitive linker is roughly divided into two short strands, -D(65)GTTSP(70)- and -G(71)LK(73)-, which are bent around -T(67)TSPG(71)- at an angle of approximately 60 degrees. This bending motif of the linker may function to orient the two ice-binding sites of the N- and C-domains of RD3 in the same direction, leading to their simultaneous interactions with the ice crystal surface.
Miura
K
K
Division of Biological Sciences, Graduate School of Science, Hokkaido University, Kita-ku, Sapporo, 060-0808, Japan.
Ohgiya
S
S
Hoshino
T
T
Nemoto
N
N
Odaira
M
M
Nitta
K
K
Tsuda
S
S
D000595:Amino Acid Sequence; D000818:Animals; D000864:Antarctic Regions; D021343:Antifreeze Proteins, Type III; D001483:Base Sequence; D004524:Eels / Q000097:blood*; D004926:Escherichia coli / Q000378:metabolism; D005615:Freezing; D006023:Glycoproteins / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification
D021343:Antifreeze Proteins, Type III; D006023:Glycoproteins; D011506:Proteins; C092813:RD3 protein, eel; D011994:Recombinant Proteins