Determination of the Solution Structure of the N-Domain Plus Linker of Antarctic Eel Pout Antifreeze Protein RD3

• Published in: Journal of biochemistry (Tokyo) 1999-08, Vol.126 (2), p.387-394
• Main Authors: Miura, Kazunori, Ohgiya, Satoru, Hoshino, Tamotsu, Nemoto, Nobuaki, Odaira, Masato, Nitta, Ratsutoshi, Tsuda, Sakae
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Antarctic Regions; antifreeze protein RD3; Antifreeze Proteins, Type III; Base Sequence; Eels - blood; Escherichia coli - metabolism; Freezing; Glycoproteins - chemistry; linker; Magnetic Resonance Spectroscopy; Marine; Models, Molecular; Molecular Sequence Data; NMR structure; Protein Structure, Tertiary; Proteins - chemistry; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification
• ISSN: 0021-924X
• DOI: 10.1093/oxfordjournals.jbchem.a022462

RD3, a new antifreeze protein (AFP) extracted from antarctic eel pout is a single polypep-tide divided into homologous N-terminal (residues Asn1-Glu64) and C-terminal (residues Ser74-Glu134) domains, each of which has a high sequence identity with Type III AFP. A 9-residue linker (-D65GTTSPGLK73-) 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 15N-NMR backbone dynamics data of the N-domain plus the linker of recombinant RD3 protein (RD3-N1: residues 1–73) by employing homo- and heteronuclear multidimensional NMR spectros-copy. Forty converged structures of RD3-N1 were successfully calculated by using a total of 958 NMR-derived structural restraints. It was found that the N-domain of RD3-N1 has a globular form comprising six β-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, -D65GTTSP70- and -G71LK73-, which are bent around -T67TSPG71- at an angle of approximately 604. 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.