Biomolecular Engineering 16 (1999) 5–12 The X-ray three-dimensional structure of avidin Camillo Rosano a , Paolo Arosio b , Martino Bolognesi a,c, * a Centro Biotecnologie Aanzate -IST and Dipartimento di Fisica -INFM, Uniersity of Genoa. Largo Rosanna Benzi, 10 I -16132 Genoa, Italy b Dibit, Department of Biological and Technological Research, San Raffaele Scientific Institute, Via Olgettina, 60 I -20132 Milan, Italy c Department of Genetics and Microbiology, Uniersity of Paia, Via Abbiategrasso, 207 I -27100 Paia, Italy Abstract Avidin is a basic, highly stable, homotetrameric protein, isolated from bird egg-white, binding up to four molecules of D-biotin with extremely high affinity (K d 10 -15 M). The protein has been the object of different crystallographic investigations. In all the crystal structures, the four avidin subunits display almost exact 222 symmetry. Each avidin chain (128 amino acids) is arranged in a eight-stranded antiparallel -barrel, whose inner region defines the D-biotin binding site. The molecular bases of D-biotin affinity can be recognised in a fairly rigid binding site, which is sterically complementary to the shape and polarity of the incoming vitamin, and is readily accessible in the apoprotein structure. Avidin displays remarkable structural and functional relationships to the acidic protein sretpavidin, isolated from Streptomyces aidinii. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Hen egg-white avidin; Crystal structure; Biotin; Calycins; Avidin biotechnology www.elsevier.com/locate/geneanabioeng 1. Avidin Avidin is a glycosylated and positively charged protein, found as a minor component in the egg-white of birds, reptiles and amphibia. It is secreted as a highly stable homo-tetrameric protein, which, in hen egg- white, is composed of 4 ×128 amino acid residue chains (M r 62 400) [1–3]. Avidin binds up to four molecules of vitamin H, D-biotin, in a non-covalent interaction which is extremely tight, the affinity for the vitamin (K d 10 -15 M) being three to six orders of magnitude higher than that of typical antigen:antibody complexes [1,3,4]. Due to such a strong avidity for biotin, avidin has been proposed to act as an antibiotic protein inhibiting bacterial growth. However, other bio- logical functions, including weak esterolytic activity, have been proposed [5,6]. Together with two other families of small-ligand binding proteins, the lipocalins and the fatty-acid binding proteins, avidin belongs to the ‘calycins’ protein structural superfamily [7]. Avidin is structurally and functionally related to streptavidin, a slightly acidic tetrameric protein from Streptomyces aidinii, composed of four identical chains, each 159 amino acid residues long [8] (see articles by Weber and by Hendrickson in this issue). A segment of 124 core residues of streptavidin shares 32% amino acid identities, when compared to the full chain of avidin. The conserved regions are distributed through six primary structure short segments, which are partly related to biotin recognition and binding in both proteins, as shown by chemical protection studies and spectroscopic investigations [1,9 – 11] (see Fig. 1). Avidin is a substantially thermostable protein; the t m for the unfolding transition of the biotin-depleted protein is 85°C, in the 7–9 pH range. Biotin binding increases substantially the protein t m (132°C) and the denaturation enthalpy [12,13]. Moreover, the avidin – biotin complex is stable under strong chemically denat- urant conditions (9 M urea or 7 M guanidinium hydrochloride) over a wide pH range [14]. The interest in avidin (and streptavidin) stems from the rapid and almost irreversible binding to biotin, which supports several applications for in vitro and in vivo research. Indeed, when biotin is covalently linked through its valeric acid moiety and a suitable spacer arm to almost any compound, it is still available for specific and tight binding to tetrameric avidin. Thus, Abbreiations: Avidin, hen egg-white avidin; Biotin, D-biotin; FTIR, Fourier transform infrared spectroscopy; NAG, N-acetylglu- coseamine; PEG, polyethylene glycol; rmsd, root mean square devia- tion. * Corresponding author. Tel./fax: +39-10-5737306. E-mail address: bolognes@fisica.unige.it (M. Bolognesi) 1389-0344/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII:S1050-3862(99)00047-9