Crystal structure of apo-avidin from hen egg-white

Pugliese, Luisa; Malcovati, Massimo; Coda, Alessandro; Bolognesi, Martino

doi:10.1016/s0022-2836(05)80010-5

Cited by 50 publications

(56 citation statements)

References 14 publications

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“…The limited proteolytic attack of avidin by proteinase K, the total prevention of this event by biotin, and the enhancement of the process by HABA can be interpreted in terms of the structure of apo-and holoavidin (Livnah et al, 1993a;Pugliese et al, 1994). The factors that influence susceptibility of native proteins to proteolytic attack are largely unknown.…”

Section: Discussionmentioning

confidence: 99%

Limited proteolysis of native proteins: The interaction between avidin and proteinase K

et al. 1995

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Avidin is a tetramer of 16-kDa subunits that have a high affinity for biotin. Proteolysis of native apoavidin by proteinase K results in a limited attack at the loop between P-strands 3 and 4, involving amino acids 38-43. Specifically, sites of proteolysis are at Thr 40-Ser 41 and Asn 42-Glu 43. The limited proteolysis results in an avidin product that remains otherwise intact and which has enhanced binding for 4'-hydroxyazobenzene-2-benzoic acid (HABA), a chromogenic reporter that can occupy the biotin-binding site. Saturation of the biotin-binding site with the natural ligand protects avidin from proteolysis, but saturation with HABA enhances the rate of proteolysis of the same site.Analysis of the three-dimensional structures of apoavidin and holoavidin reveals that the 3-4 loop is accessible to solvent and scores highly in an algorithm developed to identify sites of proteolytic attack. The structure of holoavidin is almost identical to the apoprotein. In particular, the 3-4 loop has the same structure in the apo and holo forms, yet there are marked differences in proteolytic susceptibility of this region. Evidence suggests that the 3-4 loop is rather mobile and flexible in the apoprotein, and that it becomes constrained upon ligand binding. In one crystal structure of the apoprotein, this loop appears constrained by contacts with symmetry-related molecules. Structural analyses suggest that the "lid" to the biotin-binding site, formed by the 3-4 loop, is displaced and made more accessible by HABA binding, thereby enhancing its proteolytic susceptibility.

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Section: Discussionmentioning

confidence: 99%

Limited proteolysis of native proteins: The interaction between avidin and proteinase K

et al. 1995

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“…Structure solution of recombinant avidin was achieved through molecular-replacement techniques, using the atomic coordinates of hen egg-white apo-avidin dimer as search model [6,15]. Both the rotational search (run in the 0.8Ϫ0.3 nm resolution range; final correlation coefficient 29.1) as well as the transrational search (run in the 0.75Ϫ0.4-nm resolution range; final correlation coefficient 67.7) provided unambiguous solutions (final R factor 0.385).…”

Section: Methodsmentioning

confidence: 99%

Biochemical characterization and crystal structure of a recombinant hen avidin and its acidic mutant expressed in Escherichia coli

Nardone

Rosano

Santambrogio

et al. 1998

European Journal of Biochemistry

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The mature hen avidin encoded by a synthetic cDNA was expressed in Escherichia coli in an insoluble form. After resolubilization, renaturation and purification, a recovery of about 20 mg/l cell culture was obtained. ELISA assays indicated no apparent differences in biotin binding between the natural and recombinant avidins. In addition, an acidic avidin mutant, bearing the substitutions Lys3→Glu, Lys9→ Glu, Arg26→Asp and Arg124→Leu of four exposed basic residues, was produced. The protein, expressed and renatured as wild-type avidin, showed unaltered biotin-binding activity. The acidic pI (Ϸ5.5) and lack of aggregation of the mutant allowed easy electrophoretic analysis under non-denaturing conditions of the protein alone and of its complexes with biotin, biotinylated transferrin or peroxidase. Analysis of the sera from sensitized subjects revealed that the avidin mutant has altered antigenicity. Both recombinant avidins were crystallized and the three-dimensional structures solved by molecular replacement and refined to 0.22 nm resolution. The three-dimensional structures of the two recombinant molecules, in the absence of biotin and of glycosylation, are fully comparable with those of the natural hen avidin previously reported.Keywords : avidin; crystal structure ; mutagenesis ; recombinant protein.Avidin, a minor constituent of egg white of reptiles, amphibia and birds, is a glycosylated and positively charged protein which can bind up to four molecules of vitamin H, D-biotin [1]. The interaction with biotin is non-covalent but extremely tight, the dissociation constant of about 1 fM being about 10 3 Ϫ10 6 times higher than that of a typical antigen-antibody interaction. The cDNA-derived and protein-derived sequences [2,3] and the crystallographic structures of avidin [4Ϫ6] are known. Functional avidin is a tetramer of identical subunits and each subunit is folded into an eight-stranded anti-parallel β-barrel displaying up-and-down topology. The biotin-binding site is located in the core of the barrel, and is built by residues of the barrel itself and partly by a loop of an adjacent subunit. The interest in avidin derives mainly from several applications based on its rapid and almost irreversible binding to biotin and to practically any biotin-based molecular construct [7,8]. Avidin is used in vivo for the targeting of solid tumors [9] in applications where proteinsurface properties are determinants for proper biodistribution, serum clearance and immunological response [10]. The production of recombinant avidin and its mutated forms could increase the already remarkable versatility of this protein and may improve its properties for in vivo use.

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“…The avidin binding site is also relatively hidden, as can be seen in Figure 1. Crystal structures of the apo state shows a varying number of water molecules in the ligand-free binding site: 69 two in one subunit, one in another, and no water molecules in the other two subunits. The reason for this could be the rather poor quality of the structure, that the water molecules in the binding site are disordered, or that water molecules diffuse readily between the binding site and the bulk solvent.…”

Section: Ps Lmentioning

confidence: 99%

Accurate Predictions of Nonpolar Solvation Free Energies Require Explicit Consideration of Binding-Site Hydration

Genheden

Mikulskis

et al. 2011

J. Am. Chem. Soc.

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Continuum-solvation methods are frequently used to increase the efficiency of computational methods to estimate free energies. In this paper, we have evaluated how well such methods estimate the non-polar solvation free-energy change when a ligand binds to a protein. Three different continuum methods at various levels of approximation were considered, viz. the polarised continuum model (PCM), a method based on cavity and dispersion terms (CD), and a method based on a linear relation to the solvent-accessible surface area (SASA). Formally rigorous double-decoupling thermodynamic integration was used as a benchmark for the continuum methods. We have studied four protein-ligand complexes with binding sites of varying solvent exposure, namely the binding of phenol to ferritin, a biotin analogue to avidin, 2-aminobenzimidazole to trypsin, and a substituted galactoside to galectin-3. For ferritin and avidin, which have relatively hidden binding sites, rather accurate non-polar solvation free energies could be obtained with the continuum methods if the binding site is prohibited to be filled by continuum water in the unbound state, even though experiments show that the ligand replaces several water molecules upon binding. For the more solvent exposed binding sites of trypsin and galectin-3, no accurate continuum estimates could be obtained, even if the binding site was allowed or prohibited to be filled by continuum water. This shows that continuum methods fail to give accurate free energies on a wide range of systems with varying solvent exposure, because they lack a microscopic picture of bindingsite hydration as well as information about the entropy of water molecules that are in the binding site before the ligand binds. Consequently, binding-affinity estimates based upon continuum solvation methods will give absolute binding energies that may differ by up to 200 kJ/mol depending on the method used. Moreover, even relative energies between ligands with the same scaffold may differ by up to 75 kJ/mol. We have tried to improve the continuumsolvation methods by adding information about the solvent exposure of the binding site or of the hydration of the binding site and the results are promising at least for this small set of complexes.2

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Crystal structure of apo-avidin from hen egg-white

Cited by 50 publications

References 14 publications

Limited proteolysis of native proteins: The interaction between avidin and proteinase K

Limited proteolysis of native proteins: The interaction between avidin and proteinase K

Biochemical characterization and crystal structure of a recombinant hen avidin and its acidic mutant expressed in Escherichia coli

Accurate Predictions of Nonpolar Solvation Free Energies Require Explicit Consideration of Binding-Site Hydration

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