Structural and functional characteristics of xenavidin, the first frog avidin from Xenopus tropicalis

<p>Abstract</p> <p>Background</p> <p>Avidins are proteins with extraordinarily high ligand-binding affinity, a property which is used in a wide array of life science applications. Even though useful for biotechnology and nanotechnology, the biological function of avidins is not fully understood. Here we structurally and functionally characterise a novel avidin named xenavidin, which is to our knowledge the first reported avidin from a frog.</p> <p>Results</p> <p>Xenavidin was identified from an EST sequence database for <it>Xenopus tropicalis </it>and produced in insect cells using a baculovirus expression system. The recombinant xenavidin was found to be homotetrameric based on gel filtration analysis. Biacore sensor analysis, fluorescently labelled biotin and radioactive biotin were used to evaluate the biotin-binding properties of xenavidin - it binds biotin with high affinity though less tightly than do chicken avidin and bacterial streptavidin. X-ray crystallography revealed structural conservation around the ligand-binding site, while some of the loop regions have a unique design. The location of structural water molecules at the entrance and/or within the ligand-binding site may have a role in determining the characteristic biotin-binding properties of xenavidin.</p> <p>Conclusion</p> <p>The novel data reported here provide information about the biochemically and structurally important determinants of biotin binding. This information may facilitate the discovery of novel tools for biotechnology.</p

Chemical Linkage to Injected Tissues Is a Distinctive Property of Oxidized Avidin

We recently reported that the oxidized avidin, named AvidinOX®, resides for weeks within injected tissues as a consequence of the formation of Schiff's bases between its aldehyde groups and tissue protein amino groups. We also showed, in a mouse pre-clinical model, the usefulness of AvidinOX for the delivery of radiolabeled biotin to inoperable tumors. Taking into account that AvidinOX is the first oxidized glycoprotein known to chemically link to injected tissues, we tested in the mouse a panel of additional oxidized glycoproteins, with the aim of investigating the phenomenon. We produced oxidized ovalbumin and mannosylated streptavidin which share with avidin glycosylation pattern and tetrameric structure, respectively and found that neither of them linked significantly to cells in vitro nor to injected tissues in vivo, despite the presence of functional aldehyde groups. The study, extended to additional oxidized glycoproteins, showed that the in vivo chemical conjugation is a distinctive property of the oxidized avidin. Relevance of the high cationic charge of avidin into the stable linkage of AvidinOX to tissues is demonstrated as the oxidized acetylated avidin lost the property. Plasmon resonance on matrix proteins and cellular impedance analyses showed in vitro that avidin exhibits a peculiar interaction with proteins and cells that allows the formation of highly stable Schiff's bases, after oxidation

Bifunctional Avidin with Covalently Modifiable Ligand Binding Site

The extensive use of avidin and streptavidin in life sciences originates from the extraordinary tight biotin-binding affinity of these tetrameric proteins. Numerous studies have been performed to modify the biotin-binding affinity of (strept)avidin to improve the existing applications. Even so, (strept)avidin greatly favours its natural ligand, biotin. Here we engineered the biotin-binding pocket of avidin with a single point mutation S16C and thus introduced a chemically active thiol group, which could be covalently coupled with thiol-reactive molecules. This approach was applied to the previously reported bivalent dual chain avidin by modifying one binding site while preserving the other one intact. Maleimide was then coupled to the modified binding site resulting in a decrease in biotin affinity. Furthermore, we showed that this thiol could be covalently coupled to other maleimide derivatives, for instance fluorescent labels, allowing intratetrameric FRET. The bifunctional avidins described here provide improved and novel tools for applications such as the biofunctionalization of surfaces

Crystal structure of rhizavidin: insights into the enigmatic high-affinity interaction of an innate biotin-binding protein dimer

Rhizavidin, from the proteobacterium Rhizobium etli, exhibits high affinity towards biotin but maintains an inherent dimeric quaternary structure and thus, differs from all other known tetrameric avidins. Rhizavidin also differs from the other avidins, since it lacks the characteristic tryptophan residue positioned in the L7,8 loop that plays a crucial role in high-affinity binding and oligomeric stability of the tetrameric avidins. The question is, therefore, how does the dimer exist and how is the high biotin-binding affinity retained? For this purpose, the crystal structures of apo- and biotin-complexed rhizavidin were determined. The structures reveal that the rhizavidin monomer exhibits a topology similar to those of other members of the avidin family, that is, eight antiparallel β-strands that form the conventional avidin β-barrel. The quaternary structure comprises the sandwich-like dimer, in which the extensive 1–4 intermonomer interface is intact, but the 1–2 and 1–3 interfaces are nonexistent. Consequently, the biotin-binding site is partially accessible, due to the lack of the tryptophan “lid” that distinguishes the tetrameric structures. In rhizavidin, a disulfide bridge connecting the L3,4 and L5,6 loops restrains the L3,4 loop conformation, leaving the binding-site residues essentially unchanged upon biotin binding. Our study suggests that in addition to the characteristic hydrogen bonding and hydrophobic interactions, the preformed architecture of the binding site and consequent shape complementarity play a decisive role in the high-affinity biotin binding of rhizavidin. The structural description of a novel dimeric avidin-like molecule will greatly contribute to the design of improved and unique avidin derivatives for diversifying the capabilities of avidin–biotin technology

Structural characterization of core-bradavidin in complex with biotin

<div><p>Bradavidin is a tetrameric biotin-binding protein similar to chicken avidin and bacterial streptavidin, and was originally cloned from the nitrogen-fixing bacteria <i>Bradyrhizobium diazoefficiens</i>. We have previously reported the crystal structure of the full-length, wild-type (wt) bradavidin with 138 amino acids, where the C-terminal residues Gly129-Lys138 (“Brad-tag”) act as an intrinsic ligand (<i>i</i>.<i>e</i>. Gly129-Lys138 bind into the biotin-binding site of an adjacent subunit within the same tetramer) and has potential as an affinity tag for biotechnological purposes. Here, the X-ray structure of core-bradavidin lacking the C-terminal residues Gly114-Lys138, and hence missing the Brad-tag, was crystallized in complex with biotin at 1.60 Å resolution [PDB:4BBO]. We also report a homology model of rhodavidin, an avidin-like protein from <i>Rhodopseudomonas palustris</i>, and of an avidin-like protein from <i>Bradyrhizobium sp</i>. Ai1a-2, both of which have the Brad-tag sequence at their C-terminus. Moreover, core-bradavidin V1, an engineered variant of the original core-bradavidin, was also expressed at high levels in <i>E</i>. <i>coli</i>, as well as a double mutant (Cys39Ala and Cys69Ala) of core-bradavidin (CC mutant). Our data help us to further engineer the core-bradavidin–Brad-tag pair for biotechnological assays and chemical biology applications, and provide deeper insight into the biotin-binding mode of bradavidin.</p></div