Määttä JA et al. (2009), Structural and functional characteristics of xe...

XB-ART-40334

BMC Struct Biol 2009 Sep 29;9:63. doi: 10.1186/1472-6807-9-63.

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

Määttä JA , Helppolainen SH , Hytönen VP , Johnson MS , Kulomaa MS , Airenne TT , Nordlund HR .

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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. Xenavidin was identified from an EST sequence database for Xenopus tropicalis 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. 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.

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Species referenced: Xenopus tropicalis
Genes referenced: avd tbx2

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	Figure 1. Comparison of avidin, streptavidin and xenavidin. A, Structural alignment of avidin [PDB: 1AVD] (red), streptavidin [PDB: 1MK5] (cyan) and xenavidin (reported here; [PDB: 2UYW] (blue)). The A subunits of each protein are shown as ribbons. The bound biotin of xenavidin is shown as sticks. Loops connecting β-strands (for example, L1,2 connecting strands β1 and β2) as well as the amino (N) and carboxyl (C) termini are labelled. The α1-helix (H1) is also indicated. B, Selected residues around the biotin-binding pocket are shown as sticks and are labelled according to xenavidin. Biotin molecules are shown (1, xenavidin; 2 avidin; and 3 streptavidin). C, Structure-based sequence alignment created based on the ensemble of aligned structures shown in A. The secondary structure elements of avidin (β-strands 1-8, arrows; α-helix, H1) are indicated, and residues are numbered according to avidin (top) and xenavidin (bottom). Identical (red background) and physicochemically similar amino acid residues (red letters) are boxed. Potential N-glycosylation sites are indicated with an asterisk. Residues hydrogen bonded to biotin are indicated by triangles (side-chain interaction) or squares (main-chain interaction). Spheres denote residues involved in hydrophobic effect or van der Waals interactions with biotin. Interacting residues from a neighbouring subunit are indicated with an open circle. Colouring scheme for the symbols: black, conserved in all three proteins; green, conserved in xenavidin and avidin only; blue, unique to xenavidin; red, unique to avidin; and cyan, unique to streptavidin. For additional information, see Methods.
	Figure 2. Molecular surfaces of chicken avidin (A and D; [PDB: 1AVD]), xenavidin (B and E; [PDB: 2UYW]) and streptavidin (C and F; [PDB: 1MK5]). The surfaces were colored by electro potentials. The views in A, B and C are rotated 90 degrees around the x-axis in D, E and F, respectively. The subunits (I-IV) are numbered according to Livnah et al. [18]. The yellow arrows pinpoint the entry sites for biotin-binding pockets (A-C) and water channels (D-F). The valeric acid moiety end of bound biotin molecules (green spheres) is seen in C.
	Figure 3. Ligand binding to xenavidin. Simplified presentation of the ligand-binding sites: A, subunit A of unliganded xenavidin; B, subunit D of unliganded xenavidin; C, subunit A of the xenavidin-BTN complex; and D, subunit D of the xenavidin-BTN complex. Amino acid residues are numbered according to xenavidin and avidin (in brackets). Colouring scheme for the stick models: red, oxygen atoms; blue, nitrogen atoms; green, carbon atoms. Water molecules are shown as red spheres. ACT, acetate ion; BTN, D-biotin. Difference Fo-Fc electron density map (blue) contoured at 3σ around the bound ligands is depicted; the maps were calculated in the absence of ligands. An asterisk indicates the conserved water molecule found at the entrance of the water channel, at site 2 [33], and an arrow pinpoints the water molecule at site 1 [33] that was present only in the unliganded xenavidin structure (see text for details).
	Figure 4. Limited proteolysis of xenavidin and avidin by proteinase K. Samples from left to right: LWM, a molecular weight standard (weights in kDa, Fermentas Life Sciences); Avidin control sample; Avidin treated with proteinase K in the absence of biotin; Avidin treated with proteinase K in the presence of biotin; Proteinase K control sample (four times higher concentration of proteinase as compared to the other samples); Xenavidin control sample; Xenavidin treated with Proteinase K in the absence of biotin; Xenavidin treated with proteinase K in the presence of biotin. Black arrowheads indicate the putative single- (lower arrow) and double-glycosylated (upper arrow) xenavidin forms.
	Figure 5. Thermal stability of the tetrameric forms of xenavidin. SDS-PAGE -based analysis of xenavidin either in the absence of added biotin (-BTN) or saturated with biotin (+BTN) prior to analysis. The measurement temperatures, 22 (RT, room temperature), 30, 65, 70 and 75°C, are shown, and the molecular weights of the standard proteins (LWM; BioLabs) are indicated (kDa). This figure was created from the images of two different gels; the uninformative parts of the gel images are not shown.
	Figure 6. [3H]biotin dissociation analysis. The dissociation rate constant of d-[8,9-3H]biotin was measured at different temperatures. The values for bradavidin, streptavidin and avidin are from references [9,63] and [14], respectively.
	Figure 7. Dissociation analysis of fluorescent biotin. The dissociation of Bf560-labelled biotin was measured at 50°C.

References [+] :

Airenne, Production of biologically active recombinant avidin in baculovirus-infected insect cells. 1997, Pubmed