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.2014 Jan 9;426(1):199-214.
doi: 10.1016/j.jmb.2013.09.016. Epub 2013 Sep 19.

Plug-and-play pairing via defined divalent streptavidins

Affiliations

Plug-and-play pairing via defined divalent streptavidins

Michael Fairhead et al. J Mol Biol..

Abstract

Streptavidin is one of the most important hubs for molecular biology, either multimerizing biomolecules, bridging one molecule to another, or anchoring to a biotinylated surface/nanoparticle. Streptavidin has the advantage of rapid ultra-stable binding to biotin. However, the ability of streptavidin to bind four biotinylated molecules in a heterogeneous manner is often limiting. Here, we present an efficient approach to isolate streptavidin tetramers with two biotin-binding sites in a precise arrangement, cis or trans. We genetically modified specific subunits with negatively charged tags, refolded a mixture of monomers, and used ion-exchange chromatography to resolve tetramers according to the number and orientation of tags. We solved the crystal structures of cis-divalent streptavidin to 1.4Å resolution and trans-divalent streptavidin to 1.6Å resolution, validating the isolation strategy and explaining the behavior of the Dead streptavidin variant. cis- and trans-divalent streptavidins retained tetravalent streptavidin's high thermostability and low off-rate. These defined divalent streptavidins enabled us to uncover how streptavidin binding depends on the nature of the biotin ligand. Biotinylated DNA showed strong negative cooperativity of binding to cis-divalent but not trans-divalent streptavidin. A small biotinylated protein bound readily to cis and trans binding sites. We also solved the structure of trans-divalent streptavidin bound to biotin-4-fluorescein, showing how one ligand obstructs binding to an adjacent biotin-binding site. Using a hexaglutamate tag proved a more powerful way to isolate monovalent streptavidin, for ultra-stable labeling without undesired clustering. These forms of streptavidin allow this key hub to be used with a new level of precision, for homogeneous molecular assembly.

Keywords: 2-methyl-2,4-pentanediol; LDLR; MPD; PBS; PDB; Protein Data Bank; avidin; bivalent; low-density lipoprotein receptor; nanotechnology; phosphate-buffered saline; protein design; supramolecular.

© 2013. Published by Elsevier Ltd. All rights reserved.

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Figures

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Graphical abstract
Fig. 1
Fig. 1
Principle of defined divalent streptavidins. Numbering of subunits in the streptavidin tetramer, with each monomer colored separately and biotin in space-fill (from PDB ID:3RY2). Below are arrangements of 1,2 or 1,3 biotin-binding sites (biotin-bound monomers, green; non-binding monomers, blue), showing relative orientations and distances between the biotin carboxyl group carbons.
Fig. 2
Fig. 2
Generation of the 1,3trans-divalent streptavidin. (a) Amino acid sequence of streptavidin-E6 (SAe) with E6 tag red and underlined, along with Dead (D) streptavidin showing residues impairing biotin binding in blue. (b) Generation of the different valency forms of SAe/D by mixed refold and ion exchange, with predicted pI of monomer and tetramer indicated. (c) Ion-exchange chromatogram of a mixture of tetramers containing different proportions of SAe and D. (d) Analysis of peaks from ion exchange by SDS-PAGE with Coomassie staining, unboiled (left) to show tetramer mobility, or boiled (right) to show subunit composition.
Fig. 3
Fig. 3
Generation ofcis-divalent streptavidin. (a) Amino acid sequence of core streptavidin (SA), along with Dead (Dd) containing a polyaspartate insertion in the 3/4 loop (red and underlined) and residues impairing biotin binding in blue. (b) Generation of the different valency forms of SA/Dd by mixed refold and ion exchange, with predicted pI of monomer and tetramer indicated. (c) Ion-exchange chromatogram of a mixture of tetramers containing different proportions of SA and Dd. (d) Analysis of peaks from ion exchange by SDS-PAGE with Coomassie staining, unboiled (left) to show tetramer mobility, or boiled (right) to show subunit composition.
Fig. 4
Fig. 4
Crystal structures of divalent streptavidins. (a) Electron density (2Fo − Fc contoured at 1 rmsd) at residues 23, 27, and 45 in the 1,3trans-divalent structure shown for chains b/c (SAe, green) and chains a/d (D, blue), with the side chain found indicated below. (b) Crystal structure of the 1,3trans-divalent streptavidin, with green showing the biotin-binding SAe subunits and blue showing the non-biotin-binding D subunits. (c) Electron density in thecis-divalent structure, with chains b/d (SA, green) and chains a/c (Dd, blue). (d) Crystal structure ofcis-divalent streptavidin, with green showing the biotin-binding SA subunits and blue showing the non-biotin-binding Dd subunits.
Fig. 5
Fig. 5
Structures of streptavidin interaction with ligands. (a) Structure of the biotin-binding pocket in the Dead subunit (N23A, S27D, and S45A), showing chain a of the 1,3trans-divalent streptavidin (carbon atoms in blue) overlaid with biotin-bound wild-type streptavidin (PDB ID:3RY2, carbon atoms in yellow). Hydrogen bonds from residues 23, 27, and 45 to biotin in wild-type streptavidin are shown as broken lines. (b) Crystal structure of the 1,3trans-divalent streptavidin bound to biotin-4-fluorescein (space-fill, carbons in cyan), with green for SAe and blue for D subunits. (c) Residues surrounding the fluorescein tail (carbon atoms in cyan) in the 1,3trans-divalent:biotin-4-fluorescein structure, labeled according to chain (b, carbon atoms in green; d, carbon atoms in blue), and with putative interactions as broken lines. Electron density for biotin-4-fluorescein is shown as blue mesh and contoured at 1 rmsd. (d) Clash forcis-bound biotin-4-fluorescein, generated from the 1,3trans-divalent biotin-4-fluorescein structure, if two biotin-4-fluorescein molecules (carbon atoms of one in yellow and the other in cyan) were to bind in the same conformation incis as they do in 1,3trans-divalent.
Fig. 6
Fig. 6
Off-rate and thermostability of divalent streptavidins. (a) Biotin-4-fluorescein dissociation over time at 37 °C in the presence of competing biotin, for a tetramer of wild-type streptavidin subunits (SA4), a tetramer of SAe subunits (SAe4), andcis-divalent or 1,3trans-divalent streptavidin (mean of triplicate + 1 SD). (b) Tetramer thermostability for streptavidin variants determined by SDS-PAGE with Coomassie staining, following incubation in PBS for 3 min at the indicated temperatures. In B, the protein was boiled in the presence of SDS to achieve complete conversion to monomers. The mobilities of the starting tetramer and the resulting monomers are marked.
Fig. 7
Fig. 7
Differential binding of biotinylated molecules by divalent streptavidins. (a) A monobiotinylated PCR product (50 nM) was incubated with 1,3trans- orcis-divalent streptavidin (100 nM) for the indicated times at 37 °C and then analyzed by agarose gel electrophoresis, with ethidium bromide visualization of DNA. C is a control of DNA without streptavidin. (b) Titration of the biotinylated PCR product (50 nM) with varying amounts of 1,3trans-divalent (upper panel) orcis-divalent (lower panel) streptavidin for 16 h at 25 °C, analyzed by electrophoresis as in (a). (c) Native PAGE of monobiotinylated affibody titrated with 1,3trans-divalent (upper panel) orcis-divalent (lower panel) streptavidin, analyzed by Coomassie staining. (d) Comparison of the relative sizes of an affibody (PDB ID:2KZJ, space-fill), a short segment of B-form DNA (PDB ID:1BNA, space-fill), and the separation betweencis-binding sites on streptavidin (PDB ID:3RY2, protein in gray, biotin in red, Connolly surface).
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