Figure 1.
Biotin entry and exit paths in wild-type streptavidin (panels a and b) and models of the engineered mobile loop7–8 in streptavidin (panels c and d).
Subunits A, B, C and D in wild-type streptavidin are colored in blue, red, yellow and green, respectively. Loop3–4 in subunit D is in the open conformation as shown in panel a to allow biotin binding. Trp-120 (blue) from subunit A (blue) is required to form a complete biotin binding pocket in subunit D (green). With biotin binding, loop3–4 is in the closed position as shown in panel b. To develop an engineered streptavidin (8-aa-loop-H127C) with reversible biotin binding, loop7–8 in wild-type streptavidin was replaced by an engineered mobile loop to function as a dynamic gateway for the exit of the bound biotin. The engineered mobile loop7–8 in subunit A is modeled in 10 different positions to illustrate its dynamic nature (panel c). The blue loop (loop7–8) in the upper positions will form a wall as part of the biotin binding pocket for the subunit D (green). At these stages, this gate is in the closed state. When loop7–8 is in the lower position (panels c and d), the gate is in the open state. With loop3–4 in subunit D in the closed conformation, biotin still can possibly escape from the biotin binding pocket at this stage (panel d).
Table 1.
Features of the loop7–8 muteins.
Figure 2.
SDS-PAGE showing purification of 8-aa-loop-H127C mutein and its oligomeric state.
In panel a, 8-aa-loop-H127C mutein was affinity purified from B. subtilis culture supernatant using biotin-agarose matrix. S represents the concentrated culture supernatant before purification. FT, W and E are the flow-through, wash and elution fractions, respectively. 5 µl from each fraction (800 µl per fraction, each fraction contained the same volume of sample) was reduced with β–mercaptoethanol and boiled before loading onto the gel. The arrowhead marks the position of the 8-aa-loop-H127C mutein. Panel b shows the tetrameric state of 8-aa-loop-H127C (MH127C). 8-aa-loop (M) and wild-type (wt) streptavidin were included as reference. B and U represent the boiled and unboiled samples respectively. ME represents β–mercaptoethanol (used at 0.7 M final concentration). M shows the position of the molecular weight markers (numbers on the left of both panels are expressed in kDa).
Table 2.
Kinetic parameters of biotin binding in streptavidin muteins.
Figure 3.
Purification of biotinylated protein G and IgG using 8-aa-loop-H127C mutein-agarose matrix.
Excess amounts of biotinylated proteins were loaded to the column. Panel a: Biotinylated protein G with an apparent molecular mass of 37 kDa is marked by arrowhead. Some biotinylated protein G molecules are in oligomeric state with higher molecular masses. Panel b: The 54- and 25-kDa protein bands are the biotinylated heavy (marked by closed arrowhead) and light chains (marked by gray arrowhead) of IgG, respectively. Panel c: HeLa cell extract was applied to the 8-aa-loop-H127C mutein-agarose matrix. Panel d: Biotinylated IgG mixed with HeLa cell extract. S: Sample before purification, FT, W, and E: Flow-through, wash and elution fractions. M: Molecular weight markers.
Figure 4.
Electron density map (2|Fo|-|Fc| coefficients, contoured at 1.1 sigma) contoured around the region expected to be occupied by residues 114–121 of loop7–8.
The model for residues 110–112 and 122–124 fit the electron density quite well, but there is insufficient electron density to model residues 113–121. The structure of wild-type streptavidin (PDB code 1SWE) is superimposed and drawn in a thinner, gray line representation. Coefficients and phases were calculated using Refmac and the figure was prepared using PyMOL.
Figure 5.
Comparison of quaternary structures of the 8aa-loop-H127C mutein and wild-type streptavidin (1SWE).
Subunits A, B, C and D are colored blue, cyan, red and orange for both the mutein and wild-type structures. Biotin is drawn in space-filling representation and colored black. In all panels, only subunits A and B have been aligned. As a result, the cartoon diagrams and superimposed ribbon diagram at the far right show how the C/D dimer of the mutein is rotated by ∼10° relative to the C/D dimer of wild-type streptavidin when the A/B dimers of the mutein and wild-type streptavidin are superimposed. Loop7–8 (highlighted by the solid, black ovals in the front view) lies at the interface between dimer A/B and dimer C/D of wild-type streptavidin and is fixed in orientation in part by interactions between W120 and the biotin molecule bound to the opposing dimer. Loop7–8 is dynamically disordered in the mutein and hence is not shown in the cartoon model; the expected location of loop7–8 is denoted by the dashed, black ovals in the front view, and the location of the ends of the ordered parts of both loops (residues 112 and 122) are denoted by small circles. The side chains of Cys-127 from each subunit are drawn in space-filling representation, showing the formation of disulfide bonds between subunits A/C and B/D.
Figure 6.
Electron density map (2|Fo|-|Fc| coefficients, contoured at 1.1 sigma) contoured around the model of the disulfide bond formed by Cys-127 residues from adjacent subunits (A and C or B and D in the tetramer shown in Figure 5).
Coefficients and phases were calculated using Refmac and the figure was prepared using PyMOL.
Table 3.
Interaction of biotin with residues in the biotin binding pocket of subunit A.
Table 4.
Crystallographic statistics.