Crystal Structure of Rhizavidin: Insights into the Enigmatic High-Affinity Interaction of an Innate Biotin-Binding Protein Dimer

Structural Adaptation of a Thermostable Biotin-binding Protein in a Psychrophilic Environment

“…Because the shwanavidin F43A mutant and the disulfide bridge mutants of shwanavidin and rhizaividin did not bind 2-iminobiotin, SPR measurements were conducted using biotin. Protein samples were injected over the chip surface for 240 s at a flow rate of 20 l/min and then washed with elution buffer without protein for 300 s. Shwanavidin sensograms are similar to those reported previously for rhizavidin (25). The disulfide bridge mutants from shwanavidin and rhizavidin show similar sensograms, although in rhizavidin the K d is 1 order of magnitude higher than that of shwanavidin.…”

“…In addition to the polar interactions, there are critical hydrophobic interactions consisting mainly of conserved aromatic residues forming the canonical hydrophobic box. In this regard, there are three Trp residues (Trp-67, Trp-80, and Trp-97) in shwanavidin, which are also conserved in rhizavidin (25) (Fig. 3).…”

“…2). Hence, further affinity assays were conducted using SPR as described previously for rhizavidin (25). The binding of rhizavidin to the biotin-coated chip essentially resulted in irreversible binding, even in the presence of 7.2 M urea, and affinity assays were therefore performed using 2-iminobiotin at pH 11.0 where its affinity toward the avidins is maximal.…”

“…A sensor chip was prepared by activating the surface of CM5 chip (carboxymethyl dextran-derivatized surface; Biacore AB) with a mixture of 0.4 M EDC and 0.1 M N-hydroxysuccinimide (NHS) followed by attachment of ethylenediamine. In the resultant chip one chan- nel was treated by injecting a mixture of 0.4 M EDC and 10 mM 2-iminobiotin, and another with NHS-biotin as previously described (25). For the affinity measurements of 2-iminobiotin, 80 -800 nM shwanavidin and 5-500 nM rhizavidin were injected over sensor surfaces in 1 M NaCl, 50 mM Na 2 CO 3 , pH 11.0 buffer.…”

“…The 1-2 and 1-3 monomer-monomer interactions are lacking in rhizavidin, and the crucial Trp from the L7,8 loop is redundant and missing in the sequence (12,13,24). The crystal structure of rhizavidin provided indications of how the high affinity is maintained in the absence of the critical Trp residue (6,25). In this context, a disulfide bridge connecting L3,4 and L5,6 contributes to the rigidity of the loops and also forms interactions with biotin; thus, the bridge was assumed to contribute to the high affinity (25).…”