Wilavidin* — a novel member of the avidin family that forms unique biotin‐binding hexamers

Abstract

Nature's optimization of protein functions is a highly intricate evolutionary process. In addition to optimal tertiary folding, the intramolecular recognition among the monomers that generate higher‐order quaternary arrangements is driven by stabilizing interactions that have a pivotal role for ideal activity. Homotetrameric avidin and streptavidin are regularly utilized in many applications, whereby their ultra‐high affinity toward biotin is dependent on their quaternary arrangements. In recent years, a new subfamily of avidins was discovered that comprises homodimers rather than tetramers, in which the high affinity toward biotin is maintained. Intriguingly, several of the respective dimers have been shown to assemble into higher‐order cylindrical hexamers or octamers that dissociate into dimers upon biotin binding. Here, we present wilavidin, a newly discovered member of the dimeric subfamily, forming hexamers in the apo form, which are uniquely maintained upon biotin binding with six high‐affinity binding sites. Removal of the short C‐terminal segment of wilavidin resulted in the presence of the dimer only, thus emphasizing the role of this segment in stabilizing the hexamer. Utilization of a hexavalent biotin‐binding form of avidin would be beneficial for expanding the biotechnological toolbox. Additionally, this unique family of dimeric avidins and their propensity to oligomerize to hexamers or octamers can serve as a basis for protein oligomerization and intermonomeric recognition as well as cumulative interactions that determine molecular assemblies.

Self‐assembly of a dimeric avidin into unique higher‐order oligomers

“…A primary conserved feature of the dimeric avidins is the presence of a disulfide bridge which connects loops L3,4 and L5,6, located in the vicinity of the biotin carboxylate (in the biotin-complexed state). A second critical molecular feature that characterizes the dimeric avidins involves either a narrowing of the binding site upon binding biotin, designated as the pinching effect (observed in agroavidin, rhizavidin and hoefavidin) [17,21] or the presence of an aromatic residue (Phe or Tyr) in the L3,4 loop (present in shawnavidin, afifavidin and wilavidin) that emulates the position of the intermonomeric L7,8 Trp of the tetrameric avidins [18,19,21]. These critical features are now regarded as signature markers of the dimeric avidin family, which are beneficial for identifying new family members.…”

“…hoefavidin and afifavidin form octamers [17,[19][20][21][22]. In addition, most of these dimeric avidins form the corresponding oligomers in solution as well [19][20][21].…”

“…One critical feature is the C-terminal segment of the different dimeric avidins that in most cases crosses over to interact with the entrance of the respective biotin-binding site of a non-canonical monomer. For rhizavidin, hoefavidin, afifavidin and wilavidin, the C-terminal segments penetrate the entrance of the respective biotinbinding site and are considered to stabilize the oligomers [17,[19][20][21]. In this regard, removal of the Cterminal segments of the latter avidins resulted in the absence of oligomers and exclusive presence of dimers.…”

“…In the past several years, a new sub-family of avidins was identified whose members exhibit a dimeric (1-4 dimer) rather than a tetrameric structure. All of its five current members originate from bacterial sources: rhizavidin from Rhizobium etli, shwanavidin from Shewanella denitrificans, hoefavidin from Hoeflea phototrophica DFL-43, afifavidin from Afifella pfennigii and wilavidin from a member of the class Gammaproteobacteria [17,[19][20][21][22][23]. In all cases, the tertiary structure is similar to that of the monomers of the tetrameric avidins.…”

“…Upon binding biotin, the multimers dissociate into dimers. Intriguingly, wilavidin, the most recently described dimeric avidin, forms hexamers even in the presence of biotin [19]. Additionally, for several members of the dimeric sub-family, the C-terminal segment was shown to be essential to the oligomeric assembly.…”