High-resolution crystal structure of an avidin-related protein: insight into high-affinity biotin binding and protein stability

Abstract

The chicken avidin gene belongs to an extended gene family encoding seven avidin-related genes (AVRs), of which only avidin is expressed in the chicken. The sequences of AVR4 and AVR5 are identical and the common protein (AVR4) has been expressed both in insect and bacterial systems. The recombinant proteins are similarly hyperthermostable and bind biotin with similarly high affinities. AVR4 was crystallized in the apo and biotin-complexed forms and their structures were determined at high resolution. Its tertiary and quaternary structures are very similar to those of avidin and streptavidin. Its biotin-binding site shows only a few alterations compared with those of avidin and streptavidin, which account for the observed differences in binding affinities. The increased hyperthermostability can be attributed to the conformation of the critical L3,4 loop and the extensive network of 1-3 inter-monomeric interactions. The loop contains a tandem Pro-Gly sequence and an Asp-Arg ion pair that collectively induce rigidity, thus maintaining its closed and ordered conformation in both the apo and biotin-complexed forms. In addition, Tyr115 is present on the AVR4 1-3 monomer-monomer interface, which is absent in avidin and streptavidin. The interface tyrosine generates inter-monomeric interactions, i.e. a tyrosine-tyrosine pi-pi interaction and a hydrogen bond with Lys92. The resultant network of interactions confers a larger 1-3 dimer-dimer contact surface on AVR4, which correlates nicely with its higher thermostability compared with avidin and streptavidin. Several of the proposed thermostability-determining factors were found to play a role in strengthening the tertiary and quaternary integrity of AVR4.

Essentials of biorecognition: The (strept)avidin–biotin system as a model for protein–protein and protein–ligand interaction

“…The quaternary structure of avidin and its bacterial analogue streptavidin consists of a dimer of dimers ( Fig. 1A) [18,19]. Each avidin dimer consists of an intimate set of intramolecular interactions that occur at the interface between two monomers, referred to as the 1-4 monomer-monomer interaction ( Fig.…”

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

“…In the tetrameric avidins the biotin ligand inhabits a hydrophobic box, formed by either 3 or 4 conserved aromatic residues, which is then sealed by the additional Trp contributed by a neighboring monomer. The biotin molecule is also stabilized by an intricate network of H-bond interactions in which its bicyclic segment and the carboxylate moiety are highly conserved throughout the avidin family (11,17).…”

A model system for investigating lineshape/structure correlations in RNA site-directed spin labeling

“…Tethering was achieved utilizing a biotin moiety that is covalently linked to the 5′ phosphate group of the RNA via a (CH 2 ) 6 linker. The (CH 2 ) 6 linker allows the biotin to reach into the deep binding pocket in avidin [31] without generating excess relative motion between RNA and avidin. The biotin-avidin interaction, located at the RNA terminus that is distal to the labeling site, has no interactions with the local RNA environment being studied.…”