title: 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.
authors.affiliation: Department of Biological Chemistry, The Institute of Life Sciences, The Wolfson Centre for Applied Structural Biology, The Hebrew University of Jerusalem, Givat Ram, Jerusalem 91904, Israel.
authors.forename: Yael
authors.initials: Y
authors.lastname: Eisenberg-Domovich
authors.forename: Vesa P
authors.initials: VP
authors.lastname: Hytönen
authors.forename: Meir
authors.initials: M
authors.lastname: Wilchek
authors.forename: Edward A
authors.initials: EA
authors.lastname: Bayer
authors.forename: Markku S
authors.initials: MS
authors.lastname: Kulomaa
authors.forename: Oded
authors.initials: O
authors.lastname: Livnah
mesh_terms: D000818:Animals; D001360:Avidin / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D001419:Bacteria / Q000378:metabolism; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism; D001710:Biotin / Q000737:chemistry*; D002645:Chickens / Q000378:metabolism*; D003460:Crystallization; D003627:Data Interpretation, Statistical; D006358:Hot Temperature; D006860:Hydrogen Bonding; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D019809:Streptavidin / Q000737:chemistry
chemical_list: D001360:Avidin; D001710:Biotin; D019809:Streptavidin