Controlling Quaternary Structure Assembly: Subunit Interface Engineering and Crystal Structure of Dual Chain Avidin Vesa P. Hytönen 1 , Jarno Hörhä 1 , Tomi T. Airenne 2 Einari A. Niskanen 1 , Kaisa J. Helttunen 1 , Mark S. Johnson 2 Tiina A. Salminen 2 , Markku S. Kulomaa 3 and Henri R. Nordlund 3 ⁎ 1 NanoScience Center , Department of Biological and Environmental Science, P.O. Box 35 FI-40014 University of Jyväskylä, Finland 2 Department of Biochemistry and Pharmacy, Åbo Akademi University, FI-20520 Turku, Finland 3 Institute of Medical Technology, FI-33014 University of Tampere, Finland Dual chain avidin (dcAvd) is an engineered avidin form, in which two circularly permuted chicken avidin monomers are fused into one polypeptide chain. DcAvd can theoretically form two different pseudote- trameric quaternary assemblies because of symmetry at the monomer- monomer interfaces. Here, our aim was to control the assembly of the quaternary structure of dcAvd. We introduced the mutation I117C into one of the circularly permuted domains of dcAvd and scanned residues along the 1-3 subunit interface of the other domain. Interestingly, V115H resulted in a single, disulfide locked quaternary assembly of dcAvd, whereas I117H could not guide the oligomerisation process even though it stabilised the protein. The modified dcAvd forms were found to retain their characteristic pseudotetrameric state both at high and low pH, and were shown to bind D- biotin at levels comparable to that of wild-type chicken avidin. The crystal structure of dcAvd-biotin complex at 1.95 Å resolution demonstrates the formation of the functional dcAvd pseudotetramer at the atomic level and reveals the molecular basis for its special properties. Altogether, our data facilitate further engineering of the biotechnologically valuable dcAvd scaffold and gives insights into how to guide the quaternary structure assembly of oligomeric proteins. © 2006 Elsevier Ltd. All rights reserved. *Corresponding author Keywords: avidin-biotin technology; interface engineering; protein-protein interaction Introduction We have previously described the construction of dual chain avidin (dcAvd), in which two circularly permuted chicken avidin monomers were fused in one polypeptide chain. 1 These circularly permuted molecules were generated by genetically fusing the original termini via a short peptide linker and by introducing new termini at selected new locations on the avidin monomer structure. Two of such dcAvd chains were shown to spontaneously form a pseudotetrameric (i.e. four biotin-binding domains per two polypeptide chains) quaternary structure comparable to that of wild-type (wt) avidin. Moreover, dcAvd was shown to be active in terms of biotin binding and showed good thermal stability. Instead of the four subunits present in the tetrameric avidin, dcAvd contains only two subunits, both of which are equipped with two biotin-binding domains. Because of the subunit fusion, further engineering can be performed to a selected domain of dcAvd and thus avidin molecules displaying different properties on the neighbouring binding sites can be obtained. This was recently demonstrated by Present address: V. P. Hytönen, Department of Materials, ETH Zürich, Hönggerberg, CH-8093 Zürich, Switzerland. Abbreviations used: cpAvd5→4, circularly permuted avidin wherein the new N terminus is before β-strand 5 and new C terminus after β-strand 4; cpAvd6→5, circularly permuted avidin wherein the new N terminus is before β-strand 6 and new C terminus after β-strand 5; dcAvd, dual-chain avidin, wherein the circularly permuted avidins cpAvd5→4 and cpAvd6→5 are joined together in a single polypeptide chain; wt, wild-type. E-mail address of the corresponding author: henri.nordlund@uta.fi doi:10.1016/j.jmb.2006.04.044 J. Mol. Biol. (2006) 359, 1352–1363 0022-2836/$ - see front matter © 2006 Elsevier Ltd. All rights reserved.