F-box only protein 7 (Fbxo7), a member of the Fbxo subfamily of FBPs, is a biologically and pathophysiologically important human protein that assumes many critical functions. The different functions of Fbxo7 depend on the formation of various multi-protein complexes. Possible interplay between different Fbxo7 functions further complicate the protein-protein interaction networks involved in Fbxo7 biology. The best known function of Fbxo7 is the substrate-recognition subunit of the SCFFbxo7 (Skp1-Cul1-F-box protein) E3 ubiquitin ligase that catalyzes the ubiquitination of hepatoma up-regulated protein (HURP) and inhibitor of apoptosis protein (IAP). Fbxo7 contains a ubiquitin-like domain (Ubl; amino acids 1�78), a Cdk6-interacting motif (cdk; amino acids 129�169), an Fbxo7/PI31 domain (FP; amino acids 180�324), an F-box motif (amino acids 332�375) and a proline-rich region (PRR; amino acids 423�522)[1]. Recently, We have determined the crystal structure of the FP domain (residues 181-335) of human Fbxo7 by SAD phasing method at a resolution of 2.0 �[2, 3]. The Fbxo7 FP domain adopts an a/� fold similar to the PI31 FP domain reported by Kirk[1]. Most of the secondary structural elements are comparable in the two structures. However, the lengths of helix a5, and strands �3, �4, and �5 are longer in the FP domain of Fbxo7. More significantly, structure of the Fbxo7 FP domain reveals a novel mode of inter-domain protein-protein interaction. The inter-domain interface of the Fbxo7 FP domain is best described as a a� interface because the contact surfaces in the two interacting FP domains are mainly defined by the a helices in one protomer and the � strands in the other protomer (Figure1). The a� interface buries 1934 �2 of solvent accessible molecular surface area from the two domains, which is significantly larger than the estimated minimal area (1200 �2) for a stable protein-protein complex. While the inter-domain interface of PI31 FP domain can be described as a/� fold because the two adjacent FP domains interact with each other through either the �a interface� or �� interface� which buries 1114 �2 and 560 �2of solvent accessible molecular surface area respectively, much smaller than in the structure of the Fbxo7 FP domain. Currently, we are trying to determine the crystal structure of F-box motif in Fbxo7 and study the interactions with its protein partners.Figure 1 . comparison of the inter-domain interfaces in the structures of the FP domains of Fbxo7 and PI31. Three adjacent domains ( in red, green, and blue respectively) are shown in cartoon and surface modes.1. Kirk R, Laman H, Knowles PP, Murray-Rust J, Lomonosov M, Meziane el K, McDonald NQ: Structure of a conserved dimerization domain within the F-box protein Fbxo7 and the PI31 proteasome inhibitor  . J Biol Chem 2008, 283 (32):22325-22335.2. Shang J, Wang G, Yang Y, Huang X, Du Z: Expression, purification and crystallization of the FP domain of the human F-box protein Fbxo7  . Acta Crystallogr Sect F Struct Biol Cryst Commun 2013, 69 (Pt 10):1097-1099.3. Shang J, Wang G, Yang Y, Huang X, Du Z: Structure of the FP domain of Fbxo7 reveals a novel mode of protein-protein interaction  . Acta Crystallographica Section D-Biological Crystallography 2014, D70  :155-164.

Structural and functional studies of Fbxo7 and its interactions with protein partners

248th National Meeting (2014)

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