Abstract
Monobodies, engineered protein scaffolds derived from the fibronectin type III domain, are powerful alternatives to conventional antibodies. While the native scaffold is robust, engineering the variable loops can often compromise solubility and promote aggregation. Here, we report the crystallization and structure determination at 2.57 Å resolution of a monobody (Mb-P') engineered to bind the synthetic small molecule HPPU [1-(4-hydroxyphenyl)-3-phenylurea] with nanomolar affinity. Although Mb-P' exhibited severe polydispersity and heterogeneous oligomerization in solution, N-terminal fusion with maltose-binding protein (MBP) using an optimized linker successfully yielded monodisperse species and diffraction-quality crystals. The crystal structure exhibited pseudo-D(3) symmetry in the asymmetric unit, in which the MBP moiety interacts with and partially covers the F and G β-strands of the monobody. This steric masking suggests that MBP acts as a solubility enhancer by shielding the aggregation-prone surface patches generated by loop engineering. Our results demonstrate that this fusion strategy effectively enables structural studies of aggregation-prone proteins obtained from engineered scaffolds.