De novo design of buttressed loops for sculpting protein functions

Abstract

In natural proteins, structured loops play central roles in molecular recognition, signal transduction and enzyme catalysis. However, because of the intrinsic flexibility and irregularity of loop regions, organizing multiple structured loops at protein functional sites has been very difficult to achieve by de novo protein design. Here we describe a solution to this problem that generates structured loops buttressed by extensive hydrogen bonding interactions with two neighboring loops and with secondary structure elements. We use this approach to design tandem repeat proteins with buttressed loops ranging from 9 to 14 residues in length. Experimental characterization shows the designs are folded and monodisperse, highly soluble, and thermally stable. Crystal structures are in close agreement with the computational design models, with the loops structured and buttressed by their neighbors as designed. We demonstrate the functionality afforded by loop buttressing by designing and characterizing binders for extended peptides in which the loops form one side of an extended binding pocket. The ability to design multiple structured loops should contribute quite generally to efforts to design new protein functions.

Fig. 1.. Computational design of repeat proteins with buttressed loops.

(A) Design strategy for generating and stabilizing multiple loops in helical repeat proteins. (B) A gallery of diverse designed proteins that pass the in silico design filters. (C) Loop buttressing bidentate hydrogen bonds in the designed proteins.

Fig. 2.. Biophysical characterization of designed helical repeat proteins with buttressed loops.

(A) Structural models of six representative designs. (B) Traces of UV280 in size-exclusion chromatography. (C) Circular dichroism spectra collected at 25 °C (blue), 95 °C (orange) and 25 °C after heating (green). (D) Overlay of experimental (black) and theoretical (red) small angle X-ray scattering profiles.

Fig. 3.. Structural characterization by X-ray crystallography.

(A) Superimposition of crystal structure (yellow) onto the design model of RBL4 (gray). (B) Alignment of individual repeat units. (C-E) Accurately designed loop buttressing interactions. (F) Superimposition of crystal structure (blue) onto the design model of RBL7_C2_3 (gray). (G) Overlay of a monomer unit in the crystal structure onto the design model. (H-J) Accurately designed loop buttressing interactions.

Fig. 4.. Designed repeat peptide binding RBLs.

(A, D and G) Design models of binding proteins for peptides with 6 repeats of DLP, KLP and DLS respectively. (B, E and H) Sequence-specific interactions between the designed binders and the repeat peptides. (C, F and I) Fluorescence polarization measurement of binding of designs to repeat peptides. For each binder, a titration curve is plotted for the binding of each peptide (Blue: DLPx6, Orange: KLPx6, Green: DLSx6).