De novo design of modular peptide-binding proteins by superhelical matching

Abstract

General approaches for designing sequence-specific peptide-binding proteins would have wide utility in proteomics and synthetic biology. However, designing peptide-binding proteins is challenging, as most peptides do not have defined structures in isolation, and hydrogen bonds must be made to the buried polar groups in the peptide backbone1–3. Here, inspired by natural and re-engineered protein–peptide systems4–11, we set out to design proteins made out of repeating units that bind peptides with repeating sequences, with a one-to-one correspondence between the repeat units of the protein and those of the peptide. We use geometric hashing to identify protein backbones and peptide-docking arrangements that are compatible with bidentate hydrogen bonds between the side chains of the protein and the peptide backbone12. The remainder of the protein sequence is then optimized for folding and peptide binding. We design repeat proteins to bind to six different tripeptide-repeat sequences in polyproline II conformations. The proteins are hyperstable and bind to four to six tandem repeats of their tripeptide targets with nanomolar to picomolar affinities in vitro and in living cells. Crystal structures reveal repeating interactions between protein and peptide interactions as designed, including ladders of hydrogen bonds from protein side chains to peptide backbones. By redesigning the binding interfaces of individual repeat units, specificity can be achieved for non-repeating peptide sequences and for disordered regions of native proteins.

Design of stimulus-responsive two-state hinge proteins

“…Like transistors in electronic circuits, we can couple the switches to external outputs and inputs to create sensing devices and incorporate them into larger protein systems to address a wide range of outstanding design challenges. Hinges containing a disulfide that locks them in state X couple the input “red/ox state” to the output “target binding,” where the target can be a peptide or a protein, and our FRET-labeled hinges couple the input “target binding” to the output “FRET signal.” Our approach can be readily extended such that state switching is driven by naturally occurring rather than designed peptides: recently designed extended peptide binding proteins ( 39 ) resemble the state X of our hinges, and recent designs that bind glucagon, secretin, or neuropeptide Y ( 40 ) resemble the state Y of our hinges. Hinges based on such designs could thus provide new routes to applications in sensing and detection.…”

Adjuvant physiochemistry and advanced nanotechnology for vaccine development

“…For example, Bateriophae VLPs (28 nm in diameter) that are assembled in an icosahedral structure by 180 copies of a subunit protein incorporate an RNA adjuvant (TLR7/8 agoinst), triggering potent humoral response. 450 The Baker group recently reported several innovative strategies of self-assembled protein nanosystems, 451,452 offering a great potential for vaccine development. VLP-based preventive vaccines, such as Engerix and Recombivax HB against HBV, Cervarix and Gardasil against for HPV, is commercially available for many years.…”

Is Drosophila Dpp/BMP morphogen spreading required for wing patterning and growth?

“…Indeed, a recent proof of principle study successfully designed such sequence-specific peptide-binding proteins. [90]…”