Rational design of α-helical tandem repeat proteins with closed architectures

Abstract

Tandem repeat proteins, which are formed by repetition of modular units of protein sequence and structure, play important biological roles as macromolecular binding and scaffolding domains, enzymes, and building blocks for the assembly of fibrous materials1,2. The modular nature of repeat proteins enables the rapid construction and diversification of extended binding surfaces by duplication and recombination of simple building blocks3,4. The overall architecture of tandem repeat protein structures – which is dictated by the internal geometry and local packing of the repeat building blocks – is highly diverse, ranging from extended, super-helical folds that bind peptide, DNA, and RNA partners5–9, to closed and compact conformations with internal cavities suitable for small molecule binding and catalysis10. Here we report the development and validation of computational methods for de novo design of tandem repeat protein architectures driven purely by geometric criteria defining the inter-repeat geometry, without reference to the sequences and structures of existing repeat protein families. We have applied these methods to design a series of closed alpha-solenoid11 repeat structures (alpha-toroids) in which the inter-repeat packing geometry is constrained so as to juxtapose the N- and C-termini; several of these designed structures have been validated by X-ray crystallography. Unlike previous approaches to tandem repeat protein engineering12–20, our design procedure does not rely on template sequence or structural information taken from natural repeat proteins and hence can produce structures unlike those seen in nature. As an example, we have successfully designed and validated closed alpha-solenoid repeats with a left-handed helical architecture that – to our knowledge – is not yet present in the protein structure database21.

Computationally Designed Armadillo Repeat Proteins for Modular Peptide Recognition

“…Internal repeats were constrained to adopt the same primary sequence, side-chain, and backbone conformations by using symmetric sequence design and conformation sampling during all modeling moves. Symmetric structure prediction [18] and design has been used extensively in Rosetta [11] yielding atomic-accuracy predictions for large homomeric oligomers, designed cage-like assemblies or repeat proteins [12][13][14][15][19][20][21]. Our calculations were restricted to three internal repeats and two capping repeats.…”

“…Using this template, the relative orientations between subsequent repeats were extracted and imposed as symmetric modeling constraints during backbone and side chain sampling simulations using the Rosetta software suite [11]. Similar design protocols have been used for the computational design of repeat proteins, first with sequence and structural information obtained from natural repeat protein families [12,13] and then for de novo designed repeat proteins with open [14] and closed [15] architectures. Using such approaches, typically >50% of the designed constructs can be expressed as soluble, folded, monomeric proteins and determined structures agree well with the design models (typical RMSD of Cα atoms 0.5 -2.5 Å).…”

X-ray Crystallographic Structure and Solution Behavior of an Antiparallel Coiled-Coil Hexamer Formed by de Novo Peptides

“…[1][2][3] The formation of coiled coils from the assembly of α-helices was first predicted over 60 years ago for naturally occurring proteins and peptides. 4 De novo peptides that fold into coiled coils have proven to be a robust scaffold with interesting properties.…”

Computational Redesign of Thioredoxin Is Hypersensitive toward Minor Conformational Changes in the Backbone Template

“…However, designing larger (>70 aa) globular αβ proteins with irregular contact patterns is a highly complex task and has only been achieved by employing computational methods [10–14]. In addition to this unique achievement, computational methods have been employed to full-sequence design of a variety of protein structures including early mini-proteins [15–17], tandem repeats [18,19], and ligand binders [20–21]. Despite much effort, however, the total number of full-sequence designed proteins for which an atomic resolution structure has been solved still remains low; to our knowledge, less than 10 larger globular αβ proteins have been reported in the literature [10,12–14,22].…”