Biology subjects

Mylemans, B.

Publications and source records attributed to Mylemans, B..

6 recordsLinked to original sources

bioRxiv2026

Expanding all-α-helical protein space through rational computational design

De novo protein design is advancing rapidly1,2. This is being driven by AI to generate protein backbones, sequences, and structural models3-7. As a result, de novo designed proteins are becoming larger and more complex8-10, and increasingly explore new protein structures11,12. By contrast, natural proteins have evolved structural and functional complexity by modular combination of recurring protein domains13. Approximately 25% of these natural domains are mostly -helical structures14. Here we show how these can be expanded using rational computational design. Following the domain classification scheme CATH15, we build complex all- de novo proteins hierarchically using sequence-to-structure relationships for helix-helix interactions, systematic rules to connect helices, computational tools to design loops, and in silico evaluation. The pipeline starts with a target architecture of free-standing helices. These are connected into a topology by considering local arrangements of helical bundles using understood sequence-to-structure relationships for helix packing. Single-chain sequences are completed using template- and AI-based methods. Finally, AlphaFold models are assessed to give small numbers of designs for experimental validation. We test 31 designs for 14 different architectures and 25 topologies. 75% of these express as stable, monomeric, water-soluble proteins; and >30% yield X-ray crystal structures matching the designs to atomic accuracy and with new-to-nature structures. Finally, several of the scaffolds are functionalised through one-shot designs to deliver ion, small-molecule and protein binders.

synthetic biology↗

bioRxiv2025

Coiled-coil scaffolds for Targeted Protein Degradation

In targeted protein degradation (TPD) the cells quality control machinery is co-opted to degrade proteins of interest. Currently TPD is limited by the availability of small-molecule binders for targets and the many potential human E3 ubiquitin ligases. Consequently, other approaches are needed to tackle new targets and to exploit degradation pathways fully. The natural binding epitopes for E3s and other degradation machineries are often short linear peptide motifs, which offer routes to new biologics-based degraders. Here, targeting the anti-apoptotic BCL-xL and initiation of apoptosis, we show that several protein-degradation pathways--ubiquitination, direct proteasome, autophagy-lysosome recruitment--can be leveraged by integrating target-binding and degradation motifs into single-polypeptide constructs. This exploits versatile and varied de novo coiled-coil scaffolds as adaptable molecular glues by presenting motifs multivalently and in diverse arrangements. The constructs are small ({approx}80 residues) and provide a plug-and-play platform to map and optimise target-degradation space using rational design. We call this Coiled Coils for induced Proximity (CoCoProx) and propose its use for targeted degradation and other induced-proximity reactions.

synthetic biology↗

bioRxiv2025

De novo designed bifunctional proteins for targeted protein degradation

Targeted protein degradation (TPD) is a therapeutic strategy to remove disease-causing proteins by routing them to the ubiquitin-proteasome, autophagy, or lysosme machineries. For instance, proteolysis-targeting chimeras (PROTACs) are synthetic hetero-bifunctional small molecules that simultaneously bind the target and an E3 ubiquitin ligase to drive ubiquitination and degradation by the proteasome. Despite considerable success, designing such molecules is challenging and the number of currently addressable ubiquitin E3 ligases is limited. Here we demonstrate hetero-bifunctional de novo designed proteins as alternatives for TPD to access more targets and ligases. First, we develop a stable and highly adaptable helix-turn-helix scaffold for presenting different binding sites. Next, we use computational protein design to incorporate and embellish hot-spot-binding sites to target BCL-xL, plus short linear motifs (SLiMs) for KLHL20 ligase recruitment. The resulting mono- and bi-functionalised proteins bind the targets in vitro, and the latter degrade BCL-xL in cells leading to apoptosis.

synthetic biology↗

bioRxiv2022

From peptides to proteins: coiled-coil tetramers to single-chain 4-helix bundles

The design of completely synthetic proteins from first principles--de novo protein design--is challenging. This is because, despite recent advances in computational protein-structure prediction and design, we do not understand fully the sequence-to-structure relationships for protein folding, assembly, and stabilization. Antiparallel 4-helix bundles are amongst the most studied scaffolds for de novo protein design. We set out to re-examine this target, and to determine clear sequence-to-structure relationships, or design rules, for the structure. Our aim was to determine a common and robust sequence background for designing multiple de novo 4-helix bundles, which, in turn, could be used in chemical and synthetic biology to direct protein-protein interactions and as scaffolds for functional protein design. Our approach starts by analyzing known antiparallel 4-helix coiled-coil structures to deduce design rules. In terms of the heptad repeat, abcdefg--i.e., the sequence signature of many helical bundles--the key features that we identify are: a = Leu, d = Ile, e = Ala, g = Gln, and the use of complementary charged residues at b and c. Next, we implement these rules in the rational design of synthetic peptides to form antiparallel homo- and heterotetramers. Finally, we use the sequence of the homotetramer to derive a single-chain 4-helix-bundle protein for recombinant production in E. coli. All of the assembled designs are confirmed in aqueous solution using biophysical methods, and ultimately by determining high-resolution X-ray crystal structures. Our route from peptides to proteins provides an understanding of the role of each residue in each design.

synthetic biology↗

bioRxiv2021

Crystal sturctures of Scone, pseudosymmetric folding of a symmetric designer protein

Recent years have seen a raise in the development of computational proteins including symmetric ones. We recently developed a nine-fold symmetric {beta}-propeller protein named Cake. Here we wanted to further engineer this protein to a three-fold symmetric nine-bladed propeller using computational design. Two nine-bladed propeller proteins were designed, named Scone-E and Scone-R. Crystallography however revealed the structure of both designs to adopt an eight-fold conformation with distorted termini, leading to a pseudo-symmetric protein. One of the proteins could only be crystallized upon addition of a polyoxometalate highlighting the usefulness of these molecules as a crystallisation additive.

systems biology↗

bioRxiv2021

Structure and Stability of the designer protein WRAP-T and its permutants

{beta}-Propeller proteins are common natural disc-like pseudo-symmetric proteins that contain multiple repeats ( blades) each consisting of a 4-stranded anti-parallel {beta}-sheet. So far, 4- to 12-bladed {beta}-propellers have been discovered in nature showing large functional and sequential variation. Using computational design approaches, we created perfectly symmetric {beta}-propellers out of natural pseudo-symmetric templates. These proteins are useful tools to study protein evolution of this very diverse fold. While the 7-bladed architecture is the most common, no symmetric 7-bladed monomer has been created and characterized so far. Here we describe such a engineered protein, based on a highly symmetric natural template, and test the effects of circular permutation on its stability. Geometrical analysis of this protein and other artificial symmetrical proteins reveals no systematic constraint that could be used to help in engineering of this fold, and suggests sequence constraints unique to each {beta}-propeller sub-family.

biophysics↗