A topological refactoring design strategy yields highly stable granulopoietic proteins

Abstract

Protein therapeutics frequently face major challenges, including complicated production, instability, poor solubility, and aggregation. De novo protein design can readily address these challenges. Here, we demonstrate the utility of a topological refactoring strategy to design novel granulopoietic proteins starting from the granulocyte-colony stimulating factor (G-CSF) structure. We change a protein fold by rearranging the sequence and optimising it towards the new fold. Testing four designs, we obtain two that possess nanomolar activity, the most active of which is highly thermostable and protease-resistant, and matches its designed structure to atomic accuracy. While the designs possess starkly different sequence and structure from the native G-CSF, they show specific activity in differentiating primary human haematopoietic stem cells into mature neutrophils. The designs also show significant and specific activity in vivo. Our topological refactoring approach is largely independent of sequence or structural context, and is therefore applicable to a wide range of protein targets.

Tackling Severe Neutrophilic Inflammation in Airway Disorders with Functionalized Nanosheets

“…After decalcification in ethylenediaminetetraacetic acid (EDTA), the MBs were embedded in paraffin and sectioned . Then, staining for lymphocyte antigen 6G (Ly6G) and CitH3, which are biomarkers for neutrophil and NET immunostaining, respectively, was performed. , LPS instillation induced approximately 6.5-fold more neutrophil recruitment, as well as 5.4-fold more NET formation in the nasal mucosa of mice (Figure I and J). In line with its anti-inflammatory effect, DNase treatment (20 U/mouse) also significantly reduced neutrophil recruitment (by approximately 60%) and NET formation (by 55%) in the nasal mucosa, thereby ameliorating the subsequent inflammatory cascade (Figure I and J).…”

“…We next investigated the mechanism by which BP-PGA 50 nanosheets ameliorate nasal inflammation. Neutrophil infiltration and NET formation were assessed through Ly6G and CitH3 immunostaining of the nasal mucosa from experimental mice. , Examination of CLSM images revealed a 70% reduction in the number of infiltrated neutrophils in BP-PGA 50 -treated inflammatory mice compared with untreated model mice. In contrast, the PGA 50 NP group exhibited a higher percentage of infiltrated neutrophils, with values exceeding 60% (Figure A and B).…”

Towards de novo design of transmembrane α-helical assemblies using structural modelling and molecular dynamics simulation

“…2,7,8 These processes often involve generating hundreds to thousands of sequences/structures followed by rapid scoring. Therefore, these are aided by computational design suites such as Rosetta, 39 ISAMBARD, 40 OSPREY, 41 Damietta, 42 and dTERMen. 43 In the de novo design of membrane proteins, the implementation of the effect of a lipid bilayer into an amino acid sequence is one more important key for successful designs.…”

Tuning of granulopoietic signaling byde novodesigned agonists

“…Depending on the construct, up to 14 positions (compare Supplementary Methods) were mutated utilizing the few-to-many-to-few combinatorial sampler of Damietta (v0.32, [43]). Analysis of the designs' structural stability was done using a tempering molecular dynamics routine, and candidates ranked by their conformational homogeneity score, as previously described in [42]. This molecular dynamics analysis was performed for all unique variants that were generated by Damietta and the most stable variant of each construct was considered as the final design.…”

“…S8 and S15). * The SPR parameters of rhG-CSF (self-made purified from E. coli) were taken from Skokowa et al 2022 [42]. (1.7 ± 0.4) × 10 0.371 10.9 ± 5.5 bv6_st2 (4.9 ± 0.9) × 10 2: Composition of PCR reaction mix used for all reactions with the following specifications; for all reactions 5 ng/µ L template was inputted but for the ones whose products were used for assembly reactions, 0.3 ng/mL were inputted.…”