Molecular analysis of the type III interferon complex and its applications in protein engineering.

Type III interferons (IFNλs) are cytokines with critical roles in the immune system and are attractive therapeutic candidates due to their tissue-specific activity. Despite entering several clinical trials, results have demonstrated limited efficacy and potency, partially attributed to low-affinity protein-protein interactions (PPIs) responsible for receptor complex formation. Subsequently, structural studies of the native IFNλ signaling complexes remain inaccessible. While protein engineering can overcome affinity limitations, tools to investigate low-affinity systems like these remain limited. To provide insights into previous efforts to strengthen the PPIs within this complex, we perform a molecular analysis of the extracellular ternary complexes of IFNλ3 using both computational and experimental approaches. We first use molecular simulations and modeling to quantify differences in PPIs and residue strain fluctuations, generate detailed free energy landscapes, and reveal structural differences between an engineered, high-affinity complex, and a model of the wild-type, low-affinity complex. This analysis illuminates distinct behaviors of these ligands, yielding mechanistic insights into IFNλ complex formation. We then apply these computational techniques in protein engineering and design by utilizing simulation data to identify hotspots of interaction to rationally engineer the native cytokine-receptor complex for increased stability. These simulations are then validated by experimental techniques, showing that a single mutation at a computationally predicted site of interaction between the two receptors increases PPIs and improves complex formation for all IFNλs. This study highlights the power of molecular dynamics simulations for protein engineering and design as applied to the IFNλ family but also presents a potential tool for analysis and engineering of other systems with low-affinity PPIs.

Recent and future perspectives on engineering interferons and other cytokines as therapeutics.

As crucial mediators and regulators of our immune system, cytokines are involved in a broad range of biological processes and are implicated in various disease pathologies. The field of cytokine therapeutics has gained much momentum from the maturation of conventional protein engineering methodologies such as structure-based designs and/or directed evolution, which is further aided by the advent of in silico protein designs and characterization. Just within the past 5 years, there has been an explosion of proof-of-concept, preclinical, and clinical studies that utilize an armory of protein engineering methods to develop cytokine-based drugs. Here, we highlight the key engineering strategies undertaken by recent studies that aim to improve the pharmacodynamic and pharmacokinetic profile of interferons and other cytokines as therapeutics.

Distinct molecular phenotypes involving several human diseases are induced by IFN-λ3 and IFN-λ4 in monocyte-derived macrophages.

Human Interferon (IFN) lambda 3 (IFN-λ3) and IFN-λ4 are closely linked at the IFNL locus and show association with several diseases in genetic studies. Since they are only ~30% identical to each other, to better understand their roles in disease phenotypes, comparative studies are needed. Monocytes are precursors to macrophages (monocyte-derived macrophages; MDMs) that get differentiated under the influence of various immune factors, including IFNs. In a recent study, we characterized lipopolysaccharide-activated M1 and M2-MDMs that were differentiated in presence of IFN-λ3 or IFN-λ4. In this study, we performed transcriptomics on these M1 and M2-MDMs to further understand their molecular phenotypes. We identified over 760 genes that were reciprocally regulated by IFN-λ3 and IFN-λ4, additionally we identified over 240 genes that are significantly affected by IFN-λ4 but not IFN-λ3. We observed that IFN-λ3 was more active in M2-MDMs while IFN-λ4 showed superior response in M1-MDMs. Providing a structural explanation for these functional differences, molecular modeling showed differences in expected interactions of IFN-λ3 and IFN-λ4 with the extracellular domain of IFN-λR1. Further, pathway analysis showed several human infectious diseases and even cancer-related pathways being significantly affected by IFN-λ3 and/or IFN-λ4 in both M1 and M2-MDMs.

Cell-free styrene biosynthesis at high titers.

Styrene is an important petroleum-derived molecule that is polymerized to make versatile plastics, including disposable silverware and foamed packaging materials. Finding more sustainable methods, such as biosynthesis, for producing styrene is essential due to the increasing severity of climate change as well as the limited supply of fossil fuels. Recent metabolic engineering efforts have enabled the biological production of styrene in Escherichia coli, but styrene toxicity and volatility limit biosynthesis in cells. To address these limitations, we have developed a cell-free styrene biosynthesis platform. The cell-free system provides an open reaction environment without cell viability constraints, which allows exquisite control over reaction conditions and greater carbon flux toward product formation rather than cell growth. The two biosynthetic enzymes required for styrene production were generated via cell-free protein synthesis and mixed in defined ratios with supplemented L-phenylalanine and buffer. By altering the time, temperature, pH, and enzyme concentrations in the reaction, this approach increased the cell-free titer of styrene from 5.36 ± 0.63 mM to 40.33 ± 1.03 mM, the highest amount achieved using biosynthesis without process modifications and product removal strategies. Cell-free systems offer a complimentary approach to cellular synthesis of small molecules, which can provide particular benefits for producing toxic molecules.

In vitro prototyping and rapid optimization of biosynthetic enzymes for cell design.

The design and optimization of biosynthetic pathways for industrially relevant, non-model organisms is challenging due to transformation idiosyncrasies, reduced numbers of validated genetic parts and a lack of high-throughput workflows. Here we describe a platform for in vitro prototyping and rapid optimization of biosynthetic enzymes (iPROBE) to accelerate this process. In iPROBE, cell lysates are enriched with biosynthetic enzymes by cell-free protein synthesis and then metabolic pathways are assembled in a mix-and-match fashion to assess pathway performance. We demonstrate iPROBE by screening 54 different cell-free pathways for 3-hydroxybutyrate production and optimizing a six-step butanol pathway across 205 permutations using data-driven design. Observing a strong correlation (r = 0.79) between cell-free and cellular performance, we then scaled up our highest-performing pathway, which improved in vivo 3-HB production in Clostridium by 20-fold to 14.63 ± 0.48 g l-1. We expect iPROBE to accelerate design-build-test cycles for industrial biotechnology.