RESUMEN
De novo macrocyclic peptides, derived using selection technologies such as phage and mRNA display, present unique and unexpected solutions to challenging biological problems. This is due in part to their unusual folds, which are able to present side chains in ways not available to canonical structures such as α-helices and ß-sheets. Despite much recent interest in these molecules, their folding and binding behavior remains poorly characterized. In this work, we present cocrystallization, docking, and solution NMR structures of three de novo macrocyclic peptides that all bind as competitive inhibitors with single-digit nanomolar Ki to the active site of human pancreatic α-amylase. We show that a short stably folded motif in one of these is nucleated by internal hydrophobic interactions in an otherwise dynamic conformation in solution. Comparison of the solution structures with a target-bound structure from docking indicates that stabilization of the bound conformation is provided through interactions with the target protein after binding. These three structures also reveal a surprising functional convergence to present a motif of a single arginine sandwiched between two aromatic residues in the interactions of the peptide with the key catalytic residues of the enzyme, despite little to no other structural homology. Our results suggest that intramolecular hydrophobic interactions are important for priming binding of small macrocyclic peptides to their target and that high rigidity is not necessary for high affinity.
Asunto(s)
Inhibidores Enzimáticos/metabolismo , alfa-Amilasas Pancreáticas/antagonistas & inhibidores , alfa-Amilasas Pancreáticas/metabolismo , Péptidos Cíclicos/metabolismo , Dominio Catalítico , Cristalización , Humanos , Simulación del Acoplamiento Molecular , alfa-Amilasas Pancreáticas/química , Unión Proteica , Conformación Proteica , Pliegue de ProteínaRESUMEN
Decoupling biorefineries from land use and agriculture is a major challenge. As formate can be produced from various sources, e.g., electrochemical reduction of CO2, microbial formate-assimilation has the potential to become a sustainable feedstock for the bioindustry. However, organisms that naturally grow on formate are limited by either a low biomass yield or by a narrow product spectrum. The engineering of a model biotechnological microbe for growth on formate via synthetic pathways represents a promising approach to tackle this challenge. Here, we achieve a critical milestone for two such synthetic formate-assimilation pathways in Escherichia coli. Our engineering strategy involves the division of the pathways into metabolic modules; the activity of each module-providing at least one essential building block-is selected for in an appropriate auxotrophic strain. We demonstrate that formate can serve as a sole source of all cellular C1-compounds, including the beta-carbon of serine. We further show that by overexpressing the native threonine cleavage enzymes, the entire cellular glycine requirement can be provided by threonine biosynthesis and degradation. Together, we confirm the simultaneous activity of all pathway segments of the synthetic serine-threonine cycle. We go beyond the formate bioeconomy concept by showing that, under anaerobic conditions, formate produced endogenously by pyruvate formate-lyase can replace exogenous formate. The resulting prototrophic strain constitutes a substantial rewiring of central metabolism in which C1, glycine, and serine metabolism proceed via a unique set of pathways. This strain can serve as a platform for future metabolic-engineering efforts and could further pave the way for investigating the plasticity of metabolic networks.