De novo protein design has been successful in expanding the natural protein repertoire. However, most de novo proteins lack biological function, presenting a major methodological challenge. In vaccinology, the induction of precise antibody responses remains a cornerstone for next-generation vaccines. Here, we present a protein design algorithm called TopoBuilder, with which we engineered epitope-focused immunogens displaying complex structural motifs. In both mice and nonhuman primates, cocktails of three de novo–designed immunogens induced robust neutralizing responses against the respiratory syncytial virus. Furthermore, the immunogens refocused preexisting antibody responses toward defined neutralization epitopes. Overall, our design approach opens the possibility of targeting specific epitopes for the development of vaccines and therapeutic antibodies and, more generally, will be applicable to the design of de novo proteins displaying complex functional motifs.
De novo protein design has enabled the creation of novel protein structures. To design novel functional proteins, state-of-the-art approaches use natural proteins or first design protein scaffolds that subsequently serve as templates for the transplantation of functional motifs. In these approaches, the templates are function-agnostic and motifs have been limited to those with regular secondary structure. Here, we present a bottom-up approach to build de novo proteins tailored to structurally complex functional motifs. We applied a bottom-up strategy to design scaffolds for four different binding motifs, including one bi-functionalized protein with two motifs. The de novo proteins were functional as biosensors to quantify epitope-specific antibody responses and as orthogonal ligands to activate a signaling pathway in engineered mammalian cells. Altogether, we present a versatile strategy for the bottom-up design of functional proteins, applicable to a wide range of functional protein design challenges.
UDP-glucose pyrophosphorylase (UGP) is a nucleotidyltransferase of central importance in all organisms and considered an attractive drug target in the human pathogens Leishmania and Trypanosoma. Here, we used wild-type and mutant Leishmania major UGP to solve the crystal structures of postreactive, UTP, and UDP-Glc bound states and performed kinetic and theoretical chemistry analysis of the enzymatic reaction. The new data filled critical gaps in the knowledge of the UGP mechanism and allowed reconstructing the complete enzymatic cycle on three levels: global (movements of molecular functional blocks), local (behavior of separate residues), and chemical (quantum mechanical description of enzymatic reaction). Results were integrated into a model of UGP activity describing structural changes along the cycle, the mechanisms of substrate binding, UGP catalysis, and product release. Our study revealed the mechanisms of allosteric regulation common for nucleotidyltransferases and, in particular, the mechanical control of the chemical reaction in the active site.
In mammals, UDP-glucose pyrophosphorylase (UGP) is the only enzyme capable of activating glucose-1-phosphate (Glc-1-P) to UDP-glucose (UDP-Glc), a metabolite located at the intersection of virtually all metabolic pathways in the mammalian cell. Despite the essential role of its product, the molecular basis of UGP function is poorly understood. Here we report the crystal structure of human UGP in complex with its product UDP-Glc. Beyond providing first insight into the active site architecture, we describe the substrate binding mode and intermolecular interactions in the octameric enzyme that are crucial to its activity. Importantly, the quaternary mechanism identified for human UGP in this study may be common for oligomeric sugar-activating nucleotidyltransferases. Elucidating such mechanisms is essential for understanding nucleotide sugar metabolism and opens the perspective for the development of drugs that specifically inhibit simpler organized nucleotidyltransferases in pathogens.
Biocatalysts utilize allosteric mechanisms to control selectivity, catalytic activity, and the transport of reaction components. The allosteric control of catalysis has a high potential for the development of drugs and technologies. In particular, it opens the way to specific regulation of vital enzymes with conserved active sites. Using the central metabolic enzyme UDP-glucose pyrophosphorylase from the pathogen Leishmania major (LmUGP), we demonstrate how specific allosteric inhibition sites and their links to the catalytic center can be revealed rationally, through analysis of molecular interfaces along the enzymatic reaction cycle. Two previously unknown specific allosteric inhibition sites in LmUGP were rationally identified and experimentally verified. The molecular scaffold for allosteric inhibitor targeting the pathogen's enzyme was developed. This led to the identification of murrayamine-I as an allosteric inhibitor that selectively blocks LmUGP. The presented approach opens up the possibility of using central metabolic enzymes with highly conserved active sites as allosteric drug targets, thus solving the cross-reactivity problem. In particular, it paves the ways to antimicrobial treatments.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.