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Characterizing the function of domain linkers in regulating the dynamics of multi-domain fusion proteins by microsecond molecular dynamics simulations and artificial intelligence.

ABSTRACT: Multi-domain proteins are not only formed through natural evolution but can also be generated by recombinant DNA technology. Because many fusion proteins can enhance the selectivity of cell targeting, these artificially produced molecules, called multi-specific biologics, are promising drug candidates, especially for immunotherapy. Moreover, the rational design of domain linkers in fusion proteins is becoming an essential step toward a quantitative understanding of the dynamics in these biopharmaceutics. We developed a computational framework to characterize the impacts of peptide linkers on the dynamics of multi-specific biologics. Specifically, we first constructed a benchmark containing six types of linkers that represent various lengths and degrees of flexibility and used them to connect two natural proteins as a test system. We then projected the microsecond dynamics of these proteins generated from Anton onto a coarse-grained conformational space. We further analyzed the similarity of dynamics among different proteins in this low-dimensional space by a neural-network-based classification model. Finally, we applied hierarchical clustering to place linkers into different subgroups based on the classification results. The clustering results suggest that the length of linkers, which is used to spatially separate different functional modules, plays the most important role in regulating the dynamics of this fusion protein. Given the same number of amino acids, linker flexibility functions as a regulator of protein dynamics. In summary, we illustrated that a new computational strategy can be used to study the dynamics of multi-domain fusion proteins by a combination of long timescale molecular dynamics simulation, coarse-grained feature extraction, and artificial intelligence.

SUBMITTER: Wang B

PROVIDER: S-EPMC8500615 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Diphtheria toxin translocation (T) domain undergoes conformational changes in acidic solution and associates with the lipid membranes, followed by refolding and transmembrane insertion of two nonpolar helices. This process is an essential step in delivery of the toxic catalytic domain of the diphtheria toxin to the infected cell, yet its molecular determinants are poorly characterized and understood. Therefore, an atomistic model of the T-domain-membrane interaction is needed to help characterize factors responsible for such association. In this work, we present atomistic model structures of T-domain membrane-bound conformations and investigate structural factors responsible for T-domain affinity with the lipid bilayer in acidic solution using all-atom molecular dynamics (MD) simulations. The initial models of the protein conformations and protein-membrane association that serve as starting points in the present work were developed using atomistic simulations of partial unfolding of the T-domain in acidic solution (Kurnikov, I. V.; et al. J. Mol. Biol. 2013, 425, 2752-2764), and coarse-grained simulations of the T-domain association with the membranes of various compositions (Flores-Canales, J. C.; et al. J. Membr. Biol. 2015, 248, 529-543). In this work we present atomistic level modeling of two distinct configurations of the T-domain in association with the anionic lipid bilayer. In microsecond-long MD simulations both conformations retain their compact structure and gradually penetrate deeper into the bilayer interface. One membrane-bound conformation is stabilized by the protein contacts with the lipid hydrophobic core. The second modeled conformation is initially inserted less deeply and forms multiple contacts with the lipid at the interface (headgroup) region. Such contacts are formed by the charged and hydrophilic groups of partially unfolded terminal helixes and loops. Neutralization of the acidic residues at the membrane interface allows for deeper insertion of the protein and reorientation of the protein at the membrane interface, which corroborates that acidic residue protonation as well as presence of the anionic lipids may play a role in the membrane association and further membrane insertion of the T-domain as implicated in experiments. All simulations reported in this work were performed using AMBER force-field on Anton supercomputer. To perform these reported simulations, we developed and carefully tested a force-field for the anionic 1-palmitoyl-2-oleoyl-phosphatidyl-glycerol (POPG) lipid, compatible with the Amber 99SB force-field and stable in microsecond-long MD simulations in isothermal-isobaric ensemble.

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Characterizing the function of domain linkers in regulating the dynamics of multi-domain fusion proteins by microsecond molecular dynamics simulations and artificial intelligence.

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