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Enzyme–enzyme interactions are fundamental to the function of cells. Their atomistic mechanisms remain elusive mainly due to limitations of in-cell measurements. We address this challenge by atomistically modeling, for a total of ≈80 μs, a slice of the human cell cytoplasm that includes three successive enzymes along the glycolytic pathway: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), and phosphoglycerate mutase (PGM). We tested the model for nonspecific protein stickiness, an artifact of current atomistic force fields in crowded environments. The simulations reveal that the human enzymes co-organize in-cell into transient submetabolon complexes, consistent with previous experimental results. Our data both reiterate known specificity between GAPDH and PGK and reveal extensive direct interactions between GAPDH and PGM. Our simulations further reveal, through force field benchmarking, the critical role of protein solvation in facilitating these enzyme–enzyme interactions. Transient interenzyme interactions with μs lifetime occur repeatedly in our simulations via specific sticky protein surface patches, with interactions often mediated by charged patch residues. Some of the residues that interact frequently with one another lie in or near the active site of the enzymes. We show that some of these patches correspond to a general mode to interact with several partners for promiscuous enzymes like GAPDH. We further show that the non-native yeast PGK is stickier than human PGK in our human cytoplasm model, supporting the idea of evolutionary pressure to reduce sticking. Our cytoplasm modeling paves the way toward capturing the atomistic dynamics of an entire enzymatic pathway in-cell. 

Protein–protein and protein–water hydrogen bonding interactions play essential roles in the way a protein passes through the transition state during folding or unfolding, but the large number of these interactions in molecular dynamics (MD) simulations makes them difficult to analyze. Here, we introduce a state space representation and associated “rarity” measure to identify and quantify transition state passage (transit) events. Applying this representation to a long MD simulation trajectory that captured multiple folding and unfolding events of the GTT WW domain, a small protein often used as a model for the folding process, we identified three transition categories: Highway (faster), Meander (slower), and Ambiguous (intermediate). We developed data sonification and visualization tools to analyze hydrogen bond dynamics before, during, and after these transition events. By means of these tools, we were able to identify characteristic hydrogen bonding patterns associated with “Highway” versus “Meander” versus “Ambiguous” transitions and to design algorithms that can identify these same folding pathways and critical protein–water interactions directly from the data. Highly cooperative hydrogen bonding can either slow down or speed up transit. Furthermore, an analysis of protein–water hydrogen bond dynamics at the surface of WW domain shows an increase in hydrogen bond lifetime from folded to unfolded conformations with Ambiguous transitions as an outlier. In summary, hydrogen bond dynamics provide a direct window into the heterogeneity of transits, which can vary widely in duration (by a factor of 10) due to a complex energy landscape. 

Abstract We examine the influence of cellular interactions in all‐atom models of a section of theHomo sapienscytoplasm on the early folding events of the three‐helix bundle protein B (PB). While genetically engineered PB is known to fold in dilute water box simulations in three microseconds, the three initially unfolded PB copies in our two cytoplasm models using a similar force field did not reach the native state during 30‐microsecond simulations. We did however capture the formation of all three helices in a compact native‐like topology. Folding in vivo is delayed because intramolecular contact formation within PB is in direct competition with intermolecular contacts between PB and surrounding macromolecules. In extreme cases, intermolecular beta‐sheets are formed. Interactions with other macromolecules are also observed to promote structure formation, for example when a PB helix in our simulations is shielded from solvent by macromolecular crowding. Sticking and crowding in our models initiate sampling of helix/sheet structural plasticity of PB. Relatedly, in past in vitro experiments, similar GA domains were shown to switch between two different folds. Finally, we also observed that stickiness between PB and the cellular environment can be modulated in our simulations through the reduction in protein hydrophobicity when we reversed PB back to the wild‐type sequence. This study demonstrates that even fast‐folding proteins can get stuck in non‐native states in the cell, making them useful models for protein–chaperone interactions and early stages of aggregate formation relevant to cellular disease.