Meredith M. Rickard

WRN helicase is an established synthetic lethal target for inhibition in the treatment of microsatellite instability-high (MSI-H) and mismatch repair deficient (MMRd) cancers. The identification of helicase inhibitors is challenging as high-throughput biochemical screening campaigns typically return few validated hits that are often inactive in cell-based assays. Herein, we highlight the power of non-covalent fragment-based lead discovery in locating new druggable allosteric sites on WRN, enabling us to bypass the challenging behavior of WRN during high-throughput screening hampering hit identification. During the fragment optimization process, structures of WRN with key prioritized fragments reveal multiple conformations of WRN with significant domain rotations up to 180°, including a WRN conformation not previously described. Rooted in a combination of biochemical, biophysical, and structural approaches, we present the detailed analyses of optimized chemical matter evolved from screening hits and the unique ability of WRN to accommodate diverse conformations as detailed by structural characterization.

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.

Many enzymes undergo major conformational changes to function in cells, particularly when they bind to more than one substrate. We quantify the large-amplitude hinge-bending landscape of human phosphoglycerate kinase (PGK) in a human cytoplasm. Approximately 70 μs of all-atom simulations, upon coarse graining, reveal three metastable states of PGK with different hinge angle distributions and additional substates. The "open" state was more populated than the "semi-open" or "closed" states. In addition to free energies and barriers within the landscape, we characterized the average transition state passage time of ≈0.3 μs and reversible substrate and product binding. Human PGK in a dilute solution simulation shows a transition directly from the open to closed states, in agreement with previous SAXS experiments, suggesting that the cell-like model environment promotes stability of the human PGK semi-open state. Yeast PGK also sampled three metastable states within the cytoplasm model, with the closed state favored in our simulation.

We examine the influence of cellular interactions in all‐atom models of a section of the Homo sapiens cytoplasm 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.

We examine how cellular interactions in an all-atom model of the Homo sapiens cytoplasm influence the early folding events of Protein B (PB), a three-helix bundle protein. While PB is known to fold during in vitro simulations in three microseconds, all three initially unfolded PB copies in our cytoplasm model never completely reached their native topology across our 31 microsecond simulation. We were able to capture initial formation of all three helices and a compact topology similar to the native state. Sticking interactions between PB and surrounding macromolecules, as well as other unfolded PBs, became competitive with PB folding. Interaction between PB copies seeded beta-strand formation, modeling initial events of protein aggregation. Finally, the fold-switching potential of PB related GA domains has been explored in previous studies, and the sticking and crowding in our model thus initiates sampling of helix/sheet structural plasticity of PB.

The cytoplasm is an environment crowded by macromolecules and filled with metabolites and ions. Recent experimental and computational studies have addressed how this environment affects protein stability, folding kinetics, and protein-protein and protein-nucleic acid interactions, though its impact on metabolites remains largely unknown. Here we show how a simulated cytoplasm affects the conformation of adenosine triphosphate (ATP), a key energy source and regulatory metabolite present at high concentrations in cells. Analysis of our all-atom model of a small volume of the Escherichia coli cytoplasm when contrasted with ATP modeled in vitro or resolved with protein structures deposited in the Protein Data Bank reveals that ATP molecules bound to proteins in cell form specific pitched conformations that are not observed at significant concentrations in the other environments. We hypothesize that these interactions evolved to fulfill functional roles when ATP interacts with protein surfaces.

How do enzymes form metabolons inside cells? To answer that question, we created an all-atom model of a section of the human cytoplasm and simulated it for over 30 microseconds. Among other proteins, nucleic acids, and metabolites, the model contains three successive members of the glycolytic cycle: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), and phosphoglycerate mutase (PGM). These enzymes interact to form transient, but long-lived, multi-enzyme complexes with characteristic lifetimes in the 1 to 5 μs range, thus modeling the functional metabolon structures that facilitate compartmentalization of metabolic pathways and substrate channeling in cell. We analyze the quinary structure between enzymes down to the formation of specific hydrogen-bonded interactions between side chains, together with the movement, in concert, of water molecules in or out between interacting amino acids to mediate contact formation and dissolution. We also observed large-scale enzymatic domain motion that has been proposed to convert between substrate-accessible and catalytically functional states: a direct hinge-bending motion of up to 28° changes the relative orientation of the N- and C-terminal domains of PGK, causing the initially open, and presumably inactive, conformation of PGK to sample both “semi-closed” and “closed” conformations. Although classical molecular dynamics (MD) cannot simulate enzymatic activity, closed structures are the functionally active forms of PGK, and their equilibrium with open structures opens the door for future quantum mechanics/molecular mechanics (QM/MM) and other reactive simulations of the cytoplasm.

The protein folding reaction is one of the most important chemical reactions in the human body. Yet, despite its importance, it is sometimes omitted from undergraduate courses due to the challenging nature of some of the underlying concepts. To help make key concepts of the protein folding reaction accessible to our undergraduate students, we implemented three, simplified 2D lattice models of various amino acid chains, and we used these models to generate sound-enhanced animations that allow students to see and hear the dynamics of protein folding in action. In spring of 2021, we used these videos in remote learning biophysics and music courses to introduce four key concepts of the folding reaction: solvation and hydrophobicity; energy and conformational entropy; funneled energy landscape; and frustration and traps. Our lattice model animations and sonifications helped provide insight into protein folding dynamics for undergraduate and graduate biophysical chemistry students, undergraduate musicians, and even authors who are experts in this field. We plan to incorporate these and additional animations, along with enhancements to the 2D lattice models, in our future courses.

Recent experimental data has shown that protein folding in the cytoplasm can differ from in vitro folding with respect to speed, stability, and residual structure. Here we investigate the all-atom molecular dynamics (MD) simulations of 9 copies of the model protein GTT WW domain in a small bacterial cytoplasm model using three force fields. GTT has been well-studied by MD in aqueous solution for comparison. We find that folded copies remain folded for up 25 μs, whereas unfolded copies do not fold for up to 190 μs. Unfolded GTT in our cytoplasm model does populate partly folded intermediates with one of the two hairpins formed. Relative to aqueous solution, GTT gets stuck in metastable states with a small RMSD and radius of gyration and extensive burial of surface area against other macromolecules. In particular, GTT is even able to form transient intermolecular β-sheets with other proteins, resulting in a “chimeric structure” that could be a precursor to oligomeric β-aggregates. We conclude that sticking, enhanced by the non-native mutations of GTT, is largely responsible, and we propose, on the basis of our result as well as recent experiments, that coevolution of protein surfaces with their solvation environment (including chaperones) is important for folding and diffusion of proteins in the cytoplasm.