A platform to accelerate optimization of proteolysis targeting chimeras (PROTACs) has been developed using a direct-to-biology (D2B) approach with a focus on linker effects. A large number of linker analogs—with varying length, polarity, and rigidity—were rapidly prepared and characterized in four cell-based assays by streamlining time-consuming steps in synthesis and purification. The expansive dataset informs on linker structure–activity relationships (SAR) for in-cell E3 ligase target engagement, degradation, permeability, and cell toxicity. Unexpected aspects of linker SAR was discovered, consistent with literature reports on “linkerology”, and the method dramatically speeds up empirical optimization. Physicochemical property trends emerged, and the platform has the potential to rapidly expand training sets for more complex prediction models. In-depth validation studies were carried out and confirm the D2B platform is a valuable tool to accelerate PROTAC design–make–test cycles.

Proteins commonly sample a number of conformational states to carry out their biological function, often requiring transitions from the ground state to higher-energy states. Characterizing the mechanisms that guide these transitions at the atomic level promises to impact our understanding of functional protein dynamics and energy landscapes. The leucine-99-toalanine (L99A) mutant of T4 lysozyme is a model system that has an experimentally well characterized excited sparsely populated state as well as a ground state. Despite the exhaustive study of L99A protein dynamics, the conformational changes that permit transitioning to the experimentally detected excited state (~3%, DG~2 kcal/mol) remain unclear. Here, we describe the transitions from the ground state to this sparsely populated excited state of L99A as observed through a single molecular dynamics (MD) trajectory on the Anton supercomputer. Aside from detailing the ground-to-excited-state transition, the trajectory samples multiple metastates and an intermediate state en route to the excited state. Dynamic motions between these states enable cavity surface openings large enough to admit benzene on timescales congruent with known rates for benzene binding. Thus, these fluctuations between rare protein states provide an atomic description of the concerted motions that illuminate potential path(s) for ligand binding. These results reveal, to our knowledge, a new level of complexity in the dynamics of buried cavities and their role in creating mobile defects that affect protein dynamics and ligand binding.

Atmospheric aerosols have long been known to alter climate by scattering incoming solar radiation and acting as seeds for cloud formation. These processes have vast implications for controlling the chemistry of our environment and the Earth’s climate. Sea spray aerosol (SSA) is emitted over nearly three-quarters of our planet, yet precisely how SSA impacts Earth’s radiation budget remains highly uncertain. Over the past several decades, studies have shown that SSA particles are far more complex than just sea salt. Ocean biological and physical processes produce individual SSA particles containing a diverse array of biological species including proteins, enzymes, bacteria, and viruses and a diverse array of organic compounds including fatty acids and sugars. Thus, a new frontier of research is emerging at the nexus of chemistry, biology, and atmospheric science. In this Outlook article, we discuss how current and future aerosol chemistry research demands a tight coupling between experimental (observational and laboratory studies) and computational (simulation-based) methods. This integration of approaches will enable the systematic interrogation of the complexity within individual SSA particles at a level that will enable prediction of the physicochemical properties of real-world SSA, ultimately illuminating the detailed mechanisms of how the constituents within individual SSA impact climate.

The atomic-level mechanisms that coordinate ligand release from protein pockets are only known for a handful of proteins. Here, we report results from accelerated molecular dynamics simulations for benzene dissociation from the buried cavity of the T4 lysozyme Leu99Ala mutant (L99A). In these simulations, benzene is released through a previously characterized, sparsely populated room-temperature excited state of the mutant, explaining the coincidence for experimentally measured benzene off rate and apo protein slow-timescale NMR relaxation rates between ground and excited states. The path observed for benzene egress is a multistep ligand migration from the buried cavity to ultimate release through an opening between the F/G-, H-, and I-helices and requires a number of cooperative multiresidue and secondary-structure rearrangements within the C-terminal domain of L99A. These rearrangements are identical to those observed along the ground state to excited state transitions characterized by molecular dynamic simulations run on the Anton supercomputer. Analyses of the molecular properties of the residues lining the egress path suggest that protein surface electrostatic potential may play a role in the release mechanism. Simulations of wild-type T4 lysozyme also reveal that benzene-egress-associated dynamics in the L99A mutant are potentially exaggerations of the substrate-processivity-related dynamics of the wild type. FIGURE 5 WT T4 lysozyme transition from ground state to a high-energy-like state. (A) Concerted side-chain motion and backbone breakages occurring at $2600 aMD time steps for the same residues as involved with ground state to high-energy state transition in L99A mutant. (B and C) Structures from the crystal structure (green) compared to the high-energy-like state (cyan) for F114, L133, F153, and V111. (D) Helices F, G, H, and I in the ground state (cyan) and WT* high-energy-like state (orange) with the peptidoglycan substrate (purple). To see this figure in color, go online.

The universally conserved N6-threonylcarbamoyladenosine (t6A) modification of tRNA is essential for translational fidelity. In bacteria, t6A biosynthesis starts with the TsaC/TsaC2-catalyzed synthesis of the intermediate threonylcarbamoyl adenylate (TC–AMP), followed by transfer of the threonylcarbamoyl (TC) moiety to adenine-37 of tRNA by the TC-transfer complex comprised of TsaB, TsaD and TsaE subunits and possessing an ATPase activity required for multi-turnover of the t6A cycle. We report a 2.5-Å crystal structure of the T. maritima TC-transfer complex (TmTsaB2D2E2) bound to Mg2+-ATP in the ATPase site, and substrate analog carboxy-AMP in the TC-transfer site. Site directed mutagenesis results show that residues in the conserved Switch I and Switch II motifs of TsaE mediate the ATP hydrolysis-driven reactivation/reset step of the t6A cycle. Further, SAXS analysis of the TmTsaB2D2-tRNA complex in solution reveals bound tRNA lodged in the TsaE binding cavity, confirming our previous biochemical data. Based on the crystal structure and molecular docking of TC–AMP and adenine-37 in the TC-transfer site, we propose a model for the mechanism of TC transfer by this universal biosynthetic system.

Mutations in isocitrate dehydrogenase 1 (IDH1) drive most low-grade gliomas and secondary glioblastomas and many chondrosarcomas and acute myeloid leukemia cases. Most tumor-relevant IDH1 mutations are deficient in the normal oxidization of isocitrate to α-ketoglutarate (αKG), but gain the neomorphic activity of reducing αKG to D-2-hydroxyglutarate (D2HG), which drives tumorigenesis. We found previously that IDH1 mutants exhibit one of two reactivities: deficient αKG and moderate D2HG production (including commonly observed R132H and R132C) or moderate αKG and high D2HG production (R132Q). Here, we identify a third type of reactivity, deficient αKG and high D2HG production (R132L). We show that R132Q IDH1 has unique structural features and distinct reactivities towards mutant IDH1 inhibitors. Biochemical and cell-based assays demonstrate that while most tumor-relevant mutations were effectively inhibited by mutant IDH1 inhibitors, R132Q IDH1 had up to a 16 300-fold increase in IC50 versus R132H IDH1. Only compounds that inhibited wild-type (WT) IDH1 were effective against R132Q. This suggests that patients with a R132Q mutation may have a poor response to mutant IDH1 therapies. Molecular dynamics simulations revealed that near the NADP+/NADPH-binding site in R132Q IDH1, a pair of α-helices switches between conformations that are more wild-type-like or more mutant-like, highlighting mechanisms for preserved WT activity. Dihedral angle changes in the dimer interface and buried surface area charges highlight possible mechanisms for loss of inhibitor affinity against R132Q. This work provides a platform for predicting a patient’s therapeutic response and identifies a potential resistance mutation that may arise upon treatment with mutant IDH inhibitors.

Triacylglycerol lipases have recently been shown to be transferred from the ocean to the atmosphere in atmospheric sea spray aerosol (SSA). Lipases have the potential to alter the composition of SSA; however, the structure and properties of enzymes in the high salt, high ionic strength, and low pH conditions found in SSA have never been explored. Here, we study the dynamics of Burkholderia cepacia triacylglycerol lipase (BCL) at SSA model surfaces comprised of palmitic acid and dipalmitoylphosphatidic acid (DPPA), two commonly found lipids at SSA surfaces. Surface adsorption Langmuir isotherm experiments and all-atom explicit solvent molecular dynamics simulations together illuminate how and why BCL expands the ordering of lipids at palmitic acid surfaces the most at pH < 4 and the least in DPPA surfaces at pH 6. Taken together, these results represent a first glimpse into the complex interplay between lipid surface structure and protein dynamics within enzyme-containing aerosols.

The ankyrin repeat and SOCS box (ASB) family is composed of 18 proteins and belongs to the suppressor of cytokine signaling (SOCS) box protein superfamily. The ASB proteins function as the substrate-recognition subunits of ECS-type (ElonginBC-Cullin-SOCS-box) Cullin RING E3 ubiquitin ligase (CRL) complexes that specifically transfer ubiquitin to cellular proteins targeting them for degradation by the proteasome. ASB9 binds to creatine kinase (CK) and targets it for degradation, however the way in which ASB9 interacts with CK is not yet known. We present complete characterization of the binding of ASB9 to CK. One ASB9 molecule binds to a dimer of CK. The binding affinity of ASB9(1-252) was extremely tight and no dissociation could be observed. Deletion of the N-terminal 34 amino acids forming ASB9(35-252) resulted in weakening of the binding so that a binding affinity of 2.6 nM could be measured. Amide hydrogen/deuterium exchange (HDXMS) experiments showed that both ASB9(1-252) and ASB9(35-252) protected the same region of CK, residues 182-203, which forms one side of the active site. The HDXMS experiments indicated that the N-terminal disordered region and first ankyrin repeat of ASB9 are protected from exchange in the complex. Molecular docking yielded a structural model consistent with all of the data that suggested the N-terminal residues of ASB9(1-252) may lie in one CK active site. This model was corroborated by enzymatic activity assays and mutational analysis.