California Science Center

With the goal of accelerating the discovery of small molecule-protein complexes, we leverage fast, low-dose, event-based electron counting microcrystal electron diffraction (MicroED) data collection and native mass spectrometry. This approach, which we term electron diffraction with native mass spectrometry (ED-MS), allows assignment of protein target structures bound to ligands with data obtained from crystal slurries soaked with mixtures of known inhibitors and crude biosynthetic reactions. This extends to libraries of printed ligands dispensed directly onto TEM grids for later soaking with microcrystal slurries, and complexes with noncovalent ligands. ED-MS resolves structures of the natural product, epoxide-based cysteine protease inhibitor E-64, and its biosynthetic analogs bound to the model cysteine protease, papain. It further identifies papain binding to its preferred natural products, by showing that two analogs of E-64 outcompete others in binding to papain crystals, and by detecting papain bound to E-64 and an analog from crude biosynthetic reactions, without purification. ED-MS also resolves binding of the CTX-M-14 β-lactamase, a target of active drug development, to the non-β-lactam inhibitor, avibactam, alone or in a cocktail of unrelated compounds. These results illustrate the utility of ED-MS for natural product ligand discovery and for structure-based screening of small molecule binders to macromolecular targets, promising utility for drug discovery.

Ecosystems function in a series of feedback loops that can change or maintain vegetation structure. Vegetation structure influences the ecological niche space available to animals, shaping many aspects of behaviour and reproduction. In turn, animals perform ecological functions that shape vegetation structure. However, most studies concerning three-dimensional vegetation structure and animal ecology consider only a single direction of this relationship. Here, we review these separate lines of research and integrate them into a unified concept that describes a feedback mechanism. We also show how remote sensing and animal tracking technologies are now available at the global scale to describe feedback loops and their consequences for ecosystem functioning. An improved understanding of how animals interact with vegetation structure in feedback loops is needed to conserve ecosystems that face major disruptions in response to climate and land-use change.

Naled, an organophosphate insecticide, is applied aerially at ultra-low volumes over aquatic ecosystems near Sacramento, California, USA, during summer months for mosquito control. Two ecosystem types (rice fields and a flowing canal) were sampled in 2020 and 2021. Naled and its primary degradation product (dichlorvos) were measured in water, biofilm, grazer macroinvertebrates, and omnivore/predator macroinvertebrates (predominantly crayfish). Maximum naled and dichlorvos concentrations detected in water samples one day after naled application were 287.3 and 5647.5 ng/L, respectively, which were above the U.S. Environmental Protection Agency’s aquatic life benchmarks for invertebrates. Neither compound was detected in water more than one day after the application. Dichlorvos, but not naled, was detected in composite crayfish samples up to 10 days after the last aerial application. Detections in water from the canal showed that the compounds were transported downstream of the target application area. Factors such as vector control flight paths, dilution, and transport through air and water likely affected concentrations of naled and dichlorvos in water and organisms from these aquatic ecosystems.Graphical

Deoxyhypusine synthase (DHPS) utilizes spermidine and NAD as cofactors to incorporate a hypusine modification into the eukaryotic translation initiation factor 5A (eIF5A). Hypusine is essential for eIF5A activation, which, in turn, plays a key role in regulating protein translation of selected mRNA that are associated with the synthesis of oncoproteins, thereby enhancing tumor cell proliferation. Therefore, inhibition of DHPS is a promising therapeutic option for the treatment of cancer. To discover novel lead compounds that target DHPS, we conducted synthetic studies with a hit obtained via high-throughput screening. Optimization of the ring structures of the amide compound (2) led to bromobenzothiophene (11g) with potent inhibitory activity against DHPS. X-ray crystallographic analysis of 11g complexed with DHPS revealed a dramatic conformational change in DHPS, which suggests the presence of a novel allosteric site. These findings provide the basis for the development of novel therapy distinct from spermidine mimetic inhibitors.

Elucidating the conformational heterogeneity of proteins is essential for understanding protein function and developing exogenous ligands. With the rapid development of experimental and computational methods, it is of great interest to integrate these approaches to illuminate the conformational landscapes of target proteins. SETD8 is a protein lysine methyltransferase (PKMT), which functions in vivo via the methylation of histone and nonhistone targets. Utilizing covalent inhibitors and depleting native ligands to trap hidden conformational states, we obtained diverse X-ray structures of SETD8. These structures were used to seed distributed atomistic molecular dynamics simulations that generated a total of six milliseconds of trajectory data. Markov state models, built via an automated machine learning approach and corroborated experimentally, reveal how slow conformational motions and conformational states are relevant to catalysis. These findings provide molecular insight on enzymatic catalysis and allosteric mechanisms of a PKMT via its detailed conformational landscape.