Molecular assemblies of the catalytic domain of SOS with KRas and oncogenic mutants

Ras is regulated by a specific guanine nucleotide exchange factor Son of Sevenless (SOS), which facilitates the exchange of inactive, GDP-bound Ras with GTP. The catalytic activity of SOS is also allosterically modulated by an active Ras (Ras–GTP). However, it remains poorly understood how oncogenic Ras mutants interact with SOS and modulate its activity. Here, native ion mobility–mass spectrometry is employed to monitor the assembly of the catalytic domain of SOS (SOS^cat) with KRas and three cancer-associated mutants (G12C, G13D, and Q61H), leading to the discovery of different molecular assemblies and distinct conformers of SOS^cat engaging KRas. We also find KRas^G13D exhibits high affinity for SOS^cat and is a potent allosteric modulator of its activity. A structure of the KRas^G13D•SOS^cat complex was determined using cryogenic electron microscopy providing insight into the enhanced affinity of the mutant protein. In addition, we find that KRas^G13D–GTP can allosterically increase the nucleotide exchange rate of KRas at the active site more than twofold compared to KRas–GTP. Furthermore, small-molecule Ras•SOS disruptors fail to dissociate KRas^G13D•SOS^cat complexes, underscoring the need for more potent disruptors. Taken together, a better understanding of the interaction between oncogenic Ras mutants and SOS will provide avenues for improved therapeutic interventions.

Ras, a member of the small G-protein family, represents important signaling molecules with diverse cellular roles, such as cell differentiation and proliferation (1–4). Different isoforms of Ras (HRas, KRas, and NRas) have high overall sequence identity and are the most commonly mutated of all discovered oncogenes (5, 6). Of the three Ras isoforms, KRas is the most frequently mutated isoform in cancers, such as pancreatic cancer (70–90%), colon cancer (30–50%), and lung cancer (20–30%) (7, 8). Ras proteins regulate cell signaling pathways by cycling between inactive, GDP-bound, and active, GTP-bound states that is accompanied by remodeling of three key regions within Ras: p-loop (residues 10–17), switch I (residues 30–38), and switch II (residues 60–76) (9–11).As Ras proteins possess slow guanine nucleotide exchange rates, their activation is regulated by guanine nucleotide exchange factors (GEFs) that reload Ras with GTP (12, 13). The multidomain protein, Son of Sevenless (SOS), is a GEF with the cdc25 and Ras exchanger motif domains representing the minimal, functionally competent unit, termed SOS^cat (14). Structural studies have revealed two Ras binding sites to SOS^cat, leading to the discovery that binding of Ras–GTP at the distal (or allosteric) site allosterically modulates SOS^cat activity, which markedly increases the nucleotide exchange rate at the active site (14, 15). In addition, the degree of this allosteric modulation greatly depends on the nucleotide-bound state of Ras (14, 16, 17). Moreover, SOS is conformationally dynamic and binding of Ras at the allosteric site appears to shift the population to active conformation(s) of SOS (18). In addition, SOS^cat samples a broad range of turnover rates by fluctuating between distinct, long-lived functional states (19). Despite these advances, the nucleotide specificity of Ras bound to the active and allosteric sites of SOS and assembly with RAS, including oncogenic mutants, at the molecular level is poorly understood.Targeting oncogenic Ras mutants presents significant challenges because of their relatively smooth surface that lacks potentially druggable pockets (6). Nevertheless, the discovery of KRas inhibitors, particularly those that form irreversible covalent bonds with Cys-12, comprises one of the most active areas of cancer research (6, 20). As there are few windows of opportunity to specifically target Ras mutants (6), apart from covalent binding to Cys-12, other approaches have also been explored, such as designing molecules to disrupt Ras•SOS interactions, thereby preventing activation of Ras (20). An increasing number of small-molecule disruptors and peptide mimetics have been designed to disrupt the Ras•SOS interaction (21). Potent small-molecule disruptors have recently been discovered that inhibit the formation of the Ras•SOS complex and demonstrate antiproliferative activity, representing a viable approach for targeting Ras-driven tumors (22, 23). These results highlight the therapeutic importance of disrupting the interaction between Ras and SOS.Over the past three decades, native ion mobility–mass spectrometry (IM-MS) has evolved as a powerful analytical technique to investigate protein complexes and their interaction with other molecules (24–26). In native MS, biological samples in aqueous ammonium acetate are ionized using nanoelectrospray ionization and introduced into a mass spectrometer tuned to preserve noncovalent interactions and structure (24, 27). Native IM-MS can provide information on protein complexes, such as subunit stoichiometry and topology (28), and, unlike other biophysical techniques, resolve individual ligand-binding events (29). In combination with an apparatus to control temperature, native MS can be used to determine equilibrium binding constants and thermodynamics for protein–ligand and protein–protein that are in direct agreement with traditional biophysical approaches, such as isothermal calorimetry (30–35). Recently, native MS has been employed to determine transition state thermodynamics for the intrinsic GTPase activity of KRas and several oncogenic mutants (36). Notably, intrinsic GTPase activity rates determined using traditional solution-based assays mirrored those obtained using native MS (36).Although the interaction of Ras and SOS has been the subject of numerous studies, the interaction of oncogenic Ras mutants with SOS remains poorly described. To better understand the role of these interactions in cancer, native IM-MS is used to characterize the molecular assemblies formed between SOS^cat and mutants of KRas associated with cancer. IM spectrometry shows conformational heterogeneity of SOS^cat with specific conformers engaging KRas. Three selected oncogenic mutants of KRas form distinct molecular assemblies with SOS^cat, such as KRas^G13D forming exclusively a ternary complex with SOS^cat. The cryogenic electron microscopy (cryo-EM) structure of the KRas^G13D•SOS^cat complex provides insight into the mechanism for the higher affinity of KRas^G13D for SOS^cat. KRas^G13D–GTP also allosterically modulates the activity of SOS^cat more than the wild-type protein. In addition, the recent inhibitors developed to disrupt the KRas•SOS^cat complex, BAY-293 and BI-3406, cannot dissociate KRas^G13D•SOS^cat complexes. Other small molecules, such as ARS-1620 and Kobee0065, display a range of efficacies in disrupting complexes formed between KRas mutants and SOS^cat.     

An engineered 4-1BBL fusion protein with “activity on demand”

Engineered cytokines are gaining importance in cancer therapy, but these products are often limited by toxicity, especially at early time points after intravenous administration. 4-1BB is a member of the tumor necrosis factor receptor superfamily, which has been considered as a target for therapeutic strategies with agonistic antibodies or using its cognate cytokine ligand, 4-1BBL. Here we describe the engineering of an antibody fusion protein, termed F8-4-1BBL, that does not exhibit cytokine activity in solution but regains biological activity on antigen binding. F8-4-1BBL bound specifically to its cognate antigen, the alternatively spliced EDA domain of fibronectin, and selectively localized to tumors in vivo, as evidenced by quantitative biodistribution experiments. The product promoted a potent antitumor activity in various mouse models of cancer without apparent toxicity at the doses used. F8-4-1BBL represents a prototype for antibody-cytokine fusion proteins, which conditionally display “activity on demand” properties at the site of disease on antigen binding and reduce toxicity to normal tissues.

Cytokines are immunomodulatory proteins that have been considered for pharmaceutical applications in the treatment of cancer patients (1–3) and other types of disease (2). There is a growing interest in the use of engineered cytokine products as anticancer drugs, capable of boosting the action of T cells and natural killer (NK) cells against tumors (3, 4), alone or in combination with immune checkpoint inhibitors (3, 5–7).Recombinant cytokine products on the market include interleukin-2 (IL-2) (Proleukin) (8, 9), IL-11 (Neumega) (10, 11), tumor necrosis factor (TNF; Beromun) (12), interferon (IFN)-α (Roferon A, Intron A) (13, 14), IFN-β (Avonex, Rebif, Betaseron) (15, 16), IFN-γ (Actimmune) (17), granulocyte colony-stimulating factor (Neupogen) (18), and granulocyte macrophage colony-stimulating factor (Leukine) (19, 20). The recommended dose is typically very low (often <1 mg/d) (21–23), as cytokines may exert biological activity in the subnanomolar concentration range (24). Various strategies have been proposed to develop cytokine products with improved therapeutic index. Protein PEGylation or Fc fusions may lead to prolonged circulation time in the bloodstream, allowing the administration of low doses of active payload (25, 26). In some implementations, cleavable polyethylene glycol polymers may be considered, yielding prodrugs that regain activity at later time points (27). Alternatively, tumor-homing antibody fusions have been developed, since the preferential concentration of cytokine payloads at the tumor site has been shown in preclinical models to potentiate therapeutic activity, helping spare normal tissues (28–34). Various antibody-cytokine fusions are currently being investigated in clinical trials for the treatment of cancer and of chronic inflammatory conditions (reviewed in refs. 2, 33, 35–37).Antibody-cytokine fusions display biological activity immediately after injection into patients, which may lead to unwanted toxicity and prevent escalation to therapeutically active dosage regimens (9, 22, 38). In the case of proinflammatory payloads (e.g., IL-2, IL-12, TNF-α), common side effects include hypotension, nausea, and vomiting, as well as flu-like symptoms (24, 39–42). These side effects typically disappear when the cytokine concentration drops below a critical threshold, thus providing a rationale for slow-infusion administration procedures (43). It would be highly desirable to generate antibody-cytokine fusion proteins with excellent tumor-targeting properties and with “activity on demand”— biological activity that is conditionally gained on antigen binding at the site of disease, helping spare normal tissues.Here we describe a fusion protein consisting of the F8 antibody specific to the alternatively spliced extra domain A (EDA) of fibronectin (44, 45) and of murine 4-1BBL, which did not exhibit cytokine activity in solution but could regain potent biological activity on antigen binding. The antigen (EDA+ fibronectin) is conserved from mouse to man (46), is virtually undetectable in normal adult tissues (with the exception of the placenta, endometrium, and some vessels in the ovaries), but is expressed in the majority of human malignancies (44, 45, 47, 48). 4-1BBL, a member of the TNF superfamily (49), is expressed on antigen-presenting cells (50, 51) and binds to its receptor, 4-1BB, which is up-regulated on activated cytotoxic T cells (52), activated dendritic cells (52), activated NK and NKT cells (53), and regulatory T cells (54). Signaling through 4-1BB on cytotoxic T cells protects them from activation-induced cell death and skews the cells toward a more memory-like phenotype (55, 56).We engineered nine formats of the F8-4-1BBL fusion protein, one of which exhibited superior performance in quantitative biodistribution studies and conditional gain of cytokine activity on antigen binding. The antigen-dependent reconstitution of the biological activity of the immunostimulatory payload represents an example of an antibody fusion protein with “activity on demand.” The fusion protein was potently active against different types of cancer without apparent toxicity at the doses used. The EDA of fibronectin is a particularly attractive antigen for cancer therapy in view of its high selectivity, stability, and abundant expression in most tumor types (44, 45, 47, 48).     

“You” speaks to me: Effects of generic-you in creating resonance between people and ideas

Creating resonance between people and ideas is a central goal of communication. Historically, attempts to understand the factors that promote resonance have focused on altering the content of a message. Here we identify an additional route to evoking resonance that is embedded in the structure of language: the generic use of the word “you” (e.g., “You can’t understand someone until you’ve walked a mile in their shoes”). Using crowd-sourced data from the Amazon Kindle application, we demonstrate that passages that people highlighted—collectively, over a quarter of a million times—were substantially more likely to contain generic-you compared to yoked passages that they did not highlight. We also demonstrate in four experiments (n = 1,900) that ideas expressed with generic-you increased resonance. These findings illustrate how a subtle shift in language establishes a powerful sense of connection between people and ideas.

Consider the feeling evoked by watching a gripping scene in a film, hearing a moving song, or coming across a quotation that seems to be written just for you. Experiencing resonance, a sense of connection, is a pervasive human experience. Prior research examining the processes that promote this experience suggests that altering a message to evoke emotion (1–7), highlighting its applicability to a person’s life (2, 6, 8–10), or appealing to a person’s beliefs (4, 8, 11) can all contribute to an idea’s resonance. Here we examine an additional route to cultivating this experience, which is grounded in a message’s form rather than its content: the use of a linguistic device that frames an idea as applying broadly.The ability to frame an idea as general rather than specific is a universal feature of language (12–15). One frequently used device is the generic usage of the pronoun “you” (15–17). Although “you” is often used to refer to a specific person or persons (e.g., “How did you get to work today?”), in many languages, it can also be used to refer to people in general (e.g., “You avoid rush hour if you can.”). This general use of “you” is comparable to the more formal “one,” but is used much more frequently (18).Research indicates that people often use “you” in this way to generalize from their own experiences. For example, a person reflecting on getting fired from their job might say, “It makes you feel betrayed” (18). Here, we propose that using “you” to refer to people in general has additional social implications, affecting whether an idea evokes resonance.Two features of the general usage of “you” (hereafter, “generic-you”) motivate this hypothesis. First, generic-you conveys that ideas are generalizable. Rather than expressing information that applies to a particular situation (e.g., “Leo broke your heart”), generic-you expresses information that is timeless and applies across contexts (e.g., “Eventually, you recover from heartbreak”; 18–23). Second, generic-you is expressed with the same word ("you") that is used in nongeneric contexts to refer to the addressee. Thus, even when “you” is used generically, the association to its specific meaning may further pull in the addressee, heightening resonance. Together, these features suggest that generic-you should promote the resonance of an idea. We tested this hypothesis across five preregistered studies (24–28), using a combination of crowd-sourced data and online experimental paradigms. Data, code, and materials are publicly available via the Open Science Framework (https://osf.io/6J2ZC/) (29). Study 1 used publicly available data from the Amazon Kindle application. Studies 2–5 were approved by the University of Michigan Health Sciences and Behavioral Sciences institutional review board (IRB) under HUM00172473 and deemed exempt from ongoing IRB review. All participants who participated in studies 2–5 provided informed consent via a checkbox presented through the online survey platform, Qualtrics.     

Chemical mutagenesis of a GPCR ligand: Detoxifying “inflammo-attraction” to direct therapeutic stem cell migration

A transplanted stem cell’s engagement with a pathologic niche is the first step in its restoring homeostasis to that site. Inflammatory chemokines are constitutively produced in such a niche; their binding to receptors on the stem cell helps direct that cell’s “pathotropism.” Neural stem cells (NSCs), which express CXCR4, migrate to sites of CNS injury or degeneration in part because astrocytes and vasculature produce the inflammatory chemokine CXCL12. Binding of CXCL12 to CXCR4 (a G protein-coupled receptor, GPCR) triggers repair processes within the NSC. Although a tool directing NSCs to where needed has been long-sought, one would not inject this chemokine in vivo because undesirable inflammation also follows CXCL12–CXCR4 coupling. Alternatively, we chemically “mutated” CXCL12, creating a CXCR4 agonist that contained a strong pure binding motif linked to a signaling motif devoid of sequences responsible for synthetic functions. This synthetic dual-moity CXCR4 agonist not only elicited more extensive and persistent human NSC migration and distribution than did native CXCL 12, but induced no host inflammation (or other adverse effects); rather, there was predominantly reparative gene expression. When co-administered with transplanted human induced pluripotent stem cell-derived hNSCs in a mouse model of a prototypical neurodegenerative disease, the agonist enhanced migration, dissemination, and integration of donor-derived cells into the diseased cerebral cortex (including as electrophysiologically-active cortical neurons) where their secreted cross-corrective enzyme mediated a therapeutic impact unachieved by cells alone. Such a “designer” cytokine receptor-agonist peptide illustrates that treatments can be controlled and optimized by exploiting fundamental stem cell properties (e.g., “inflammo-attraction”).

A transplanted stem cell’s engagement with a pathologic niche is the first step in cell-mediated restoration of homeostasis to that region, whether by cell replacement, protection, gene delivery, milieu alteration, toxin neutralization, or remodeling (1–4). Not surprisingly, the more host terrain covered by the stem cells, the greater their impact. We and others found that a propensity for neural stem cells (NSCs) to home in vivo to acutely injured or actively degenerating central nervous system (CNS) regions—a property called “pathotropism” (1–12), now viewed as central to stem cell biology—is undergirded, at least in part, by the presence of chemokine receptors on the NSC surface, enabling them to follow concentration gradients of inflammatory cytokines constitutively elaborated by pathogenic processes and expressed by reactive astrocytes and injured vascular endothelium within the pathologic niche (5–9). This engagement of NSC receptors was first described for the prototypical chemokine receptor CXCR4 (C-X-C chemokine receptor type 4; also known as fusin or cluster of differentiation-184 [CD184]) and its unique natural cognate agonist ligand, the inflammatory chemokine CXCL12 (C-X-C motif chemokine ligand-12; also known as stromal cell-derived factor 1α [SDF-1α]) (5), but has since been described for many chemokine receptor-agonist pairings (6–9). Chemokine receptors belong to a superfamily that is characterized by seven transmembrane GDP-binding protein-coupled receptors (GPCRs) (13–21). In addition to their role in mediating inflammatory reactions and immune responses (22, 23), these receptors and their agonists are components of the regulatory axes for hematopoiesis and organogenesis in other systems (21, 24). Therefore, it is not surprising that binding of CXCL12 to CXCR4 mediates not only an inflammatory response, but also triggers within the NSC a series of intracellular processes associated with migration (as well as proliferation, differentiation, survival, and, during early brain development, proper neuronal lamination) (10).A tool directing therapeutic NSCs to where they are needed has long been sought in regenerative medicine (11, 12). While it was appealing to contemplate electively directing reparative NSCs to any desired area by emulating this chemoattractive property through the targeted injection of exogenous recombinant inflammatory cytokines, it ultimately seemed inadvisable to risk increasing toxicity in brains already characterized by excessive and usually inimical inflammation from neurotraumatic or neurodegenerative processes. However, the notion of engaging the homing function of these NSC-borne receptors without triggering that receptor’s undesirable downstream inflammatory signaling [particularly given that the NSCs themselves can exert a therapeutic antiinflammatory action in the diseased region (1, 2)] seemed a promising heretofore unexplored “workaround.”There had already been an impetus to examine the structure–function relationships of CXCR4, known to be the entry route into cells for HIV-1, in order to create CXCR4 antagonists that block viral infection (25–30). Antagonists of CXCR4 were also devised to forestall hematopoietic stem cells from homing to the bone marrow, hence prolonging their presence in the peripheral blood (31) to treat blood dyscrasias. An agonist, however, particularly one with discrete and selective actions, had not been contemplated. In other words, if CXCL12 could be stripped of its undesirable actions while preserving its tropic activity, an ideal chemoattractant would be derived.Based on the concept that CXCR4’s functions are conveyed by two distinct molecular “pockets”—one mediating binding (i.e., allowing a ligand to engage CXCR4) and the other mediating signaling (i.e., enabling a ligand, after binding, to trigger CXCR4-mediated intracellular cascades that promote not only inflammation but also migration) (13–18)—we performed chemical mutagenesis that should optimize binding while narrowing the spectrum of signaling. We created a simplified de novo peptide agonist of CXCR4 that contained a strong pure binding motif derived from CXCR4’s strongest ligand, viral macrophage inflammatory protein-II (vMIP-II) and linked it to a truncated signaling motif (only 8 amino acid residues) derived from the N terminus of native CXCL12 (19, 20). This synthetic dual-moiety CXCR4 agonist, which is devoid of a large portion of CXCL12’s native sequence (presumably responsible for undesired functions such as inflammation) not only elicited (with great specificity) more extensive and long-lasting human NSC (hNSC) migration and distribution than native CXCL12 (overcoming migratory barriers), but induced no host inflammation (or other adverse effects). Furthermore, because all of the amino acids in the binding motif were in a D-chirality, rendering the peptide resistant to enzymatic degradation, persistence of this benign synthetic agonist in vivo was prolonged. The hNSC’s gene ontology expression profile was predominantly reparative in contrast to inflammatory as promoted by native CXCL12. When coadministered with transplanted human induced pluripotent stem cell (hiPSC)-derived hNSCs (hiPSC derivatives are now known to have muted migration) in a mouse model of a prototypical neurodegenerative disease [the lethal neuropathic lysosomal storage disorder (LSD) Sandhoff disease (29), where hiPSC-hNSC migration is particularly limited], the synthetic agonist enhanced migration, dissemination, and integration of donor-derived cells into the diseased cortex (including as electrophysiologically active cortical neurons), where their secreted cross-corrective enzyme could mediate a histological and functional therapeutic impact in a manner unachieved by transplanting hiPSC-derived cells alone.In introducing such a “designer” cytokine receptor agonist, we hope to offer proof-of-concept that stem cell-mediated treatments can be controlled and optimized by exploiting fundamental stem cell properties (e.g., “inflammo-attraction”) to alter a niche and augment specific actions. Additionally, when agonists are strategically designed, the various functions of chemokine receptors (and likely other GCPRs) may be divorced. We demonstrate that such a strategy might be used safely and effectively to direct cells to needed regions and broaden their chimerism. We discuss the future implications and uses within the life sciences of such a chemical engineering approach.     

Characterizing the “fungal shunt”: Parasitic fungi on diatoms affect carbon flow and bacterial communities in aquatic microbial food webs

Microbial interactions in aquatic environments profoundly affect global biogeochemical cycles, but the role of microparasites has been largely overlooked. Using a model pathosystem, we studied hitherto cryptic interactions between microparasitic fungi (chytrid Rhizophydiales), their diatom host Asterionella, and cell-associated and free-living bacteria. We analyzed the effect of fungal infections on microbial abundances, bacterial taxonomy, cell-to-cell carbon transfer, and cell-specific nitrate-based growth using microscopy (e.g., fluorescence in situ hybridization), 16S rRNA gene amplicon sequencing, and secondary ion mass spectrometry. Bacterial abundances were 2 to 4 times higher on individual fungal-infected diatoms compared to healthy diatoms, particularly involving Burkholderiales. Furthermore, taxonomic compositions of both diatom-associated and free-living bacteria were significantly different between noninfected and fungal-infected cocultures. The fungal microparasite, including diatom-associated sporangia and free-swimming zoospores, derived ∼100% of their carbon content from the diatom. By comparison, transfer efficiencies of photosynthetic carbon were lower to diatom-associated bacteria (67 to 98%), with a high cell-to-cell variability, and even lower to free-living bacteria (32%). Likewise, nitrate-based growth for the diatom and fungi was synchronized and faster than for diatom-associated and free-living bacteria. In a natural lacustrine system, where infection prevalence reached 54%, we calculated that 20% of the total diatom-derived photosynthetic carbon was shunted to the parasitic fungi, which can be grazed by zooplankton, thereby accelerating carbon transfer to higher trophic levels and bypassing the microbial loop. The herein termed “fungal shunt” can thus significantly modify the fate of photosynthetic carbon and the nature of phytoplankton–bacteria interactions, with implications for diverse pelagic food webs and global biogeochemical cycles.

Parasitism is one of the most common consumer strategies on Earth (1–3). Recently, it has also been identified as one of the dominating interactions within the planktonic interactome (4, 5), and yet parasites remain poorly considered in analyses of trophic interactions and element cycling in aquatic systems (6, 7). The foundation of trophic interactions in plankton communities is set by single-cell phytoplankton, which contributes almost half of the world’s primary production (8). According to our common understanding, the newly fixed carbon (C) is channeled either through the microbial loop, classical food web, or viral shunt, which supports the growth of heterotrophic bacteria and nanoflagellates, zooplankton and higher trophic levels, or viruses, respectively (9). However, fungi, particularly fungal microparasites, are rarely considered as contributors to C and nutrient cycling, although they are present and active in diverse aquatic environments (10–12).Members of the fungal division Chytridiomycota, referred to as chytrids, can thrive as microparasites on phytoplankton cells in freshwater (11, 13, 14) and marine systems (15–17), infecting up to 90% of the phytoplankton host population (18–21). A recent concept, called mycoloop, describes parasitic chytrids as an integral part of aquatic food webs (22). Energy and organic matter are thereby transferred from large, often inedible phytoplankton to chytrid zoospores, which are consumed by zooplankton (23–27). Hence, parasitic chytrids establish a novel trophic link between phytoplankton and zooplankton. Our understanding of element cycling and microbial interactions during chytrid epidemics, however, remains sparse. For instance, the cell-to-cell C transfer from single phytoplankton cells to their directly associated chytrids has not been quantified to date. Moreover, the relationship between parasitic chytrids and heterotrophic bacteria is largely undescribed.Phytoplankton cells release substantial amounts of dissolved organic C (DOC) (28), whereby up to 50% of photosynthetic C is consumed as DOC by bacteria (29–32). Thus, bacterial communities are intimately linked to phytoplankton abundances and production (33). Phytoplankton–bacteria interactions are particularly strong within the phycosphere, the region immediately surrounding individual phytoplankton cells (33–35), where nutrient concentrations are several-folds higher compared to the ambient water (36, 37), and nutrient assimilation rates of phytoplankton-associated bacteria are at least twice as fast as those of their free-living counterparts (38, 39). Importantly, parasitic chytrids may distort these phytoplankton–bacteria interactions within and outside the phycosphere since they modulate substrate and nutrient availabilities and presumably also bacterial activity and community composition. The effects of this distortion are virtually unresolved, but the few available data indicate that chytrid infections alter the composition and concentration of DOC (40), while abundances of free-living bacteria increase (25, 40) or remain unchanged (24).To disentangle phytoplankton–fungi–bacteria interactions at a microspatial single-cell scale—the scale at which phytoplankton, fungi, and bacteria intimately interact—we used one of the few existing model pathosystems, composed of the freshwater diatom Asterionella formosa, the chytrid Rhizophydiales sp., and coenriched populations of heterotrophic bacteria. Our methodology included dual stable-isotope incubations (¹³C-bicarbonate and ¹⁵N-nitrate), single-cell–resolution secondary ion mass spectrometry (SIMS) (IMS 1280 and NanoSIMS 50L), 16S rRNA gene/16S rRNA sequencing, microscopy (e.g., fluorescence in situ hybridization [FISH]), and nutrient analyses. We particularly focused on the initial C transfer from the phytoplankton host to parasitic chytrids, which we term the “fungal shunt,” as part of the mycoloop. The objectives were twofold: 1) quantifying the transfer of photosynthetic C from phytoplankton cells to infectious chytrids, cell-associated bacteria, and free-living bacteria and 2) characterizing the effect of parasitic fungi on bacterial populations, considering bacterial abundances, bacterial–diatom attachment, single-cell activity rates, and community composition. The obtained data challenge the common perception of aquatic microbial food webs by demonstrating the significant role that parasitic fungi can play in microbial community structure, interactions, and element cycling during phytoplankton growth.     

Increasing risk of another Cape Town “Day Zero” drought in the 21st century

Three consecutive dry winters (2015–2017) in southwestern South Africa (SSA) resulted in the Cape Town “Day Zero” drought in early 2018. The contribution of anthropogenic global warming to this prolonged rainfall deficit has previously been evaluated through observations and climate models. However, model adequacy and insufficient horizontal resolution make it difficult to precisely quantify the changing likelihood of extreme droughts, given the small regional scale. Here, we use a high-resolution large ensemble to estimate the contribution of anthropogenic climate change to the probability of occurrence of multiyear SSA rainfall deficits in past and future decades. We find that anthropogenic climate change increased the likelihood of the 2015–2017 rainfall deficit by a factor of five to six. The probability of such an event will increase from 0.7 to 25% by the year 2100 under an intermediate-emission scenario (Shared Socioeconomic Pathway 2-4.5 [SSP2-4.5]) and to 80% under a high-emission scenario (SSP5-8.5). These results highlight the strong sensitivity of the drought risk in SSA to future anthropogenic emissions.

The Day Zero Cape Town drought was one of the worst water crises ever experienced in a metropolitan area (1, 2). Droughts are a regular occurrence in southwestern South Africa (SSA), having occurred during the late 1920s, early 1970s, and, more recently, during 2003–2004 (Fig. 1 A and B). However, the extended winter (April to September [AMJJAS]) 3-y rainfall deficit (Fig. 1 A and B; SI Appendix, Fig. S1) which drove the 2015–2017 Cape Town drought (2–8) was exceptional over the last century (4, 9). Storage in reservoirs supplying water to 3.7 million people in the Cape Town metropolitan area dropped to about 20% of capacity in May 2018. As a consequence, strict water-usage restrictions were implemented to delay water levels reaching 13.5%, the level at which much of the city’s municipal supply would have been disconnected (7), a scenario referred to as “Day Zero” by the municipal water authorities (7). Above-average winter rain over the rest of the 2018 austral winter allowed Cape Town to avoid the Day Zero scenario.Open in a separate windowFig. 1.(A) Mean 2015–2017 AMJJAS rainfall anomaly relative to 1921–1970. The dashed (continuous) line denotes negative anomalies beyond 1 (1.5) SD. (B) Time series of the observed (GPCC, blue; CRU, red) 3-y running mean AMJJAS WRI (Materials and Methods) from 1901 to 2017. The 2015–2017 mean is record-breaking over the period 1901–2017. (C–E) Mean 1921–1970 AMJJAS rainfall (millimeters per month) in observations (GPCC) (C), SPEAR_MED (D), and SPEAR_LO (E). The red lines encircle the area receiving at least 65% of the total annual rainfall during AMJJAS used to define WRI. (F) Monthly WRI in observations and models. Comparison of SPEAR_MED with SPEAR_LO shows how an enhanced resolution is key to capture finer-scale regional details of winter rainfall in the relatively small SSA Mediterranean region.While poor water-management practices and infrastructure deficiencies worsened the crisis (10, 11), the 2015–2017 rainfall deficit was the main driver of the drought (5). To facilitate the improvement of water-management practices and the infrastructure necessary to make the system more resilient, it is critical to first determine how likely a meteorological drought like the one in 2015–2017 might be in the coming decades. Increased aridity is expected in most of southern Africa (12–14) as a consequence of the Hadley Cell poleward expansion (4, 15–18) and southward shift of the Southern Hemisphere jet stream (19). Second, the risk of more extreme droughts should be quantified to understand the potential for emerging risks that could make a Day Zero event in Cape Town unavoidable.Previous work (5) has suggested that the Day Zero drought may have been made 1.4 to 6.4 times more likely over the last century due to +1 K of global warming, with the risk expected to scale linearly with one additional degree of warming. Such estimates make use of statistical models of the probability distribution’s tail (e.g., the generalized extreme value) applied to observations and previous-generation [i.e., as those participating to the Coupled Model Intercomparison Project Phase 3 (CMIP3) (20) and 5 (21)] climate models. CMIP3 and CMIP5 models have been shown to have a systematically biased position of the Southern Hemisphere jet stream toward the Equator, due to insufficient horizontal resolution (19). This produces a large uncertainty in model projections of jet-stream shifts (22, 23), thus hindering realistic projections of Southern Hemisphere climate change. Furthermore, for hydroclimatic variables, a statistical extrapolation of the probability distribution’s tail might have inherent limitations in providing precise estimates of the event probability of future extreme events, although its precision profits from the use of large ensembles (24, 25).Large ensembles of comprehensive climate models provide thousands of years of data that allow direct construction of the underlying probability distribution of hydroclimatic extremes without relying on a hypothesized statistical model of extremes (25, 26). South African winter rains have high interannual and decadal variability due to El Niño–Southern Oscillation (27), the Southern Annular Mode (28), and interdecadal variability (29). A multidecade to multicentury record may be required to detect the emergence of statistically significant trends in regional precipitation extremes. A large ensemble is, thus, a powerful method to isolate, at the decadal timescale, internal natural variability (e.g., SI Appendix, Fig. S2) from the forced signal (30–32).     

CAP1 binds and activates adenylyl cyclase in mammalian cells

CAP1 (Cyclase-Associated Protein 1) is highly conserved in evolution. Originally identified in yeast as a bifunctional protein involved in Ras-adenylyl cyclase and F-actin dynamics regulation, the adenylyl cyclase component seems to be lost in mammalian cells. Prompted by our recent identification of the Ras-like small GTPase Rap1 as a GTP-independent but geranylgeranyl-specific partner for CAP1, we hypothesized that CAP1-Rap1, similar to CAP-Ras-cyclase in yeast, might play a critical role in cAMP dynamics in mammalian cells. In this study, we report that CAP1 binds and activates mammalian adenylyl cyclase in vitro, modulates cAMP in live cells in a Rap1-dependent manner, and affects cAMP-dependent proliferation. Utilizing deletion and mutagenesis approaches, we mapped the interaction of CAP1-cyclase with CAP’s N-terminal domain involving critical leucine residues in the conserved RLE motifs and adenylyl cyclase’s conserved catalytic loops (e.g., C1a and/or C2a). When combined with a FRET-based cAMP sensor, CAP1 overexpression–knockdown strategies, and the use of constitutively active and negative regulators of Rap1, our studies highlight a critical role for CAP1-Rap1 in adenylyl cyclase regulation in live cells. Similarly, we show that CAP1 modulation significantly affected cAMP-mediated proliferation in an RLE motif–dependent manner. The combined study indicates that CAP1-cyclase-Rap1 represents a regulatory unit in cAMP dynamics and biology. Since Rap1 is an established downstream effector of cAMP, we advance the hypothesis that CAP1-cyclase-Rap1 represents a positive feedback loop that might be involved in cAMP microdomain establishment and localized signaling.

CAP/srv2 was originally identified in yeast biochemically as an adenylyl cyclase–associated protein (1) and genetically as a suppressor of the hyperactive Ras2-V19 allele (2). CAP/srv2-deficient yeast cells are unresponsive to active Ras2, and adenylyl cyclase activity is no longer regulated by Ras2 in these cells (1, 2), indicating the involvement of CAP/srv2 in the Ras/cyclase pathway. However, some mutant CAP/srv2 alleles presented phenotypes not observed in strains with impaired Ras/cyclase pathway (1–3), indicating the existence of Ras/cyclase-independent functions downstream of CAP/srv2. These two phenotype groups, that is, Ras/cyclase-linked and Ras/cyclase-independent, could be suppressed by expression of an N-terminal half and a C-terminal half of CAP/srv2, respectively (4). Subsequent studies showed that the C-terminal half of CAP/srv2 was able to bind monomeric G-actin (5–8) and other actin regulators establishing a role in F-actin dynamics (9–16). Thus, CAP/srv2 is a bifunctional protein with an N-terminal domain involved in Ras/cyclase regulation and a C-terminal domain involved with F-actin dynamics regulation (16–18).CAP1 is structurally conserved in all eukaryotes (18–22); however, their functions are not. Expression of the closely related Schizosaccharomyces pombe cap or mammalian CAP1 in yeast can only suppress the phenotypes associated with deletion of CAP/srv2’s C-terminal but not its N-terminal domain (19, 20, 22), suggesting that only the F-actin dynamics function was conserved while the Ras/cyclase regulation diverged early on in evolution (16–18). CAP/srv2’s N-terminal 1 to 36 domain was sufficient for cyclase binding in yeast involving a conserved RLE motif with predicted coiled-coil folding (23). Interestingly, this domain is also involved in CAP1 oligomerization both in yeast and mammalian cells (24–26), where it purifies as a high-molecular complex of ∼600 kDa consistent with a 1:1 stoichiometric CAP1-actin hexameric organization (12, 25, 27, 28). Importantly, removal of this domain disrupted CAP1 oligomerization, reduced F-actin turnover in vitro and caused defects in cell growth, cell morphology, and F-actin organization in vivo (24, 29). However, whether the conserved RLE motif in mammalian CAP1 interacts with other coiled-coil–containing proteins is for the moment unknown.Ras2-mediated cyclase regulation in yeast requires its farnesylation (30–32). However, the lipid target involved was not identified in the original studies. We have recently shown that mammalian CAP1 interacts with the small GTPase Rap1. The interaction involves Rap1’s C-terminal hypervariable region (HVR) and its lipid moiety in a geranylgeranyl-specific manner; that is, neither the closely related Ras1 nor engineered farnesylated Rap1 interacted with CAP1 (33). Thus, we raised the question whether CAP1-Rap1, similar to CAP/srv2-Ras2 in yeast, plays a role in cAMP dynamics in mammalian cells.In this study, we report that CAP1 binds to and activates mammalian adenylyl cyclase in vitro. The interaction involves CAP1’s conserved RLE motifs and cyclase’s conserved catalytic subdomains (e.g., C1a and/or C2a). Most importantly, we show that both CAP1 and Rap1 modulate cAMP dynamics in live cells and are critical players in cAMP-dependent proliferation.     

From the Cover: A Middle Eocene lowland humid subtropical “Shangri-La” ecosystem in central Tibet

Tibet’s ancient topography and its role in climatic and biotic evolution remain speculative due to a paucity of quantitative surface-height measurements through time and space, and sparse fossil records. However, newly discovered fossils from a present elevation of ∼4,850 m in central Tibet improve substantially our knowledge of the ancient Tibetan environment. The 70 plant fossil taxa so far recovered include the first occurrences of several modern Asian lineages and represent a Middle Eocene (∼47 Mya) humid subtropical ecosystem. The fossils not only record the diverse composition of the ancient Tibetan biota, but also allow us to constrain the Middle Eocene land surface height in central Tibet to ∼1,500 ± 900 m, and quantify the prevailing thermal and hydrological regime. This “Shangri-La”–like ecosystem experienced monsoon seasonality with a mean annual temperature of ∼19 °C, and frosts were rare. It contained few Gondwanan taxa, yet was compositionally similar to contemporaneous floras in both North America and Europe. Our discovery quantifies a key part of Tibetan Paleogene topography and climate, and highlights the importance of Tibet in regard to the origin of modern Asian plant species and the evolution of global biodiversity.

The Tibetan Plateau, once thought of as entirely the product of the India–Eurasia collision, is known to have had significant complex relief before the arrival of India early in the Paleogene (1–3). This large region, spanning ∼2.5 million km², is an amalgam of tectonic terranes that impacted Asia long before India’s arrival (4, 5), with each accretion contributing orographic heterogeneity that likely impacted climate in complex ways. During the Paleogene, the Tibetan landscape comprised a high (>4 km) Gangdese mountain range along the southern margin of the Lhasa terrane (2), against which the Himalaya would later rise (6), and a Tanghula upland on the more northerly Qiangtang terrane (7). Separating the Lhasa and Qiangtang blocks is the east–west trending Banggong-Nujiang Suture (BNS), which today hosts several sedimentary basins (e.g., Bangor, Nyima, and Lunpola) where >4 km of Cenozoic sediments have accumulated (8). Although these sediments record the climatic and biotic evolution of central Tibet, their remoteness means fossil collections have been hitherto limited. Recently, we discovered a highly diverse fossil assemblage in the Bangor Basin. These fossils characterize a luxuriant seasonally wet and warm Shangri-La forest that once occupied a deep central Tibetan valley along the BNS, and provide a unique opportunity for understanding the evolutionary history of Asian biodiversity, as well as for quantifying the paleoenvironment of central Tibet.^*Details of the topographic evolution of Tibet are still unclear despite decades of investigation (4, 5). Isotopic compositions of carbonates recovered from sediments in some parts of central Tibet have been interpreted in terms of high (>4 km) Paleogene elevations and aridity (9, 10), but those same successions have yielded isolated mammal (11), fish (12), plant (13–18), and biomarker remains (19) more indicative of a low (≤3-km) humid environment, but how low is poorly quantified. Given the complex assembly of Tibet, it is difficult to explain how a plateau might have formed so early and then remained as a surface of low relief during subsequent compression from India (20). Recent evidence from a climate model-mediated interpretation of palm fossils constrains the BNS elevation to below 2.3 km in the Late Paleogene (16), but more precise paleoelevation estimates are required. Further fossil discoveries, especially from earlier in the BNS sedimentary records, would document better the evolution of the Tibetan biota, as well as informing our understanding of the elevation and climate in an area that now occupies the center of the Tibetan Plateau.Our work shows that the BNS hosted a diverse subtropical ecosystem at ∼47 Ma, and this means the area must have been both low and humid. The diversity of the fossil flora allows us to 1) document floristic links to other parts of the Northern Hemisphere, 2) characterize the prevailing paleoclimate, and 3) quantify the elevation at which the vegetation grew. We propose that the “high and dry” central Tibet inferred from some isotope paleoaltimetry (9, 10) reflects a “phantom” elevated paleosurface (20) because fractionation over the bounding mountains allowed only isotopically light moist air to enter the valley, giving a false indication of a high elevation (21).     

“Soft” oxidative coupling of methane to ethylene: Mechanistic insights from combined experiment and theory

The oxidative coupling of methane to ethylene using gaseous disulfur (2CH₄ + S₂ → C₂H₄ + 2H₂S) as an oxidant (SOCM) proceeds with promising selectivity. Here, we report detailed experimental and theoretical studies that examine the mechanism for the conversion of CH₄ to C₂H₄ over an Fe₃O₄-derived FeS₂ catalyst achieving a promising ethylene selectivity of 33%. We compare and contrast these results with those for the highly exothermic oxidative coupling of methane (OCM) using O₂ (2CH₄ + O₂ → C₂H₄ + 2H₂O). SOCM kinetic/mechanistic analysis, along with density functional theory results, indicate that ethylene is produced as a primary product of methane activation, proceeding predominantly via CH₂ coupling over dimeric S–S moieties that bridge Fe surface sites, and to a lesser degree, on heavily sulfided mononuclear sites. In contrast to and unlike OCM, the overoxidized CS₂ by-product forms predominantly via CH₄ oxidation, rather than from C₂ products, through a series of C–H activation and S-addition steps at adsorbed sulfur sites on the FeS₂ surface. The experimental rates for methane conversion are first order in both CH₄ and S₂, consistent with the involvement of two S sites in the rate-determining methane C–H activation step, with a CD₄/CH₄ kinetic isotope effect of 1.78. The experimental apparent activation energy for methane conversion is 66 ± 8 kJ/mol, significantly lower than for CH₄ oxidative coupling with O₂. The computed methane activation barrier, rate orders, and kinetic isotope values are consistent with experiment. All evidence indicates that SOCM proceeds via a very different pathway than that of OCM.

The oxidative coupling of methane (OCM) with O₂ would seem to be a concise, direct route to convert methane, one of the most Earth-abundant carbon sources (1), to ethylene (2CH₄ + O₂ → C₂H₄ + 2H₂O), a key chemical intermediate (2, 3), and this process has been extensively studied (1, 4–19) since 1982 (20). Nevertheless, the widespread use of OCM is challenged by methane overoxidation to CO₂ and other oxygenates. Furthermore, the severe reaction conditions of nonoxidative pathways (2, 21–28) typically risk carbon deposition and catalyst deactivation (2, 21–26). In preliminary studies, we reported a 2CH₄ + S₂ → C₂H₄ + 2H₂S coupling process that moderates the methane overoxidation driving force using gaseous disulfur (S₂) as a “soft” oxidant (SOCM; Fig. 1A) (29). S₂ is isoelectronic with O₂, the major sulfur vapor species at 700 to 925 °C (30–32), and is a less aggressive oxidant than O₂ (33). In this scenario, elemental sulfur is recovered from the H₂S coproduct via the known Claus process (Fig. 1B) (30), in a cycle where sulfur mediates/moderates the high nonselective O₂ reactivity. SOCM achieved promising ethylene selectivity, raising intriguing mechanistic questions and the possibility of higher selectivity. Methane + S_2(g) ethylene selectivities near ∼20% are achieved over a PdS/ZrO₂ catalyst (29), and oxide precatalysts give selectivities near 33% (34).Open in a separate windowFig. 1.Energetic comparison between the oxidative coupling of methane with O₂ (OCM) and with S₂ (SOCM) and the pathway to recover elemental sulfur from H₂S. (A) Gibbs free energy of desired and overoxidation processes in OCM and SOCM at 800 and 1,050 °C. (B) Industrialized catalytic Claus process used to recover elemental sulfur from H₂S.Nevertheless, in contrast to extensive OCM (17, 35–39) and nonoxidative CH₄ coupling studies (40), far less is known about the SOCM reaction pathway. Post-SOCM X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD), and elemental analysis (29, 34) indicate that the oxide precatalysts are predominantly sulfided. Density functional theory (DFT) analyses of molybdenum sulfide catalysts suggest that methane is activated at M–S or S–S sites to form surface-bound CH₃* species which dehydrogenate to form CH₂* (methylidene) species, which then couple to produce C₂H₄. It was proposed that CH₃* species can also desorb as methyl radicals which couple to form ethane (29). The overoxidation product, CS₂, was suggested to form via sulfur addition to methylidene surface intermediates (29).Kinetic, mechanistic, and theoretical analyses are needed to better understand the CH₄ conversion pathways to C₂H₄ and other products. In principle, there are two plausible pathways following methane activation: 1) H abstraction from adsorbed methyl species forms methylidene (CH₂*) and methylidyne (CH*) species then couple to C₂ products or undergo oxidation to CS₂ or 2) coupling of surface or gas phase methyl species form ethane, which then dehydrogenates to form ethylene or oxidizes to CS₂. For further SOCM optimization it is important to determine which pathways are operative, their relative rates, and the C₂ and CS₂ formation sites.Here we investigate SOCM pathways over a sulfided Fe₃O₄ precatalyst which affords C₂H₄ selectivities near 33%, complete oxide to sulfide conversion, minimal carbon deposition (coking), and 48-h SOCM stability at 950 °C (34). We first summarize SOCM phenomenology, followed by analysis of the Fe phases during sulfurization and SOCM. Next, kinetic/mechanistic studies focus on the methane and S₂ reaction orders, activation energetics, and isotope effects and probe the pathways governing C₂ vs. CS₂ formation. Complementary DFT calculations focus on reaction mechanisms, the active sites, and their role in product formation. The results are used in a microkinetic model to simulate reaction rates, apparent activation barriers, and reaction rate orders and to compare with experiment. Finally, SOCM and OCM are compared, revealing that they follow distinctly different pathways.     

A Sos proteomimetic as a pan-Ras inhibitor

Aberrant Ras signaling is linked to a wide spectrum of hyperproliferative diseases, and components of the signaling pathway, including Ras, have been the subject of intense and ongoing drug discovery efforts. The cellular activity of Ras is modulated by its association with the guanine nucleotide exchange factor Son of sevenless (Sos), and the high-resolution crystal structure of the Ras–Sos complex provides a basis for the rational design of orthosteric Ras ligands. We constructed a synthetic Sos protein mimic that engages the wild-type and oncogenic forms of nucleotide-bound Ras and modulates downstream kinase signaling. The Sos mimic was designed to capture the conformation of the Sos helix–loop–helix motif that makes critical contacts with Ras in its switch region. Chemoproteomic studies illustrate that the proteomimetic engages Ras and other cellular GTPases. The synthetic proteomimetic resists proteolytic degradation and enters cells through macropinocytosis. As such, it is selectively toxic to cancer cells with up-regulated macropinocytosis, including those that feature oncogenic Ras mutations.

The Ras-specific guanine nucleotide exchange factor Son of sevenless (Sos) mediates the conversion of Ras from its inactive GDP-bound form to the active GTP-bound state (1). Sos catalyzes nucleotide exchange via insertion of a critical helical segment (αH) between the conformationally dynamic Switch I and II regions that flank the Ras nucleotide binding pocket leading to disruption of water-mediated and direct interactions between the protein and the cofactor (Fig. 1A) (2). Given the biomedical importance of the conformationally dynamic Sos-binding interface of Ras, several rational design and screening strategies have been attempted to develop ligands for this interface (3, 4). Recent efforts to engage the Ras G12C (5, 6) and the G12D isoforms (7, 8) suggest that targeted screens may afford small molecules and peptide macrocycles as potential leads. The structure of the Ras–Sos complex provides a basis for the rational design of Sos helix mimics that engage the Ras switch regions. Our group has previously developed a conformationally stabilized α-helix mimic to target the Ras–Sos protein–protein interaction (PPI) (9, 10). The stabilized α-helix was shown to bind Ras at the orthosteric binding site and inhibit Sos-mediated nucleotide exchange, Ras activation, and phosphorylation of the downstream effector protein ERK (11), a well-characterized kinase implicated in cell proliferation and differentiation. However, this compound preferred to bind Ras in its nucleotide-free form, suggesting that a single Sos helix is likely insufficient to properly engage the dynamic Ras interface in its nucleotide-bound form. While wild-type Ras toggles between its two nucleotide-bound forms (Fig. 1A), the oncogenic forms of Ras remain activated in their GTP-bound states (3). Therefore, a compound that preferentially engages the nucleotide-free form of Ras may have limited biological utility.Open in a separate windowFig. 1.Overview of the Ras activation cycle and design of a Sos-based proteomimetic. (A) The cellular activity of Ras is tightly controlled as part of a balanced feedback loop. Oncogenic mutations shift this balance and increase the cellular concentration of Ras-GTP leading to aberrant downstream signaling. The molecular model shows the complex between Ras (gray ribbon) and its guanine exchange factor Sos (green). Sos inserts a helical hairpin (pink and blue helices) into the nucleotide binding pocket of Ras to mediate nucleotide exchange. The Ras nucleotide binding pocket is highlighted in yellow. Segments of Sos are not shown to highlight interactions of the helical hairpin with Ras (PDB code: 1NVW). (B) The molecular models depict critical Sos helices and the design of a constrained Sos proteomimetic as a Ras inhibitor. GDP, guanosine 5′-diphosphate; GTP, guanosine 5′-triphosphate; GAP, GTPase activating protein; GEF, guanine nucleotide exchange factor.Our prior results with the Sos helix mimic encapsulate a critical challenge in developing minimal protein secondary-structure mimics. Although mimics of protein secondary structures have proven to be a potent class of PPI inhibitors (12–15), many protein interfaces feature binding epitope complexity beyond what can be captured by reproduction of minimal elements of protein structure (16–19). We hypothesized that the introduction of additional contact residues from Sos may allow engagement of nucleotide-bound Ras (Fig. 1B). Sos inserts αH into the switch region of Ras, but analysis of the complex shows that several other residues from Sos also interact with Ras (SI Appendix, Fig. S1). The conformation of the αH helix, itself, is controlled by the αI domain as part of a hairpin helix organization. The αI helix makes important electrostatic contacts with the Ras effector loop in the Switch I region.We sought to develop a tertiary-structure mimic of Sos that encompasses critical binding residues from the helix–loop–helix motif to determine if the additional contacts allow engagement of nucleotide-bound Ras (Fig. 1B). We utilized a recently described synthetic approach from our group to capture the conformation of the Sos αH and αI helices. In prior efforts, we learned that helix dimers may be stabilized by judicious substitution of a surface salt bridge with a covalent bond and appropriate sculpting of the dimeric interface to coerce knob-into-hole helix packing (20, 21). These stabilized proteomimetics are termed crosslinked helix dimers or CHDs. Here, we show that Sos CHDs are proteolytically stable, selectively cell permeable, and engage the Sos-binding surface of nucleotide-bound Ras with high specificity in biochemical and cellular contexts. We utilized a combination of rational design principles and computational modeling to exploit previously unexplored and underutilized pockets at the target interface (22, 23). The optimized proteomimetic binds wild-type and mutant Ras forms with nanomolar to low micromolar affinities, modulates nucleotide exchange, engages Ras and other Ras subfamily GTPases as demonstrated by chemoproteomic assays, inhibits downstream activation of the Ras-mediated signaling cascade, and is selectively toxic to cancer cells with oncogenic Ras mutations.     

Trapping a cross-linked lysine–tryptophan radical in the catalytic cycle of the radical SAM enzyme SuiB

The radical S-adenosylmethionine (rSAM) enzyme SuiB catalyzes the formation of an unusual carbon–carbon bond between the sidechains of lysine (Lys) and tryptophan (Trp) in the biosynthesis of a ribosomal peptide natural product. Prior work on SuiB has suggested that the Lys–Trp cross-link is formed via radical electrophilic aromatic substitution (rEAS), in which an auxiliary [4Fe-4S] cluster (AuxI), bound in the SPASM domain of SuiB, carries out an essential oxidation reaction during turnover. Despite the prevalence of auxiliary clusters in over 165,000 rSAM enzymes, direct evidence for their catalytic role has not been reported. Here, we have used electron paramagnetic resonance (EPR) spectroscopy to dissect the SuiB mechanism. Our studies reveal substrate-dependent redox potential tuning of the AuxI cluster, constraining it to the oxidized [4Fe-4S]²⁺ state, which is active in catalysis. We further report the trapping and characterization of an unprecedented cross-linked Lys–Trp radical (Lys–Trp•) in addition to the organometallic Ω intermediate, providing compelling support for the proposed rEAS mechanism. Finally, we observe oxidation of the Lys–Trp• intermediate by the redox-tuned [4Fe-4S]²⁺ AuxI cluster by EPR spectroscopy. Our findings provide direct evidence for a role of a SPASM domain auxiliary cluster and consolidate rEAS as a mechanistic paradigm for rSAM enzyme-catalyzed carbon–carbon bond-forming reactions.

The radical S-adenosylmethionine (rSAM) enzyme superfamily is the largest known in nature, with over 570,000 annotated and predominantly uncharacterized members spanning all domains of life (1–4). The uniting feature of rSAM enzymes is a [4Fe-4S] cluster, usually bound by a CX₃CX₂C motif that catalyzes reductive cleavage of SAM to form L-Met and a strongly oxidizing 5′-deoxyadenosyl radical (5′-dA•) (5–7). Recent studies on a suite of rSAM enzymes have revealed the presence of a previously unknown organometallic intermediate in this process, termed Ω, in which the 5′-C of 5′-dA• is bound to the unique iron of the [4Fe-4S] cluster (Fig. 1A) (8, 9). Homolysis of the Fe–C bond ultimately liberates 5′-dA•, which abstracts a hydrogen atom from substrate to initiate a profoundly diverse set of chemical reactions in both primary and secondary metabolism, including DNA, cofactor, vitamin, and antibiotic biosynthesis (5, 10–13).Open in a separate windowFig. 1.(A) Accepted scheme for radical initiation in rSAM enzymes. (B) X-ray crystal structure of SuiB (PDB ID: 5V1T). The RS domain, SPASM domain, and RiPP recognition element are rendered blue, green, and pink, respectively. [4Fe-4S] clusters are shown as spheres with the distances separating them indicated. (C) Lys–Trp cross-link formation (20) catalyzed by SuiB. The carbon–carbon bond installed by SuiB is shown in red. (D and E) Previously proposed EAS (D) and rEAS (E) mechanisms for SuiB-catalyzed Lys–Trp cross-link formation.Of the 570,000 rSAM enzyme superfamily members, over a quarter (∼165,000 genes from the Enzyme Function Initiative-Enzyme Similarity Tool) possess C-terminal extensions, called SPASM and twitch domains, which bind auxiliary Fe-S clusters (4, 14–19). The SPASM domain typically binds two auxiliary Fe-S clusters and is named after the rSAM enzymes involved in the synthesis of subtilosin, pyrroloquinoline quinone, anaerobic sulfatase, and mycofactocin. The twitch domain is a truncated SPASM domain and only binds one auxiliary cluster (15). Despite the wide prevalence of these domains and the characterization of several different SPASM/twitch rSAM enzymes by spectroscopic and structural studies, direct evidence for their catalytic function(s) has remained elusive.We previously performed functional and structural characterization on the SPASM rSAM enzyme SuiB (Fig. 1B), which is involved in the biosynthesis of a ribosomal peptide natural product in human and mammalian microbiome streptococci (14, 20–22). SuiB introduces an unusual carbon–carbon bond onto its substrate peptide, SuiA, between the sidechains of Lys2 and Trp6 (Fig. 1C). The mechanism for this transformation is of broader relevance, as a number of enzymes, such as RrrB, PqqE, and MqnC (2, 23, 24), are known to join unactivated aliphatic and aromatic carbons to generate sp³-sp² cross-links. A general mechanistic paradigm for this class of transformations is not yet available. For SuiB, two pathways have been proposed (20), one through a typical electrophilic aromatic substitution (EAS) mechanism, which is involved in other enzyme-catalyzed indole modifications, such as indole prenylation or flavin adenine dinucleotide (FAD)-enzyme-dependent indole chlorination (25–27). In this pathway, the 5′-dA• generates an alkyl radical, which upon a second one-electron oxidation, creates an α,β-unsaturated amide electrophile with which the indole sidechain reacts via Michael addition (Fig. 1D). Lanthionine cross-links observed in diverse lanthipeptides are built by this general scheme, though via heterolytic chemistry, with Cys acting as the nucleophile (28, 29). Alternatively, a radical electrophilic aromatic substitution (rEAS) reaction has been proposed, wherein the alkyl radical, formed by 5′-dA•, would react with the indole sidechain to generate a radical σ complex, a cross-linked Lys–Trp radical (Lys–Trp•), which upon oxidation and rearomatization would yield product (Fig. 1E). In both mechanisms, AuxI is proposed as an oxidant. Although this role for an rSAM auxiliary cluster has been previously suggested (30, 31), it has yet to be directly demonstrated experimentally. Mechanistic studies have favored the rEAS pathway (20); however, intermediates in the reaction of SuiB and enzymes that catalyze similar reactions have not yet been detected (15).In the current work, we sought to differentiate between the proposed mechanisms by trapping intermediates in the catalytic cycle of SuiB and characterizing them using electron paramagnetic resonance (EPR) spectroscopy. We report observation of three transient reaction intermediates, most importantly the sought-after Lys–Trp•, which is fundamentally different from previously characterized Trp radicals, as it is cross-linked and carries an indole tetrahedral center. We also provide evidence for AuxI as the oxidant of the Lys–Trp• intermediate as well as insights into redox potential changes of Fe-S clusters in SuiB that accompany SuiA binding. Together, our findings support the rEAS pathway for formation of the sp³-sp² cross-link and carry important implications for other enzymes that catalyze related transformations.     

Viewing rare conformations of the β2 adrenergic receptor with pressure-resolved DEER spectroscopy

The β₂ adrenergic receptor (β₂AR) is an archetypal G protein coupled receptor (GPCR). One structural signature of GPCR activation is a large-scale movement (ca. 6 to 14 Å) of transmembrane helix 6 (TM6) to a conformation which binds and activates a cognate G protein. The β₂AR exhibits a low level of agonist-independent G protein activation. The structural origin of this basal activity and its suppression by inverse agonists is unknown but could involve a unique receptor conformation that promotes G protein activation. Alternatively, a conformational selection model proposes that a minor population of the canonical active receptor conformation exists in equilibrium with inactive forms, thus giving rise to basal activity of the ligand-free receptor. Previous spin-labeling and fluorescence resonance energy transfer experiments designed to monitor the positional distribution of TM6 did not detect the presence of the active conformation of ligand-free β₂AR. Here we employ spin-labeling and pressure-resolved double electron–electron resonance spectroscopy to reveal the presence of a minor population of unliganded receptor, with the signature outward TM6 displacement, in equilibrium with inactive conformations. Binding of inverse agonists suppresses this population. These results provide direct structural evidence in favor of a conformational selection model for basal activity in β₂AR and provide a mechanism for inverse agonism. In addition, they emphasize 1) the importance of minor populations in GPCR catalytic function; 2) the use of spin-labeling and variable-pressure electron paramagnetic resonance to reveal them in a membrane protein; and 3) the quantitative evaluation of their thermodynamic properties relative to the inactive forms, including free energy, partial molar volume, and compressibility.

Many aspects of physiology in health and disease are regulated by signal transduction through G protein coupled receptors (GPCRs). Among these, the β₂ adrenergic receptor (β₂AR) is an archetype for the subset of family A GPCRs activated by hormones and neurotransmitters, as well as a pharmaceutical target for asthma and chronic obstructive pulmonary disease. The activity of β₂AR and other ligand-binding GPCRs can be finely tuned by ligands of varying efficacy, with agonists stimulating an increase in activation of cognate G proteins and inverse agonists decreasing G protein activation below a basal level.β₂AR crystal structures (1) have defined the outward movement of transmembrane helix 6 (TM6) as the largest structural rearrangement associated with activation of the receptor, as was originally found for rhodopsin (2) and subsequently for other receptors. This movement of TM6 is required for β₂AR to productively couple to its signaling partners (3).Ligand-independent or basal activity has been observed in many GPCRs. The level of basal receptor activity is highly receptor-specific and is important in maintaining homeostasis in physiologic systems independent of agonist stimulation (4). Basal activity in the β₂AR (5–7) and other receptors (4, 8) could arise from distinct receptor conformations that promote weak activation of the G protein, possibly involving an induced fit mechanism for receptor–G protein interaction. Alternatively, the basal activity could arise from a preexisting equilibrium (9, 10) between inactive and active conformations, where a small fraction of active receptors could account for baseline levels of signaling in physiologic systems. In support of this mechanism, sparse NMR data on the unliganded adenosine receptor provides evidence for the existence of an equilibrium between the inactive and active conformations (11).Although data from single-molecule fluorescence on β₂AR (12) have been interpreted in terms of the presence of the active form of the unliganded receptor, direct structural evidence for the existence of an active-like conformation in equilibrium with the unliganded, inactive conformation is lacking for this pharmacologically important GPCR. Indeed, structural studies of equilibrium populations by single-molecule fluorescence resonance energy transfer (FRET) (13) and double electron–electron resonance (DEER) spectroscopy (5) did not detect the presence of the active conformation in the ensemble of the unliganded β₂AR. This leaves the mechanism of basal activity for β₂AR unresolved. If true conformational selection plays a role, then a population of active form must exist in the equilibrium manifold of the unliganded receptor. The goal of the present work is to investigate the possibility that an active conformation in fact exists but is too sparsely populated to detect with the methods employed.Detection and characterization of sparsely populated (rare or excited) conformations is a general problem in elucidating the molecular mechanisms of protein function. Such low-lying excited states may play important functional roles despite being sparsely populated (14). Extensive empirical evidence indicates that the application of pressure may provide a solution to this problem by reversibly increasing the population of excited states (15, 16), allowing the use of standard experimental techniques to characterize these states and elucidate their functional roles. The mechanisms underlying the pressure-dependent conformational shifts are discussed in recent literature (17–20). It is important to note that as long as the pressure-induced shift in population is reversible, the rare state must also exist at atmospheric pressure and is not an artifact of pressure itself.Recently, site-directed spin labeling (SDSL) together with variable pressure electron paramagnetic resonance (EPR) was developed to monitor protein conformational shifts due to applied hydrostatic pressure (21). Of particular interest for the present study is SDSL with DEER (22). DEER provides a probability distribution of the distances between site-specifically introduced spin labels. Thus, each conformation in an ensemble generates a different distance in the distribution with a probability proportional to the population of the associated conformation, provided that the spin labels are properly placed to monitor the conformational equilibrium. In this respect, DEER is well suited to provide a structural definition to excited states revealed by pressure because it does not require a single conformation to be fully populated but instead reveals each conformation in the ensemble that is sufficiently populated (above ∼5 to 10%). Here we employ pressure-resolved DEER (23) to identify and characterize sparsely populated states (below 1%) in the conformational ensemble of unliganded β₂AR. The data provide direct structural evidence for the presence of an active-like conformation in the equilibrium ensemble of the unliganded receptor. The addition of agonists and inverse agonists increase and decrease the equilibrium population of the active form, respectively. The results indicate a mechanism for basal activity as well as that for inverse agonists in the β₂AR. Equally important, the variable pressure data provide a thermodynamic characterization of the active state including the partial molar volume, free energy, and compressibility relative to the ground (inactive) state.     

Photoinduced hole hopping through tryptophans in proteins

Hole hopping through tryptophan/tyrosine chains enables rapid unidirectional charge transport over long distances. We have elucidated structural and dynamical factors controlling hopping speed and efficiency in two modified azurin constructs that include a rhenium(I) sensitizer, Re(His)(CO)₃(dmp)⁺, and one or two tryptophans (W₁, W₂). Experimental kinetics investigations showed that the two closely spaced (3 to 4 Å) intervening tryptophans dramatically accelerated long-range electron transfer (ET) from Cu^I to the photoexcited sensitizer. In our theoretical work, we found that time-dependent density-functional theory (TDDFT) quantum mechanics/molecular mechanics/molecular dynamics (QM/MM/MD) trajectories of low-lying triplet excited states of Re^I(His)(CO)₃(dmp)⁺–W₁(–W₂) exhibited crossings between sensitizer-localized (*Re) and charge-separated [Re^I(His)(CO)₃(dmp^•–)/(W₁^•+ or W₂^•+)] (CS1 or CS2) states. Our analysis revealed that the distances, angles, and mutual orientations of ET-active cofactors fluctuate in a relatively narrow range in which the cofactors are strongly coupled, enabling adiabatic ET. Water-dominated electrostatic field fluctuations bring *Re and CS1 states to a crossing where *Re(CO)₃(dmp)⁺←W₁ ET occurs, and CS1 becomes the lowest triplet state. ET is promoted by solvation dynamics around *Re(CO)₃(dmp)⁺(W₁); and CS1 is stabilized by Re(dmp^•–)/W₁^•+ electron/hole interaction and enhanced W₁^•+ solvation. The second hop, W₁^•+←W₂, is facilitated by water fluctuations near the W₁/W₂ unit, taking place when the electrostatic potential at W₂ drops well below that at W₁^•+. Insufficient solvation and reorganization around W₂ make W₁^•+←W₂ ET endergonic, shifting the equilibrium toward W₁^•+ and decreasing the charge-separation yield. We suggest that multiscale TDDFT/MM/MD is a suitable technique to model the simultaneous evolution of photogenerated excited-state manifolds.

Electron (hole) transport along chains of tryptophan (W) and tyrosine (Y) residues in proteins (1, 2) plays an essential role in delivering reducing or oxidizing equivalents to active sites in enzymes [ribonucleotide reductase (3–7), photolyase (PL) (8–15), cytochrome c peroxidase (16–18), methylamine utilization protein MauG (19), laccases (20–22), and respiratory complex I (23)], signaling [cryptochromes (CRY)] (8, 13, 24), as well as in protecting oxidases from self-damage (20, 25–27) by transporting high-potential holes to the protein surface where they can be disarmed by cellular reductants. It also is likely that electron hopping through multiheme bacterial cytochromes is responsible for reduction of extracellular mineral acceptors (28–31) whereas chains of FeS clusters carry electrons, for example, in the respiratory complex I (32, 33) and hydrogenases (34). Nearly all these processes are thought to involve nonadiabatic electron-tunneling steps between W, Y, heme, or FeS-cluster sites.To investigate the factors controlling multistep electron tunneling (hole hopping), we covalently attached an Re^I(CO)₃(dmp)⁺ photooxidant (abbreviated Re) (4,7-dimethyl-1,10-phenanthroline [dmp]) to surface histidines H124 or H126 in the blue copper protein Pseudomonas aeruginosa azurin and engineered an artificial hopping pathway by inserting one or two tryptophan residues between the Re and Cu^I sites, affording mutants ReH124W122 (Re124W) (35) and ReH126W124W122 (Re126WW) (36). Near- ultraviolet (UV) excitation of the appended Re complex triggered hole hopping through tryptophan(s), ultimately oxidizing the Cu^I site (35–39) (Scheme 1). While similar in operation to natural photoenzymes, such as PLs and CRYs, Re124W or Re126WW show important quantitative differences whose understanding will help designing protein-based photocatalysts, solar-energy harvesting systems, and bioelectronic devices.Importantly, electron transport from Cu^I to the electronically excited Re complex (*Re) is 300× (Re124W) and 10,000× (Re126WW) faster than estimated for single-step hole tunneling over the same distances although the much greater accelerating effect of two tryptophans comes at the expense of a lower yield (36). Such a lower yield is not observed for PLs or CRYs where charge separation between the flavin chromophore and the surface tryptophan (or tyrosine) occurs through two or three intervening tryptophans with a quantum yield of about 0.2 (12). We would like to know whether different protein and tryptophan solvation dynamics are responsible for the lower charge-separation efficiency in Re126WW compared to evolution-optimized natural photoenzymes. In addition, while electron transfer (ET) from the proximal tryptophan to the photoexcited chromophore in all these systems (and in flavodoxins) (41, 42) is influenced by electronic coupling between aromatic rings, the ET time constants depend on the nature of the chromophore, ranging from 0.4 to 0.8 ps (CRYs) (10, 24) or 30 to 45 ps (PLs) (11, 12) to ∼500 ps in Re-azurins.Open in a separate windowScheme 1.Phototriggered electron transport in Re124W (35, 37) and Re126WW (36, 38) Cu^I azurins. Optical excitation of the ground-state (GS) Re complex to the metal to ligand charge transfer (¹MLCT) state is followed (39, 40) by ∼150-fs intersystem crossing to hot triplet state(s) ^#*Re that undergo picosecond relaxation to the lowest triplet state *Re of mixed charge transfer (Re(CO)₃→dmp)/intraligand (ππ*-dmp) character (CT/IL). ET from the proximal tryptophan results in the charge-separated state (CS1) Re^I(CO)₃(dmp^•−)(H124)(W122^•+)Cu^I or Re^I(CO)₃(dmp^•−)(H126)(W124^•+)(W122)Cu^I. In Re124W azurins, Cu^I→W122^•+ ET in CS1 produces the redox product (RP) Re^I(CO)₃(dmp^•−)H124W122Cu^II. The cycle is then closed by ∼3-μs dmp^•–→Cu^II back ET. In Re126WW, the CS1 state is converted to CS2 by W124^•+←W122 ET (the second hole “hop”). RP formation and ∼120-μs back ET follow. Electron transport occurs over 19 (Re124W) and 23 Å (Re126WW).Here, we present results of a theoretical study designed to shed light on the roles of solvent/protein fluctuations that control hole hopping in Re124W and Re126WW. To this end, we have developed a multiscale quantum mechanics/molecular mechanics/molecular dynamics (QM/MM/MD) procedure to calculate the temporal evolution of a manifold of low-lying excited states of the active cofactors by time-dependent density-functional theory (TDDFT) while protein and water dynamics are simulated classically (Fig. 1). In simulations performed on reactive *Re states, we searched for conditions that would convert the systems to the charge-separated state CS1.Open in a separate windowFig. 1.Parts of Re124W (Left) and Re126WW (Right) azurins treated by quantum (QM, stick representation) and molecular (MM, cartoon representation of the protein secondary structure) mechanics. The QM regions consisted of Re(CO)₃(dmp)H124G123W122 and Re(CO)₃(dmp)H126L125W124G123W122 azurin fragments, respectively. The MM parts also included water molecules and the Cu^I atom.