CCAAT-enhancer binding proteins (C/EBPs) are transcription factors that play a central role in the differentiation of myeloid cells and adipocytes. Tribbles pseudokinases govern levels of C/EBPs by recruiting them to the COP1 ubiquitin ligase for ubiquitination. Here, we present the first crystal structure of a Tribbles protein, which reveals a catalytically inactive TRIB1 pseudokinase domain with a unique adaptation in the αC helix. A second crystal structure and biophysical studies of TRIB1 with its C-terminal extension, which includes the COP1-binding motif, show that the C-terminal extension is sequestered at a site formed by the modified TRIB1 αC helix. In addition, we have identified and characterized the TRIB1 substrate-recognition sequence within C/EBPα, which is evolutionarily conserved in C/EBP transcription factors. Binding studies indicate that C/EBPα recruitment is weaker in the presence of the C-terminal COP1-binding motif, but the magnitude of this effect suggests that the two bind distinct rather directly overlapping binding sites.
The Tribbles family of pseudokinases recruits substrates to the ubiquitin ligase COP1 to facilitate ubiquitylation. CCAAT/enhancer-binding protein (C/EBP) family transcription factors are crucial Tribbles substrates in adipocyte and myeloid cell development. We found that the TRIB1 pseudokinase was able to recruit various C/EBP family members and that the binding of C/EBPβ was attenuated by phosphorylation. To explain the mechanism of C/EBP recruitment, we solved the crystal structure of TRIB1 in complex with C/EBPα, which revealed that TRIB1 underwent a substantial conformational change relative to its substrate-free structure and bound C/EBPα in a pseudosubstrate-like manner. Crystallographic analysis and molecular dynamics and subsequent biochemical assays showed that C/EBP binding triggered allosteric changes that link substrate recruitment to COP1 binding. These findings offer a view of pseudokinase regulation with striking parallels to bona fide kinase regulation—by means of the activation loop and αC helix—and raise the possibility of small molecules targeting either the activation “loop-in” or “loop-out” conformations of Tribbles pseudokinases.
Staphylococcus aureus is a prevalent bacterial pathogen in both community and hospital settings, and its treatment is made particularly difficult by resilience within biofilms. Within this niche, serine hydrolase enzymes play a key role in generating and maintaining the biofilm matrix. Activity-based profiling has previously identified a family of serine hydrolases, designated fluorophosphonate-binding hydrolases (Fph's), some of which contribute to the virulence of S. aureus in vivo. These 10 Fph proteins have limited annotation and have few, if any, characterized bacterial or mammalian homologues. This suggests unique hydrolase functions even within bacterial species. Here we report structures of one of the most abundant Fph family members, FphF. Our structures capture FphF alone, covalently bound to a substrate analogue and bound to small molecule inhibitors that occupy the hydrophobic substrate-binding pocket. In line with these findings, we show that FphF has promiscuous esterase activity toward hydrophobic lipid substrates. We present docking studies that characterize interactions of inhibitors and substrates within the active site environment, which can be extended to other Fph family members. Comparison of FphF to other esterases and the wider Fph protein family suggest that FphF forms a new esterase subfamily. Our data suggest that other Fph enzymes, including the virulence factor FphB, are likely to have more restricted substrate profiles than FphF. This work demonstrates a clear molecular rationale for the specificity of fluorophosphonate probes that target FphF and provides a structural template for the design of enhanced probes and inhibitors of the Fph family of serine hydrolases.
Apoptosis signal-regulating kinases (ASK1-3) are apical kinases of the p38 and JNK MAP kinase pathways. They are activated by diverse stress stimuli, including reactive oxygen species, cytokines, and osmotic stress; however, a molecular understanding of how ASK proteins are controlled remains obscure. Here, we report a biochemical analysis of the ASK1 kinase domain in conjunction with its N-terminal thioredoxin-binding domain, along with a central regulatory region that links the two. We show that in solution the central regulatory region mediates a compact arrangement of the kinase and thioredoxin-binding domains and the central regulatory region actively primes MKK6, a key ASK1 substrate, for phosphorylation. The crystal structure of the central regulatory region reveals an unusually compact tetratricopeptide repeat (TPR) region capped by a cryptic pleckstrin homology domain. Biochemical assays show that both a conserved surface on the pleckstrin homology domain and an intact TPR region are required for ASK1 activity. We propose a model in which the central regulatory region promotes ASK1 activity via its pleckstrin homology domain but also facilitates ASK1 autoinhibition by bringing the thioredoxin-binding and kinase domains into close proximity. Such an architecture provides a mechanism for control of ASK-type kinases by diverse activators and inhibitors and demonstrates an unexpected level of autoregulatory scaffolding in mammalian stress-activated MAP kinase signaling.itogen-activated protein (MAP) kinase cascades transmit signals from membrane-associated receptors to intracellular targets to effect changes in cellular behavior. They form a hierarchical system in which activated upstream kinases (MAP3Ks) phosphorylate intermediate MAP kinase kinases (MAP2Ks), which in turn phosphorylate terminal MAP kinases, primarily ERK, p38, and JNK and their isoforms (1). Extensive studies have focused on the activation of RAS-RAF-MEK upstream in the ERK pathway and provided fertile ground for the discovery of new therapeutics (2). In contrast to the ERK pathway, which primarily promotes cellular proliferation, JNK and p38 phosphorylate a range of substrates to promote inflammation and cell death (1, 3). In addition, cross-regulation among the p38, JNK, and ERK pathways is important for the efficacy of various cancer therapies that are in use or in development (4, 5). Molecular details on the more diverse upstream regulation of the p38 and JNK pathways are currently less clear, however.Apoptosis signal-regulating kinases (ASK1-3) are MAP3Ks that trigger cellular responses to redox stress and inflammatory cytokines (6, 7) and play vital roles in innate immunity and viral infection (8-11). When activated, ASK1-3 activate JNK and p38 via phosphorylation of MAP2Ks (MKK3/4/6/7) (12). The key initiator role of ASK1-3 in this pathway means that either too much or too little ASK activity can have pathological effects. For instance, inhibiting ASK1 is beneficial against gastric cancer (13,14), but inactivating mutations in ASK1...
Multivalent structures can provide multiple interactions at a target site and improve binding affinity. The multivalent presentation of the anti‐tumour heptapeptide, SNTSESF, was investigated. This peptide's activity has been attributed to blockade of the PD‐1 receptor‐mediated signalling pathway. Two and four peptide units were conjugated to poly ethoxy ethyl glycinamide (PEE−G) scaffolds to prepare high‐purity products. These conjugates and the peptide were examined in a mouse model implanted with GL261 tumours that indicated that presenting more than two copies of peptide SNTSESF on the dendritic scaffold does not increase anti‐tumour activity per peptide. The fluorescent labelled peptide and most active multivalent peptide conjugate were therefore screened for their interaction with the human PD−L1 protein in a fluorescence polarisation assay. No indication of a specific SNTSESF peptide/PD−L1 interaction was observed. This finding was further supported by a molecular modelling binding study.
In bacteria and archaea, tripartite ATP-independent periplasmic (TRAP) transporters uptake essential nutrients. TRAP transporters receive their substrates via a secreted soluble substrate-binding protein. How a sodium ion-driven secondary active transporter is strictly coupled to a substrate-binding protein is poorly understood. Here we report the cryo-EM structure of the sialic acid TRAP transporter SiaQM from Photobacterium profundum at 2.97 Å resolution. SiaM comprises a “transport” domain and a “scaffold” domain, with the transport domain consisting of helical hairpins as seen in the sodium ion-coupled elevator transporter VcINDY. The SiaQ protein forms intimate contacts with SiaM to extend the size of the scaffold domain, suggesting that TRAP transporters may operate as monomers, rather than the typically observed oligomers for elevator-type transporters. We identify the Na+ and sialic acid binding sites in SiaM and demonstrate a strict dependence on the substrate-binding protein SiaP for uptake. We report the SiaP crystal structure that, together with docking studies, suggest the molecular basis for how sialic acid is delivered to the SiaQM transporter complex. We thus propose a model for substrate transport by TRAP proteins, which we describe herein as an ‘elevator-with-an-operator’ mechanism.
Abstract:The Tribbles family of pseudokinases recruit substrates to the COP1 ubiquitin ligase for ubiquitination. CCAAT-enhancer binding protein (C/EBP) family transcription factors are crucial Tribbles substrates in adipocyte and myeloid development. Here we show that the TRIB1 pseudokinase can recruit various C/EBP family members, with binding of C/EBPβ attenuated by phosphorylation. To explain the mechanism of substrate recruitment, we solved the crystal structure of TRIB1 in complex with C/EBPα. TRIB1 undergoes a significant conformational change relative to its substrate-free structure, to bind C/EBPα in a pseudo-substrate-like manner.Crucially, substrate binding triggers allosteric changes that link substrate recruitment to COP1 binding, which is consistent with molecular dynamics and biochemical studies. These findings offer a view of pseudokinase regulation with striking parallels to bona fide kinase regulationvia the activation loop and αC-helix-and raise the possibility of small molecules targeting either the activation loop-in, or loop-out, conformations of Tribbles pseudokinases.All rights reserved. No reuse allowed without permission.was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
334 Leonard St
Brooklyn, NY 11211
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.