The p53 homolog TAp63α is the transcriptional key regulator of genome integrity in oocytes. After DNA damage, TAp63α is activated by multistep phosphorylation involving multiple phosphorylation events by the kinase CK1, which triggers the transition from a dimeric and inactive conformation to an open and active tetramer that initiates apoptosis. By measuring activation kinetics in ovaries and single-site phosphorylation kinetics in vitro with peptides and full-length protein, we show that TAp63α phosphorylation follows a biphasic behavior. Although the first two CK1 phosphorylation events are fast, the third one, which constitutes the decisive step to form the active conformation, is slow. Structure determination of CK1 in complex with differently phosphorylated peptides reveals the structural mechanism for the difference in the kinetic behavior based on an unusual CK1/TAp63α substrate interaction in which the product of one phosphorylation step acts as an inhibitor for the following one.
Iron-bound cyclic tetrapyrroles (hemes) are key redox-active cofactors in membrane-integrated oxygen reductases and other bioenergetic enzymes. However, the mechanisms of heme transport and insertion into respiratory chain complexes remain unclear. Here we used a combination of cellular, biochemical, structural and computational methods to resolve ongoing controversies around the function of the heterodimeric bacterial ABC transporter CydDC. We provide multi-level evidence that CydDC is a highly specific heme transporter required for assembly and maturation of cytochrome bd, a pharmaceutically relevant drug target. Our systematic single-particle cryo-EM approach combined with atomistic molecular dynamics simulations provides detailed insight into the conformational landscape of CydDC during substrate binding and translocation. We found that heme binds laterally from the membrane space to the transmembrane region of CydDC, enabled by a highly asymmetrical inward-facing CydDC conformation. During the binding process, heme propionates interact with positively charged residues on the surface and later in the substrate-binding pocket of the transporter, causing the heme orientation to flip 180 degree. The membrane-accessible heme entry site of CydDC is primarily controlled by the conformational plasticity of CydD transmembrane helix 4, the extended cytoplasmic segment of which also couples heme confinement to a rotational movement of the CydC nucleotide-binding domain. Our cryo-EM data highlight that this signal transduction mechanism is necessary to drive conformational transitions toward occluded and outward open states.
Iron-bound cyclic tetrapyrroles (hemes) are redox-active cofactors in bioenergetic enzymes. However, the mechanisms of heme transport and insertion into respiratory chain complexes remain unclear. Here, we used cellular, biochemical, structural and computational methods to characterize the structure and function of the heterodimeric bacterial ABC transporter CydDC. We provide multi-level evidence that CydDC is a heme transporter required for functional maturation of cytochrome bd, a pharmaceutically relevant drug target. Our systematic single-particle cryogenic-electron microscopy approach combined with atomistic molecular dynamics simulations provides detailed insight into the conformational landscape of CydDC during substrate binding and occlusion. Our simulations reveal that heme binds laterally from the membrane space to the transmembrane region of CydDC, enabled by a highly asymmetrical inward-facing CydDC conformation. During the binding process, heme propionates interact with positively charged residues on the surface and later in the substrate-binding pocket of the transporter, causing the heme orientation to rotate 180°.
27 quality control, ovarian reserve, premature ovarian insufficiency, light sheet microscopy 28 29 Abbreviations: DSBs, DNA double strand breaks; POI, premature ovarian insufficiency; TAD, 30 transactivation domain; DBD, DNA binding domain; TD, tetramerization domain; SAM, sterile 31 alpha motif; PAD, phosphorylation activation domain; TID, transcriptional inhibitory domain; 32 NMR, nuclear magnetic resonance; Dox, doxorubicin; CHK2, checkpoint kinase 2; CK1, 33 casein kinase 1; Cs, Cisplatin; PARP 1, poly(ADP-ribose)-polymerase; DSBs, double strand 34 breaks; 35 36 2 Abstract 37Cell fate decisions such as apoptosis require cells to translate signaling input into a binary 38 yes/no response. A tight control of the process is required to avoid loss of cells by accidental 39 activation of cell death pathways. One particularly critical situation exists in primary oocytes 40 because their finite number determines the reproductive capacity of females. On the one hand 41 a stringent genetic quality control is necessary to maintain the genetic integrity of the entire 42 species; on the other hand an overly stringent mechanism that kills oocytes with even minor 43 DNA damage can deplete the whole primary oocyte pool leading to infertility. The p53 homolog 44 TAp63α is the key regulator of genome integrity in oocytes. After DNA damage TAp63α is 45 activated by multistep phosphorylation involving multiple phosphorylation events by the kinase 46 CK1, which triggers the transition from a dimeric and inactive conformation to an open and 47 active tetramer. By measuring activation kinetics in ovaries and single site phosphorylation 48 kinetics in vitro with peptides and full length protein we show that TAp63α phosphorylation 49 follows a biphasic behavior. While the first two CK1 phosphorylation events are fast, the third 50 one that constitutes the decisive step to form the active conformation is slow. We reveal the 51 structural mechanism for the difference in the kinetic behavior based on an unusual 52 CK1/TAp63α substrate interaction and demonstrate by quantitative simulation that the slow 53 phosphorylation phase determines the threshold of DNA damage required for induction of 54 apoptosis. 55activation mechanism has to be adjusted to a certain level of damage that on the one hand 93 must be sufficiently low to protect the integrity of the genetic pool of a species but on the other 94 hand tolerant enough not to endanger reproductive capacity. This situation requires a 95 mechanism that ideally works similar to a doorbell: an input signal (mechanical pressure) below 96 a certain threshold has no effect. If, however, this threshold is surpassed the output signal is 97 independent of the actual input signal strength (pressing harder does not make the doorbell 98 louder). Indeed, a tight dose-response curve has been measured in four-day old mice: while 99 most oocytes survive irradiation with 0.1 Gy (~three DSBs per cell), virtually all primary oocytes 100 were eliminated by 0.45 Gy irradiation (~ten DSBs per cell) 4 .101 Sw...
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
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.