Post-translational modifications (e.g., ubiquitylation) of histones play important roles in dynamic regulation of chromatin. Histone ubiquitylation has been speculated to directly influence the structure and dynamics of nucleosomes. However, structural information for ubiquitylated nucleosomes is still lacking. Here we report an alternative strategy for total chemical synthesis of homogenous histone H2B-K34-ubiquitylation (H2B-K34Ub) by using acid-cleavable auxiliary-mediated ligation of peptide hydrazides for site-specific ubiquitylation. Synthetic H2B-K34Ub was efficiently incorporated into nucleosomes and further used for single-particle cryo-electron microscopy (cryo-EM) imaging. The cryo-EM structure of the nucleosome containing H2B-K34Ub suggests that two flexible ubiquitin domains protrude between the DNA chains of the nucleosomes. The DNA chains around the H2B-K34 sites shift and provide more space for ubiquitin to protrude. These analyses indicated local and slight structural influences on the nucleosome with ubiquitylation at the H2B-K34 site.
Abstract The one-dimensional pattern of heterocyst in the model cyanobacterium Anabaena sp. PCC 7120 is coordinated by the transcription factor HetR and PatS peptide. Here we report the complex structures of HetR binding to DNA and its hood domain (HetR Hood ) binding to a PatS-derived hexapeptide (PatS6) at 2.80 and 2.10 Å, respectively. The intertwined HetR dimer possesses a couple of novel HTH motifs, each of which consists of two canonical α-helices in the DNA-binding domain and an auxiliary α-helix from the flap domain of the neighboring subunit. Two PatS6 peptides bind to the lateral clefts of HetR Hood and trigger significant conformational changes of the flap domain, resulting in dissociation of the auxiliary α-helix and eventually release of HetR from the DNA major grove. These findings provide the structural insights into a prokaryotic example of Turing model.
Acid-sensing ion channels (ASICs) are proton-gated cation channels that are involved in diverse neuronal processes including pain sensing. The peptide toxin Mambalgin1 (Mamba1) from black mamba snake venom can reversibly inhibit the conductance of ASICs, causing an analgesic effect. However, the detailed mechanism by which Mamba1 inhibits ASIC1s, especially how Mamba1 binding to the extracellular domain affects the conformational changes of the transmembrane domain of ASICs remains elusive. Here, we present single-particle cryo-EM structures of human ASIC1a (hASIC1a) and the hASIC1a-Mamba1 complex at resolutions of 3.56 and 3.90 Å, respectively. The structures revealed the inhibited conformation of hASIC1a upon Mamba1 binding. The combination of the structural and physiological data indicates that Mamba1 preferentially binds hASIC1a in a closed state and reduces the proton sensitivity of the channel, representing a closed-state trapping mechanism.
Immunosuppression associated with infections of nematode parasites has been documented. Cysteine protease inhibitor (CPI) released by the nematode parasites is identified as one of the major modulators of host immune response. In this report, we demonstrated that the recombinant CPI protein of Ascaris lumbricoides (Al-CPI) strongly inhibited the activities of cathepsin L, C, S, and showed weaker effect to cathepsin B. Crystal structure of Al-CPI was determined to 2.1 Å resolution. Two segments of Al-CPI, loop 1 and loop 2, were proposed as the key structure motifs responsible for Al-CPI binding with proteases and its inhibitory activity. Mutations at loop 1 and loop 2 abrogated the protease inhibition activity to various extents. These results provide the molecular insight into the interaction between the nematode parasite and its host and will facilitate the development of anthelmintic agents or design of anti-autoimmune disease drugs.
Plants utilize K + ions to maintain hydrostatic pressure, drive irreversible cell expansion for growth, and facilitate reversible changes in guard cell volume that cause stomatal opening or closing.KAT1 is a voltage-dependent potassium channel from Arabidopsis thaliana that is mainly expressed in guard cells.2][3] To understand the gating mechanism of plant K + channels poses several challenges, despite many structural similarities between these plant K + channels and mammalian Kv and Shaker channels. 4emarkably, most voltage-gated ion channels, such as Na + (Nav), Ca 2+ (Cav), and K + (Kv) channels, open when the cell membrane is depolarized (when the voltage is positive inside relative to outside).Comparing with conventional depolarized K + channels, KAT1 has a uniquely reversed voltage dependence: depolarization causes closing, and hyperpolarization causes opening. 3KAT1 thus falls into a rare class of hyperpolarization-activated channels, which include hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels in animals, and KAT and AKT channels in plants.Mechanistic studies have focused predominantly on the depolarization-activated ion channels.The mechanism underlying the voltage sensor control of the gate in hyperpolarizationactivated ion channels is little studied.To date, the only resolved structure of a hyperpolarization-activated channel is that of HCN1. 5,6The cryo-EM structure of the HCN1 channel in a "hyperpolarized" state reveals that the long S4 helix breaks into two helices, with one running parallel to the membrane surface, analogous to the S4-S5 linker of domain-swapped voltage-gated channels.These findings suggest a basis for allosteric communication between voltage sensors and the gate in hyperpolarization-gated ion channels.However, preliminary sequence analysis shows that KAT1 and HCN1 have low sequence similarity.The S4 helix of KAT1 is much shorter than that of HCN1.This leads to the question of whether the structure and proposed gating mechanism of HCN1 can fully recapitulate those of KAT1.The intriguing biophysical properties of KAT1 motivate us to elucidate its molecular architecture.Full-length KAT1 from Arabidopsis was cloned and transfected into Sf9 cells for expression.However, we failed to obtain KAT1 protein from the membrane fraction.Then we co-expressed KAT1 with KAB1, a structural component of some plant K + channels, 7 and eventually obtained highly stable and homogeneous KAT1 proteins uniform in composition as indicated by gel filtration and SDS-PAGE analysis (Supplementary information, Fig. S1).Unexpectedly, the corresponding band of KAB1 was not observed in the purification gel.This indicates that KAB1 could not form complex with KAT1 in vitro, but rather acts as a chaperon that facilitates KAT1 translocation to the membrane in Sf9 cells.The purified KAT1 was subjected to cryo-EM studies.A three-dimensional EM map was reconstructed to an overall resolution of 3.2 Å (Supplementary information, Figs.S2 andS3).The secondary-structure features of
Mutations in genes encoding PINK1 (PTEN-induced kinase 1) and Parkin (E3 ubiquitin ligase) are identified in familial Parkinson’s disease. However, it remains unclear whether the phosphorylated Ub ...
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