Zrt-/Irt-like proteins (ZIPs), a family of divalent metal transporters, are crucial for maintaining the homeostasis of zinc, an essential trace element involved in numerous biological processes. While extensive research on the prototypical ZIP from Bordetella bronchiseptica (BbZIP) have suggested an elevator transport mechanism, the dynamic conformational changes during the transport cycle have not been thoroughly studied. In this work, we developed a sandwich ELISA using a custom anti-BbZIP monoclonal antibody to investigate the conformational change induced by the metal binding to the transport site. This was achieved by determining the accessibility of a cysteine residue introduced at a position exposed to the solvent only when the transporter adopts an outward-facing conformation. This assay allowed us to report the dissociation constants of BbZIP for Zn2+ and Cd2+ at low and sub-micromolar levels, respectively. Notably, the addition of a positive charge at the M2 site drastically reduced metal binding at the M1 site, consistent with an auxiliary role for the M2 site in metal transport. We also demonstrated that this assay can be used to rapidly screen variants for identifying targets for additional biophysical characterization. We anticipate that other transporters where substrate binding induces large conformational changes can also be studied using this or similar method.

Tandem kinase proteins underlie the innate immune systems of cereal plants, but how they initiate plant immune responses remains unclear. This report identifies wheat protein wheat tandem NBD 1 (WTN1), a noncanonical nucleotide-binding leucine-rich repeat (NLR) receptor featuring tandem nucleotide binding adaptor shared by APAF-1, plant R proteins, and CED-4 (NB-ARC) domains, required for WTK3-mediated disease resistance. Both WTK3 and its allelic variant Rwt4-known for conferring resistance to wheat powdery mildew and blast, respectively-are capable of recognizing the blast effector PWT4. They activate WTN1 to form calcium-permeable channels, akin to ZAR1 and Sr35. Thus, tandem kinase proteins and their associated NLRs operate as "sensor-executor" pairs against fungal pathogens. Additionally, evolutionary analyses reveal a coevolutionary trajectory of the tandem kinase-NLR module, highlighting their cooperative role in triggering plant immunity.

GORK is a shaker-like potassium channel in plants that contains ankyrin (ANK) repeats. In guard cells, activation of GORK causes K+ efflux, reducing turgor pressure and closing stomata. However, how GORK is regulated remains largely elusive. Here, we solved the cryo-EM structure of Arabidopsis GORK, revealing an unusual symmetry reduction (from C4 to C2) feature within its tetrameric assembly. This symmetry reduction in GORK channel is driven by ANK dimerization, which disrupts the coupling between transmembrane helices and cytoplasmic domains, thus maintaining GORK in an autoinhibited state. Electrophysiological and structural analyses further confirmed that ANK dimerization inhibits GORK, and its removal restores C4 symmetry, converting GORK to an activatable state. This dynamic switching between C2 and C4 symmetry, mediated by ANK dimerization, presents a GORK target site that guard cells regulate to switch the plant K+ channel between inhibited and activatable states, thus controlling stomatal movement in response to environmental stimuli.

Porcine epidemic diarrhoea virus (PEDV) is a highly pathogenic porcine enteric coronavirus that causes severe watery diarrhoea and mortality in piglets. The nucleocapsid protein (N) is the most abundant viral protein and is highly phosphorylated, with the phosphorylation level directly affecting infection and proliferation. Here, we characterized the phosphorylation level of the N protein and found that its SR (Ser and Arg) motif was highly phosphorylated. The phosphorylation level significantly decreased after mutation of threonine (Thr) to serine (Ser). Through screening, it was determined that GSK3α/β plays a major role in phosphorylating the SR motif. Using GSK3α/β inhibitors or directly knocking out the GSK3α/β gene significantly inhibit PEDV proliferation. Finally, we used yeast recombination technology to develop a reverse genetics system for assessing PEDV and confirmed that no differences existed between the wild-type strain and the rescued virus. Using this platform, we generated a PEDV N protein SR motif mutant strain. We found that, compared to the wild-type strain, the proliferation of the mutant strain was significantly weakened, confirming that the phosphorylation of the SR motif is crucial for PEDV proliferation. In summary, we verified the phosphorylation sites of the PEDV N protein and the associated protein kinases, providing new insights into the development of relevant therapeutic strategies.

Stomata in leaves regulate gas (carbon dioxide and water vapor) exchange and water transpiration between plants and the atmosphere. SLow Anion Channel 1 (SLAC1) mediates anion efflux from guard cells and plays a crucial role in controlling stomatal aperture. It serves as a central hub for multiple signaling pathways in response to environmental stimuli, with its activity regulated through phosphorylation via various plant protein kinases. However, the molecular mechanism underlying SLAC1 phosphoactivation has remained elusive. Through a combination of protein sequence analyses, AlphaFold-based modeling and electrophysiological studies, we unveiled that the highly conserved motifs on the N- and C-terminal segments of SLAC1 form a cytosolic regulatory domain (CRD) that interacts with the transmembrane domain(TMD), thereby maintaining the channel in an autoinhibited state. Mutations in these conserved motifs destabilize the CRD, releasing autoinhibition in SLAC1 and enabling its transition into an activated state. Our further studies demonstrated that SLAC1 activation undergoes an autoinhibition-release process and subsequent structural changes in the pore helices. These findings provide mechanistic insights into the activation mechanism of SLAC1 and shed light on understanding how SLAC1 controls stomatal closure in response to environmental stimuli.

Hereditary defects in the function of the Kir7.1 in the retinal pigment epithelium are associated with the ocular diseases retinitis pigmentosa, Leber congenital amaurosis, and snowflake vitreal degeneration. Studies also suggest that Kir7.1 may be regulated by a GPCR, the melanocortin-4 receptor, in certain hypothalamic neurons. We present the first structures of human Kir7.1 and describe the conformational bias displayed by two pathogenic mutations, R162Q and E276A, to provide an explanation for the basis of disease and illuminate the gating pathway. We also demonstrate the structural basis for the blockade of the channel by a small molecule ML418 and demonstrate that channel blockade in vivo activates MC4R neurons in the paraventricular nucleus of the hypothalamus (PVH), inhibiting food intake and inducing weight loss. Preliminary purification, and structural and pharmacological characterization of an in tandem construct of MC4R and Kir7.1 suggests that the fusion protein forms a homotetrameric channel that retains regulation by liganded MC4R molecules.

Cep57, a vital centrosome-associated protein, recruits essential regulatory enzymes for centriole duplication. Its dysfunction leads to anomalies, including reduced centrioles and mosaic-variegated aneuploidy syndrome. Despite functional investigations, understanding structural aspects and their correlation with functions is partial till date. We present the structure of human Cep57 C-terminal microtubule binding (MT-BD) domain, revealing conserved motifs ensuring functional preservation across evolution. A leucine zipper, with an adjacent possible microtubule-binding region, potentially forms a stabilizing scaffold for microtubule nucleation-accommodating pulling and tension from growing microtubules. This study highlights conserved structural features of Cep57 protein, compares them with other analogous proteins, and explores how protein function is maintained across diverse organisms.

Salinity is one of the most severe abiotic stresses that adversely affect plant growth and agricultural productivity. The plant Na+/H+ antiporter Salt Overly Sensitive 1 (SOS1) located in the plasma membrane extrudes excess Na+ out of cells in response to salt stress and confers salt tolerance. However, the molecular mechanism underlying SOS1 activation remains largely elusive. Here we elucidate two cryo-electron microscopy structures of rice (Oryza sativa) SOS1, a full-length protein in an auto-inhibited state and a truncated version in an active state. The SOS1 forms a dimeric architecture, with an NhaA-folded transmembrane domain portion in the membrane and an elongated cytosolic portion of multiple regulatory domains in the cytoplasm. The structural comparison shows that SOS1 adopts an elevator transport mechanism accompanied by a conformational transition of the highly conserved Pro148 in the unwound transmembrane helix 5 (TM5), switching from an occluded conformation in the auto-inhibited state to a conducting conformation in the active state. These findings allow us to propose an inhibition-release mechanism for SOS1 activation and elucidate how SOS1 controls Na+ homeostasis in response to salt stress.

The Zrt-/Irt-like protein (ZIP) family consists of ubiquitously expressed divalent metal transporters critically involved in maintaining systemic and cellular homeostasis of zinc, iron, and manganese. Here, we present a study on a prokaryotic ZIP from Bordetella bronchiseptica (BbZIP) by combining structural biology, evolutionary covariance, computational modeling, and a variety of biochemical assays to tackle the issue of the transport mechanism which has not been established for the ZIP family. The apo state structure in an inward-facing conformation revealed a disassembled transport site, altered inter-helical interactions, and importantly, a rigid body movement of a 4-transmembrane helix (TM) bundle relative to the other TMs. The computationally generated and biochemically validated outward-facing conformation model revealed a slide of the 4-TM bundle, which carries the transport site(s), by approximately 8 Å toward the extracellular side against the static TMs which mediate dimerization. These findings allow us to conclude that BbZIP is an elevator-type transporter.

Plant roots encounter numerous pathogenic microbes that often cause devastating diseases. One such pathogen, Plasmodiophora brassicae (Pb), causes clubroot disease and severe yield losses on cruciferous crops worldwide. Here, we report the isolation and characterization of WeiTsing (WTS), a broad-spectrum clubroot resistance gene from Arabidopsis. WTS is transcriptionally activated in the pericycle upon Pb infection to prevent pathogen colonization in the stele. Brassica napus carrying the WTS transgene displayed strong resistance to Pb. WTS encodes a small protein localized in the endoplasmic reticulum (ER), and its expression in plants induces immune responses. The cryoelectron microscopy (cryo-EM) structure of WTS revealed a previously unknown pentameric architecture with a central pore. Electrophysiology analyses demonstrated that WTS is a calcium-permeable cation-selective channel. Structure-guided mutagenesis indicated that channel activity is strictly required for triggering defenses. The findings uncover an ion channel analogous to resistosomes that triggers immune signaling in the pericycle.

AbstractIn σ-dependent transcriptional pausing, the transcription initiation factor σ, translocating with RNA polymerase (RNAP), makes sequence-specific protein-DNA interactions with a promoter-like sequence element in the transcribed region, inducing pausing. It has been proposed that, in σ-dependent pausing, the RNAP active center can access off-pathway “backtracked” states that are substrates for the transcript-cleavage factors of the Gre family, and on-pathway “scrunched” states that mediate pause escape. Here, using site-specific protein-DNA photocrosslinking to define positions of the RNAP trailing and leading edges and of σ relative to DNA at the λPR’ promoter, we show directly that σ-dependent pausing in the absence of GreB in vitro predominantly involves a state backtracked by 2-4 bp, and that σ-dependent pausing in the presence of GreB in vitro and in vivo predominantly involves a state scrunched by 2-3 bp. Analogous experiments with a library of 47 (∼16,000) transcribed-region sequences show that the state scrunched by 2-3 bp--and only that state--is associated with the consensus sequence, T-3N-2Y-1G+1, (where -1 corresponds to the position of the RNA 3’ end), which is identical to the consensus for pausing in initial transcription, and which is related to the consensus for pausing in transcription elongation. Experiments with heteroduplex templates show that sequence information at position T-3 resides in the DNA nontemplate strand. A cryo-EM structure of a complex engaged in σ-dependent pausing reveals positions of DNA scrunching on the DNA nontemplate and template strands and suggests that position T-3 of the consensus sequence exerts its effects by facilitating scrunching.

SignificanceThe paradigmatic example of factor-dependent pausing in transcription elongation is σ-dependent pausing, in which sequence-specific σ-DNA interaction with a − 10 element-like sequence in a transcribed region results in pausing of a σ-containing transcription elongation complex. It has been proposed that σ-dependent pausing involves DNA scrunching, and that sequences downstream of the −10 element-like sequence modulate DNA scrunching. Here, using site-specific protein–DNA photocrosslinking, high-throughput sequencing, and cryoelectron microscopy structure determination, we show directly that σ-dependent pausing involves DNA scrunching, we define a consensus sequence for formation of a stable scrunched paused complex that is identical to the consensus sequence for pausing in initial transcription, and we identify positions of DNA scrunching on DNA nontemplate and template strands. Our results illuminate the structural and mechanistic basis of σ-dependent transcriptional pausing.

Aluminum-activated malate transporters (ALMTs) form an anion channel family that plays essential roles in diverse functions in plants. Arabidopsis ALMT12, also named QUAC1 (quick anion channel 1), regulates stomatal closure in response to environmental stimuli. However, the molecular basis of ALMT12/QUAC1 activity remains elusive. Here, we describe the cryo-EM structure of ALMT12/QUAC1 from Glycine max at 3.5-Å resolution. GmALMT12/QUAC1 is a symmetrical dimer, forming a single electropositive T-shaped pore across the membrane. The transmembrane and cytoplasmic domains are assembled into a twisted two-layer architecture, with their associated dimeric interfaces nearly perpendicular. GmALMT12/QUAC1-mediated currents display rapid kinetics of activation/deactivation and a bell-shaped voltage dependency, reminiscent of the rapid (R)–type anion currents. Our structural and functional analyses reveal a domain-twisting mechanism for malate-mediated activation. Together, our study uncovers the molecular basis for a previously uncharacterized class of anion channels and provides insights into the gating and modulation of the ALMT12/QUAC1 anion channel.

SUMMARY The rapid (R)-type anion channel plays a central role in controlling stomatal closure in plant guard cells, thus regulating the exchange of water and photosynthetic gas (CO 2 ) in response to environmental stimuli. The activity of the R- type anion channel is regulated by malate. However, the molecular basis of the R-type anion channel activity remains elusive. Here, we describe the first cryo-EM structure of the R-type anion channel QUAC1 at 3.5 Å resolution in the presence of malate. The structure reveals that the QUAC1 is a symmetrical dimer, forming a single electropositive T-shaped pore for passing anions across the membrane. The transmembrane and cytoplasmic domains are assembled into a twisted bi-layer architecture, with the associated dimeric interfaces nearly perpendicular. Our structural and functional analyses reveal that QUAC1 functions as an inward rectifying anion channel and suggests a mechanism for malate-mediated channel activation. Altogether, our study uncovers the molecular basis for a novel class of anion channels and provides insights into the gating and modulation of the R-type anion channel.

Cytochrome P450 family 102 subfamily A member 1 (CYP102A1) is a self-sufficient flavohemeprotein and a highly active bacterial enzyme capable of fatty acid hydroxylation at a >3,000 min-1 turnover rate. The CYP102A1 architecture has been postulated to be responsible for its extraordinary catalytic prowess. However, the structure of a functional full-length CYP102A1 enzyme remains to be determined. Herein, we used a cryo-EM single-particle approach, revealing that full-length CYP102A1 forms a homodimer in which both the heme and FAD domains contact each other. The FMN domain of one monomer was located close to the heme domain of the other monomer, exhibiting a trans configuration. Moreover, full-length CYP102A1 is highly dynamic, existing in multiple conformational states, including open and closed states. In the closed state, the FMN domain closely contacts the FAD domain, whereas in the open state, one of the FMN domains rotates away from its FAD domain and traverses to the heme domain of the other monomer. This structural arrangement and conformational dynamics may facilitate rapid intraflavin and trans FMN-to-heme electron transfers (ETs). Results with a variant having a 12-amino-acid deletion in the CYP102A1 linker region, connecting the catalytic heme and the diflavin reductase domains, further highlighted the importance of conformational dynamics in the ET process. Cryo-EM revealed that the Δ12 variant homodimer is conformationally more stable and incapable of FMN-to-heme ET. We conclude that closed-to-open alternation is crucial for redox partner recognition and formation of an active ET complex for CYP102A1 catalysis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Mixed Lineage Leukemia (MLL) family histone methyltransferases are the key enzymes that deposit histone H3 Lys4 (K4) mono-/di-/tri-methylation and regulate gene expression in mammals. Despite extensive structural and biochemical studies, the molecular mechanism by which the MLL complexes recognize histone H3K4 within the nucleosome core particle (NCP) remains unclear. Here, we report the single-particle cryo-electron microscopy (cryo-EM) structure of the human MLL1 core complex bound to the NCP. The MLL1 core complex anchors on the NCP through RbBP5 and ASH2L, which interacts extensively with nucleosomal DNA as well as the surface close to histone H4 N-terminal tail. Concurrent interactions of RbBP5 and ASH2L with the NCP uniquely align the catalytic MLL1SET domain at the nucleosome dyad, allowing symmetrical access to both H3K4 substrates within the NCP. Our study sheds light on how the MLL1 complex engages chromatin and how chromatin binding promotes MLL1 tri-methylation activity.

Journal Article Structural Analysis of Helicobacter pylori VacA Reveals Insights into Oligomerization Get access Amanda L Erwin, Amanda L Erwin Life Sciences Institute, University of Michigan, Ann Arbor, MI, USADepartment of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI, USA Search for other works by this author on: Oxford Academic Google Scholar Min Su, Min Su Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA Search for other works by this author on: Oxford Academic Google Scholar Anne M Campbell, Anne M Campbell Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN, USA Search for other works by this author on: Oxford Academic Google Scholar David Akey, David Akey Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA Search for other works by this author on: Oxford Academic Google Scholar D Borden Lacy, D Borden Lacy Department of Pathology, Microbiology, and Immunology, Vanderbilt University School of Medicine, Nashville, TN, USA Search for other works by this author on: Oxford Academic Google Scholar Timothy L Cover, Timothy L Cover Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN, USADepartment of Pathology, Microbiology, and Immunology, Vanderbilt University School of Medicine, Nashville, TN, USAVeterans Affairs Tennessee Valley Healthcare System, Nashville, TN, USA Search for other works by this author on: Oxford Academic Google Scholar Melanie D Ohi Melanie D Ohi Life Sciences Institute, University of Michigan, Ann Arbor, MI, USADepartment of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI, USA Corresponding author: mohi@umich.edu Search for other works by this author on: Oxford Academic Google Scholar Microscopy and Microanalysis, Volume 25, Issue S2, 1 August 2019, Pages 1290–1291, https://doi.org/10.1017/S1431927619007189 Published: 01 August 2019

The organization of protein molecules into higher-order nanoscale architectures is ubiquitous in Nature and represents an important goal in synthetic biology. Furthermore, the stabilization of enzyme activity has many practical applications in biotechnology and medicine. Here we describe the symmetry-directed design of an extremely stable, enzymatically active, hollow protein cage of Mr ≈ 2.1 MDa with dimensions similar to those of a small icosahedral virus. The cage was constructed based on icosahedral symmetry by genetically fusing a trimeric protein (TriEst) to a small pentameric de novo-designed coiled coil domain, separated by a flexible oligo-glycine linker sequence. Screening a small library of designs in which the linker length varied from 2 to 12 residues identified a construct containing 8 glycine residues (Ico8) that formed well-defined cages. Characterization by dynamic light scattering, negative stain, and cryo-EM and by atomic force and IR-photoinduced force microscopy established that Ico8 assembles into a flexible hollow cage comprising 20 copies of the esterase trimer, 60 protein subunits in total, with overall icosahedral geometry. Notably, the cages formed by Ico8 proved to be extremely stable toward thermal and chemical denaturation: whereas TriEst was unfolded by heating ( Tm ≈ 75 °C) or denatured by 1.5 M guanidine hydrochloride, the Ico8 cages remained folded even at 120 °C or in 8 M guanidine hydrochloride. The increased stability of the cages is a new property that emerges from the higher-order structure of the protein cage, rather than being intrinsic to the components from which it is constructed.