Search

The coordinated release of Ca 2+ from the sarcoplasmic reticulum (SR) is critical for excitation-contraction coupling. This release is facilitated by ryanodine receptors (RyRs) that are embedded in the SR membrane. In skeletal muscle, activity of RyR1 is regulated by metabolites such as ATP, which upon binding increase channel open probability (P o ). To obtain structural insights into the mechanism of RyR1 priming by ATP, we determined several cryo-EM structures of RyR1 bound individually to ATP-γ-S, ADP, AMP, adenosine, adenine, and cAMP. We demonstrate that adenine and adenosine bind RyR1, but AMP is the smallest ATP derivative capable of inducing long-range (>170 Å) structural rearrangements associated with channel activation, establishing a structural basis for key binding site interactions that are the threshold for triggering quaternary structural changes. Our finding that cAMP also induces these structural changes and results in increased channel opening suggests its potential role as an endogenous modulator of RyR1 conductance., Competing Interests: Declaration of interests S.S. is the founder and CEO of Gandeeva Therapeutics, a drug discovery company based in Vancouver., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The BA.2 sub-lineage of the Omicron (B.1.1.529) severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant rapidly supplanted the original BA.1 sub-lineage in early 2022. Both lineages threatened the efficacy of vaccine-elicited antibodies and acquired increased binding to several mammalian ACE2 receptors. Cryoelectron microscopy (cryo-EM) analysis of the BA.2 spike (S) glycoprotein in complex with mouse ACE2 (mACE2) identifies BA.1- and BA.2-mutated residues Q493R, N501Y, and Y505H as complementing non-conserved residues between human and mouse ACE2, rationalizing the enhanced S protein-mACE2 interaction for Omicron variants. Cryo-EM structures of the BA.2 S-human ACE2 complex and of the extensively mutated BA.2 amino-terminal domain (NTD) reveal a dramatic reorganization of the highly antigenic N1 loop into a β-strand, providing an explanation for decreased binding of the BA.2 S protein to antibodies isolated from BA.1-convalescent patients. Our analysis reveals structural mechanisms underlying the antigenic drift in the rapidly evolving Omicron variant landscape., Competing Interests: Declarations of interests S.S. is the founder and CEO of Gandeeva Therapeutics, a drug discovery company based in Vancouver., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The global spread of SARS-CoV-2 has proceeded at an unprecedented rate. Remarkably, characterization of the virus using modern tools in structural biology has also progressed at exceptional speed. Advances in electron-based imaging techniques, combined with decades of foundational studies on related viruses, have enabled the research community to rapidly investigate structural aspects of the novel coronavirus from the level of individual viral proteins to imaging the whole virus in a native context. Here, we provide a detailed review of the structural biology and pathobiology of SARS-CoV-2 as it relates to all facets of the viral life cycle, including cell entry, replication, and three-dimensional (3D) packaging based on insights obtained from X-ray crystallography, cryo-electron tomography, and single-particle cryo-electron microscopy. The structural comparison between SARS-CoV-2 and the related earlier viruses SARS-CoV and MERS-CoV is a common thread throughout this review. We conclude by highlighting some of the outstanding unanswered structural questions and underscore areas that are under rapid current development such as the design of effective therapeutics that block viral infection.

The emergence of SARS-CoV-2 variants of concern (VOCs) requires the development of next-generation biologics with high neutralization breadth. Here, we characterized a human V H domain, F6, which we generated by sequentially panning large phage-displayed V H libraries against receptor binding domains (RBDs) containing VOC mutations. Cryo-EM analyses reveal that F6 has a unique binding mode that spans a broad surface of the RBD and involves the antibody framework region. Attachment of an Fc region to a fusion of F6 and ab8, a previously characterized V H domain, resulted in a construct (F6-ab8-Fc) that broadly and potently neutralized VOCs including Omicron. Additionally, prophylactic treatment using F6-ab8-Fc reduced live Beta (B.1.351) variant viral titers in the lungs of a mouse model. Our results provide a new potential therapeutic against SARS-CoV-2 variants including Omicron and highlight a vulnerable epitope within the spike that may be exploited to achieve broad protection against circulating variants., Competing Interests: W.L, C.C, J.W.M., and D.SD, are co-inventors of a patent, filed on January 06, 2022 by the University of Pittsburgh, related to VH F6 and F6-ab8-Fc described in this article. S.S. is a founder and CEO of Gandeeva Therapeutics Inc., (© 2022 The Author(s).)

Mutations in the spike glycoproteins of SARS-CoV-2 variants of concern have independently been shown to enhance aspects of spike protein fitness. Here, we describe an antibody fragment (V H ab6) that neutralizes all major variants including the recently emerged BA.1 and BA.2 Omicron subvariants, with a unique mode of binding revealed by cryo-EM studies. Further, we provide a comparative analysis of the mutational effects within previously emerged variant spikes and identify the structural role of mutations within the NTD and RBD in evading antibody neutralization. Our analysis shows that the highly mutated Gamma N-terminal domain exhibits considerable structural rearrangements, partially explaining its decreased neutralization by convalescent sera. Our results provide mechanistic insights into the structural, functional, and antigenic consequences of SARS-CoV-2 spike mutations and highlight a spike protein vulnerability that may be exploited to achieve broad protection against circulating variants., (© 2022. The Author(s).)

Bacterial lung infections lead to greater than 4 million deaths per year with antibiotic treatments driving an increase in antibiotic resistance and a need to establish new therapeutic approaches. Recently, we have generated mouse and rat stem cell-derived alveolar-like macrophages (ALMs), which like primary alveolar macrophages (1'AMs), phagocytose bacteria and promote airway repair. Our aim was to further characterize ALMs and determine their bactericidal capabilities. The characterization of ALMs showed that they share known 1'AM cell surface markers, but unlike 1'AMs are highly proliferative in vitro. ALMs effectively phagocytose and kill laboratory strains of P. aeruginosa (P.A.), E. coli (E.C.) and S. aureus, and clinical strains of P.A. In vivo, ALMs remain viable, adapt additional features of native 1'AMs, but proliferation is reduced. Mouse ALMs phagocytose P.A. and E.C. and rat ALMs phagocytose and kill P.A. within the lung 24 h post-instillation. In a pre-clinical model of P.A.-induced lung injury, rat ALM administration mitigated weight loss and resolved lung injury observed seven days post-instillation. Collectively, ALMs attenuate pulmonary bacterial infections and promote airway repair. ALMs could be utilized as an alternative or adjuvant therapy where current treatments are ineffective against antibiotic-resistant bacteria or to enhance routine antibiotic delivery., (© 2022 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

The newly reported Omicron variant is poised to replace Delta as the most prevalent SARS-CoV-2 variant across the world. Cryo-EM structural analysis of the Omicron variant spike protein in complex with human ACE2 reveals new salt bridges and hydrogen bonds formed by mutated residues R493, S496 and R498 in the RBD with ACE2. These interactions appear to compensate for other Omicron mutations such as K417N known to reduce ACE2 binding affinity, resulting in similar biochemical ACE2 binding affinities for Delta and Omicron variants. Neutralization assays show that pseudoviruses displaying the Omicron spike protein exhibit increased antibody evasion. The increase in antibody evasion, together with retention of strong interactions at the ACE2 interface, thus represent important molecular features that likely contribute to the rapid spread of the Omicron variant.

The Delta and Kappa variants of SARS-CoV-2 co-emerged in India in late 2020, with the Delta variant underlying the resurgence of COVID-19, even in countries with high vaccination rates. In this study, we assess structural and biochemical aspects of viral fitness for these two variants using cryo-electron microscopy (cryo-EM), ACE2-binding and antibody neutralization analyses. Both variants demonstrate escape of antibodies targeting the N-terminal domain, an important immune hotspot for neutralizing epitopes. Compared to wild-type and Kappa lineages, Delta variant spike proteins show modest increase in ACE2 affinity, likely due to enhanced electrostatic complementarity at the RBD-ACE2 interface, which we characterize by cryo-EM. Unexpectedly, Kappa variant spike trimers form a structural head-to-head dimer-of-trimers assembly, which we demonstrate is a result of the E484Q mutation and with unknown biological implications. The combination of increased antibody escape and enhanced ACE2 binding provides an explanation, in part, for the rapid global dominance of the Delta variant., (© 2022. The Author(s).)

Pseudomonas aeruginosa is an opportunistic human pathogen and a leading cause of chronic infection in the lungs of individuals with cystic fibrosis. After colonization, P. aeruginosa often undergoes a phenotypic conversion to mucoidy, characterized by overproduction of the alginate exopolysaccharide. This conversion is correlated with poorer patient prognoses. The majority of genes required for alginate synthesis, including the alginate lyase, algL, are located in a single operon. Previous investigations of AlgL have resulted in several divergent hypotheses regarding the protein's role in alginate production. To address these discrepancies, we determined the structure of AlgL and, using multiple sequence alignments, identified key active site residues involved in alginate binding and catalysis. In vitro enzymatic analysis of active site mutants highlights R249 and Y256 as key residues required for alginate lyase activity. In a genetically engineered P. aeruginosa strain where alginate biosynthesis is under arabinose control, we found that AlgL is required for cell viability and maintaining membrane integrity during alginate production. We demonstrate that AlgL functions as a homeostasis enzyme to clear the periplasmic space of accumulated polymer. Constitutive expression of the AlgU/T sigma factor mitigates the effects of an algL deletion during alginate production, suggesting that an AlgU/T-regulated protein or proteins can compensate for an algL deletion. Together, our study demonstrates the role of AlgL in alginate biosynthesis, explains the discrepancies observed previously across other P. aeruginosa ΔalgL genetic backgrounds, and clarifies the existing divergent data regarding the function of AlgL as an alginate degrading enzyme., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The recently emerged severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) Beta (B.1.351) and Gamma (P.1) variants of concern (VoCs) include a key mutation (N501Y) found in the Alpha (B.1.1.7) variant that enhances affinity of the spike protein for its receptor, angiotensin-converting enzyme 2 (ACE2). Additional mutations are found in these variants at residues 417 and 484 that appear to promote antibody evasion. In contrast, the Epsilon variants (B.1.427/429) lack the N501Y mutation yet exhibit antibody evasion. We have engineered spike proteins to express these receptor binding domain (RBD) VoC mutations either in isolation or in different combinations and analyze the effects using biochemical assays and cryoelectron microscopy (cryo-EM) structural analyses. Overall, our findings suggest that the emergence of new SARS-CoV-2 variant spikes can be rationalized as the result of mutations that confer increased ACE2 affinity, increased antibody evasion, or both, providing a framework to dissect the molecular factors that drive VoC evolution., Competing Interests: Declaration of interests W.L. and D.S.D. are coinventors on a patent, filed by the University of Pittsburgh, related to ab1 and ab8 described in this paper. S.S. is founder and CEO of Gandeeva Therapeutics Inc., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

The human AAA+ ATPase p97, also known as valosin-containing protein, a potential target for cancer therapeutics, plays a vital role in the clearing of misfolded proteins. p97 dysfunction is also known to play a crucial role in several neurodegenerative disorders, such as MultiSystem Proteinopathy 1 (MSP-1) and Familial Amyotrophic Lateral Sclerosis (ALS). However, the structural basis of its role in such diseases remains elusive. Here, we present cryo-EM structural analyses of four disease mutants p97 R155H , p97 R191Q , p97 A232E , p97 D592N , as well as p97 E470D , implicated in resistance to the drug CB-5083, a potent p97 inhibitor. Our cryo-EM structures demonstrate that these mutations affect nucleotide-driven allosteric activation across the three principal p97 domains (N, D1, and D2) by predominantly interfering with either (1) the coupling between the D1 and N-terminal domains (p97 R155H and p97 R191Q ), (2) the interprotomer interactions (p97 A232E ), or (3) the coupling between D1 and D2 nucleotide domains (p97 D592N , p97 E470D ). We also show that binding of the competitive inhibitor, CB-5083, to the D2 domain prevents conformational changes similar to those seen for mutations that affect coupling between the D1 and D2 domains. Our studies enable tracing of the path of allosteric activation across p97 and establish a common mechanistic link between active site inhibition and defects in allosteric activation by disease-causing mutations and have potential implications for the design of novel allosteric compounds that can modulate p97 function., Competing Interests: Conflict of interest S. S. is Founder and CEO of Gandeeva Therapeutics Inc, a drug discovery company based in Vancouver. All other authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The recently reported "UK variant" (B.1.1.7) of SARS-CoV-2 is thought to be more infectious than previously circulating strains as a result of several changes, including the N501Y mutation. We present a 2.9-Å resolution cryo-electron microscopy (cryo-EM) structure of the complex between the ACE2 receptor and N501Y spike protein ectodomains that shows Y501 inserted into a cavity at the binding interface near Y41 of ACE2. This additional interaction provides a structural explanation for the increased ACE2 affinity of the N501Y mutant, and likely contributes to its increased infectivity. However, this mutation does not result in large structural changes, enabling important neutralization epitopes to be retained in the spike receptor binding domain. We confirmed this through biophysical assays and by determining cryo-EM structures of spike protein ectodomains bound to 2 representative potent neutralizing antibody fragments., Competing Interests: The authors have declared that no competing interests exist.

Motility in archaea is facilitated by a unique structure termed the archaellum. N -Glycosylation of the major structural proteins (archaellins) is important for their subsequent incorporation into the archaellum filament. The identity of some of these N -glycans has been determined, but archaea exhibit extensive variation in their glycans, meaning that further investigations can shed light not only on the specific details of archaellin structure and function, but also on archaeal glycobiology in general. Here we describe the structural characterization of the N -linked glycan modifications on the archaellins and S-layer protein of Methanothermococcus thermolithotrophicus , a methanogen that grows optimally at 65 °C. SDS-PAGE and MS analysis revealed that the sheared archaella are composed principally of two of the four predicted archaellins, FlaB1 and FlaB3, which are modified with a branched, heptameric glycan at all N -linked sequons except for the site closest to the N termini of both proteins. NMR analysis of the purified glycan determined the structure to be α-d- glycero -d- manno -Hep3OMe6OMe-(1-3)-[α-GalNAcA3OMe-(1-2)-]-β-Man-(1-4)-[β-GalA3OMe4OAc6CMe-(1-4)-α-GalA-(1-2)-]-α-GalAN-(1-3)-β-GalNAc-Asn. A detailed investigation by hydrophilic interaction liquid ion chromatography-MS discovered the presence of several, less abundant glycan variants, related to but distinct from the main heptameric glycan. In addition, we confirmed that the S-layer protein is modified with the same heptameric glycan, suggesting a common N -glycosylation pathway. The M. thermolithotrophicus archaellin N -linked glycan is larger and more complex than those previously identified on the archaellins of related mesophilic methanogens, Methanococcus voltae and Methanococcus maripaludis This could indicate that the nature of the glycan modification may have a role to play in maintaining stability at elevated temperatures., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Kelly et al.)

Novel COVID-19 therapeutics are urgently needed. We generated a phage-displayed human antibody V H domain library from which we identified a high-affinity V H binder ab8. Bivalent V H , V H -Fc ab8, bound with high avidity to membrane-associated S glycoprotein and to mutants found in patients. It potently neutralized mouse-adapted SARS-CoV-2 in wild-type mice at a dose as low as 2 mg/kg and exhibited high prophylactic and therapeutic efficacy in a hamster model of SARS-CoV-2 infection, possibly enhanced by its relatively small size. Electron microscopy combined with scanning mutagenesis identified ab8 interactions with all three S protomers and showed how ab8 neutralized the virus by directly interfering with ACE2 binding. V H -Fc ab8 did not aggregate and did not bind to 5,300 human membrane-associated proteins. The potent neutralization activity of V H -Fc ab8 combined with good developability properties and cross-reactivity to SARS-CoV-2 mutants provide a strong rationale for its evaluation as a COVID-19 therapeutic., Competing Interests: Declaration of Interests W.L., C.C., Z.S., J.W.M., and D.S.D. are co-inventors of a patent, filed on March 12 by the University of Pittsburgh, related to ab8 described in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

In Escherichia coli , the N-terminal domain of the essential protein FtsK (FtsK N ) is proposed to modulate septum formation through the formation of dynamic and essential protein interactions with both the Z-ring and late-stage division machinery. Using genomic mutagenesis, complementation analysis, and in vitro pull-down assays, we aimed to identify protein interaction partners of FtsK essential to its function during division. Here, we identified the cytoplasmic Z-ring membrane anchoring protein FtsA as a direct protein-protein interaction partner of FtsK. Random genomic mutagenesis of an ftsK temperature-sensitive strain of E . coli revealed an FtsA point mutation (G50E) that is able to fully restore normal cell growth and morphology, and further targeted site-directed mutagenesis of FtsA revealed several other point mutations capable of fully suppressing the essential requirement for functional FtsK. Together, this provides insight into a potential novel co-complex formed between these components during division and suggests FtsA may directly impact FtsK function.

In Escherichia coli, formation of new cells is mediated by the elongasome and divisome that govern cell elongation and septation, respectively. Proper transition between these events is essential to ensure viable progeny are produced; however, the components of each complex responsible for transmission of the cell signal to shift from elongation to septation are unclear. Recently, a region within the N-terminal domain of the essential divisome protein FtsK (FtsK N ) was identified that points to a key role for FtsK as a checkpoint of cell envelope remodeling during division. Here, we used site-specific in vivo UV cross-linking to probe the periplasmic loops of FtsK N for protein interaction partners critical for FtsK N function. Mass spectrometry analysis of five unique FtsK N periplasmic cross-links revealed a network of potential FtsK N interactors, one of which included the septal peptidoglycan binding protein rare lipoprotein A (RlpA). This protein was further verified as a novel interaction partner of FtsK N by an in vitro pull-down assay. Deletion of rlpA from an FtsK temperature-sensitive E. coli strain partially restored cell growth and largely suppressed cellular filamentation compared to the wild-type strain. This suggests that interaction with RlpA may be critical in suppressing septation until proper assembly of the divisome.

In Methanococcus maripaludis , the euryarchaeal archaellum regulator A (EarA) is required for the transcription of the fla operon, which is comprised of a series of genes which encode most of the proteins needed for the formation of the archaeal swimming organelle, the archaellum. In mutants deleted for earA (Δ earA ), there is almost undetectable transcription of the fla operon, Fla proteins are not synthesized and the cells are non-archaellated. In this study, we have isolated a spontaneous mutant of a Δ earA mutant in which the restoration of the transcription and translation of the fla operon (using flaB2 , the second gene of the operon, as a reporter), archaella formation and swarming motility were all restored even in the absence of EarA. Analysis of the DNA sequence from the fla promoter of this spontaneous mutant revealed a deletion of three adenines within a string of seven adenines in the transcription factor B recognition element (BRE). When the three adenine deletion in the BRE was regenerated in a stock culture of the Δ earA mutant, very similar phenotypes to that of the spontaneous mutant were observed. Deletion of the three adenines in the fla promoter BRE resulted in the mutant BRE having high sequence identity to BREs from promoters that have strong basal transcription level in Mc. maripaludis and Methanocaldococcus jannaschii . These data suggest that EarA may help recruit transcription factor B to a weak BRE in the fla promoter of wild-type cells but is not required for transcription from the fla promoter with a strong BRE, as in the three adenine deletion version in the spontaneous mutant.

Archaella are the swimming organelles in the Archaea. Recently, the first archaellum regulator in the Euryarchaeota, EarA Mma , was identified in Methanococcus maripaludis , one of the model organisms used for archaellum studies. EarA Mma binds to 6 bp consensus sequences upstream of the fla promoter to activate the transcription of the fla operon, which encodes most of the proteins required for archaella synthesis. In this study, synteny analysis showed that earA homologues are widely distributed in the phylum of Euryarchaeota, with the notable exception of extreme halophiles. We classified Euryarchaeota species containing earA homologues into five classes based on the genomic location of the earA genes relative to fla and chemotaxis operons. EarA homologues from Methanococcus vannielii , Methanothermococcus thermolithotrophicus and Methanocaldococcus jannaschii successfully complemented the function of EarA Mma in a ΔearAMma mutant, demonstrated by the restoration of FlaB2 expression in Western blot analysis and the appearance of archaella on the cell surface in complemented cells. Furthermore, the 6 bp consensus sequence was also found in the fla promoter region in these methanogens, indicating that the EarA homologues ly use a similar mechanism to activate transcription of the fla operons in their own hosts. Attempts to demonstrate complementation of the function of EarA Mma in a ΔearAMma mutant by the EarA homologue of Pyrococcus furiosus were unsuccessful, despite the presence of a copy of the 6 bp consensus EarA-binding sequence upstream of the fla promoter in the P. furiosus genome.

The oligosaccharyltransferase is the signature enzyme for N-linked glycosylation in all domains of life. In Archaea, this enzyme termed AglB, is responsible for transferring lipid carrier-linked glycans to select asparagine residues in a variety of target proteins including archaellins, S-layer proteins and pilins. This study investigated the ability of a variety of AglBs to compensate for the oligosaccharyltransferase activity in Methanococcus maripaludis deleted for aglB, using archaellin FlaB2 as the reporter protein since all archaellins in Mc. maripaludis are modified at multiple sites by an N-linked tetrasaccharide and this modification is required for archaellation. In the Mc. maripaludis ΔaglB strain FlaB2 runs as at a smaller apparent molecular weight in western blots and is nonarchaellated. We demonstrate that AglBs from Methanococcus voltae and Methanothermococcus thermolithotrophicus functionally replaced the oligosaccharyltransferase activity missing in the Mc. maripaludis ΔaglB strain, both returning the apparent molecular weight of FlaB2 to wildtype size and restoring archaellation. This demonstrates that AglB from Mc. voltae has a relaxed specificity for the linking sugar of the transferred glycan since while the N-linked glycan present in Mc. voltae is similar to that of Mc. maripaludis, the Mc. voltae glycan uses N-acetylglucosamine as the linking sugar. In Mc. maripaludis that role is held by N-acetylgalactosamine. This study also identifies aglB from Mtc. thermolithotrophicus for the first time by its activity. Attempts to use AglB from Methanocaldococcus jannaschii, Haloferax volcanii or Sulfolobus acidocaldarius to functionally replace the oligosaccharyltransferase activity missing in the Mc. maripaludis ΔaglB strain were unsuccessful., Competing Interests: The authors have declared that no competing interests exist.

The archaellum is the swimming organelle of the third domain, the Archaea. In the euryarchaeon Methanococcus maripaludis, genes involved in archaella formation, including the three archaellins flaB1, flaB2 and flaB3, are mainly located in the fla operon. Previous studies have shown that transcription of fla genes and expression of Fla proteins are regulated under different growth conditions. In this study, we identify MMP1718 as the first transcriptional activator that directly regulates the fla operon in M. maripaludis. Mutants carrying an in-frame deletion in mmp1718 did not express FlaB2 detected by western blotting. Quantitative reverse transcription PCR analysis of purified RNA from the Δmmp1718 mutant showed that transcription of flaB2 was negligible compared to wildtype cells. In addition, no archaella were observed on the cell surface of the Δmmp1718 mutant. FlaB2 expression and archaellation were restored when the Δmmp1718 mutant was complemented with mmp1718 in trans. Electrophoretic motility shift assay and isothermal titration calorimetry results demonstrated the specific binding of purified MMP1718 to DNA fragments upstream of the fla promoter. Four 6 bp consensus sequences were found immediately upstream of the fla promoter and are considered the putative MMP1718-binding sites. Herein, we designate MMP1718 as EarA, the first euryarchaeal archaellum regulator., (© 2016 John Wiley & Sons Ltd.)

Unlabelled: Bacterial cell division is an essential and highly coordinated process. It requires the polymerization of the tubulin homologue FtsZ to form a dynamic ring (Z-ring) at midcell. Z-ring formation relies on a group of FtsZ-associated proteins (Zap) for stability throughout the process of division. In Escherichia coli, there are currently five Zap proteins (ZapA through ZapE), of which four (ZapA, ZapB, ZapC, and ZapD) are small soluble proteins that act to bind and bundle FtsZ filaments. In particular, ZapD forms a functional dimer and interacts with the C-terminal tail of FtsZ, but little is known about its structure and mechanism of action. Here, we present the crystal structure of Escherichia coli ZapD and show it forms a symmetrical dimer with centrally located α-helices flanked by β-sheet domains. Based on the structure of ZapD and its chemical cross-linking to FtsZ, we targeted nine charged ZapD residues for modification by site-directed mutagenesis. Using in vitro FtsZ sedimentation assays, we show that residues R56, R221, and R225 are important for bundling FtsZ filaments, while transmission electron microscopy revealed that altering these residues results in different FtsZ bundle morphology compared to those of filaments bundled with wild-type ZapD. ZapD residue R116 also showed altered FtsZ bundle morphology but levels of FtsZ bundling similar to that of wild-type ZapD. Together, these results reveal that ZapD residues R116, R221, and R225 likely participate in forming a positively charged binding pocket that is critical for bundling FtsZ filaments., Importance: Z-ring assembly underpins the formation of the essential cell division complex known as the divisome and is required for recruitment of downstream cell division proteins. ZapD is one of several proteins in E. coli that associates with the Z-ring to promote FtsZ bundling and aids in the overall fitness of the division process. In the present study, we describe the dimeric structure of E. coli ZapD and identify residues that are critical for FtsZ bundling. Together, these results advance our understanding about the formation and dynamics of the Z-ring prior to bacterial cell division., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

In this study, the effects of growth conditions on archaellation in Methanococcus maripaludis were examined. Cells were grown in a variety of media, including complex, minimal and with formate as the electron donor, with different nitrogen sources, varied salinities and at a variety of growth temperatures. Of the conditions tested, Western blot results showed that major archaellin FlaB2 levels only varied detectably as a result of growth temperature. Whilst the amount of FlaB2 was similar for cells grown at < 35 °C, protein levels decreased at 38 °C and were barely detectable at 42 °C. Quantitative reverse transcription PCR experiments demonstrated that the flaB2 transcript levels were almost undetectable at 42 °C. Electron microscopy confirmed that the FlaB2 levels detected by Western blots corresponded to the state of archaellation, with cells grown at 42 °C being mostly non-archaellated. Unexpectedly, a lower apparent molecular mass for FlaB2 was observed in Western blots of cells grown at temperatures >38 °C, suggestive of a truncation in the attached N-linked tetrasaccharide at higher growth temperatures. MS analysis of archaella isolated from cells grown at 40 °C confirmed that FlaB2 was now decorated with a trisaccharide in which the third sugar was also lacking the attached threonine and acetamidino modifications found in the WT glycan.

In Methanococcus maripaludis S2, the swimming organelle, the archaellum, is composed of three archaellins, FlaB1S2, FlaB2S2 and FlaB3S2. All three are modified with an N-linked tetrasaccharide at multiple sites. Disruption of the N-linked glycosylation pathway is known to cause defects in archaella assembly or function. Here, we explored the potential requirement of N-glycosylation of archaellins on archaellation by investigating the effects of eliminating the 4 N-glycosylation sites in the wildtype FlaB2S2 protein in all possible combinations either by Asn to Glu (N to Q) substitution or Asn to Asp (N to D) substitutions of the N-glycosylation sequon asparagine. The ability of these mutant derivatives to complement a non-archaellated ΔflaB2S2 strain was examined by electron microscopy (for archaella assembly) and swarm plates (for analysis of swimming). Western blot results showed that all mutated FlaB2S2 proteins were expressed and of smaller apparent molecular mass compared to wildtype FlaB2S2, consistent with the loss of glycosylation sites. In the 8 single-site mutant complements, archaella were observed on the surface of Q2, D2 and D4 (numbers after N or Q refer to the 1st to 4th glycosylation site). Of the 6 double-site mutation complementations all were archaellated except D1,3. Of the 4 triple-site mutation complements, only D2,3,4 was archaellated. Elimination of all 4 N-glycosylation sites resulted in non-archaellated cells, indicating some minimum amount of archaellin glycosylation was necessary for their incorporation into stable archaella. All complementations that led to a return of archaella also resulted in motile cells with the exception of the D4 version. In addition, a series of FlaB2S2 scanning deletions each missing 10 amino acids was also generated and tested for their ability to complement the ΔflaB2S2 strain. While most variants were expressed, none of them restored archaellation, although FlaB2S2 harbouring a smaller 3-amino acid deletion was able to partially restore archaellation.

In Escherichia coli, FtsK is a large integral membrane protein that coordinates chromosome segregation and cell division. The N-terminal domain of FtsK (FtsKN) is essential for division, and the C terminus (FtsKC) is a well characterized DNA translocase. Although the function of FtsKN is unknown, it is suggested that FtsK acts as a checkpoint to ensure DNA is properly segregated before septation. This may occur through modulation of protein interactions between FtsKN and other division proteins in both the periplasm and cytoplasm; thus, a clear understanding of how FtsKN is positioned in the membrane is required to characterize these interactions. The membrane topology of FtsKN was initially determined using site-directed reporter fusions; however, questions regarding this topology persist. Here, we report a revised membrane topology generated by site-directed fluorescence labeling. The revised topology confirms the presence of four transmembrane segments and reveals a newly identified periplasmic loop between the third and fourth transmembrane domains. Within this loop, four residues were identified that, when mutated, resulted in the appearance of cellular voids. High resolution transmission electron microscopy of these voids showed asymmetric division of the cytoplasm in the absence of outer membrane invagination or visible cell wall ingrowth. This uncoupling reveals a novel role for FtsK in linking cell envelope septation events and yields further evidence for FtsK as a critical checkpoint of cell division. The revised topology of FtsKN also provides an important platform for future studies on essential interactions required for this process., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)