N-terminal Segment Research Articles

Highly Selective Lysine Acylation in Proteins Using a Lys-His Tag Sequence.

Chemical modification of proteins has numerous applications, but it has been challenging to achieve the required high degree of selectivity on lysine amino groups. Recently, we described the highly selective acylation of proteins with an N-terminal Gly-His6 segment. This tag promoted acylation of the N-terminal Nα -amine resulting in stable conjugates. Herein, we report the peptide sequences Hisn -Lys-Hism , which we term Lys-His tags. In combination with simple acylating agents, they facilitate the acylation of the designated Lys Nϵ -amine under mild conditions and with high selectivity over native Lys residues. We show that the Lys-His tags, which are 7 to 10 amino acids in length and still act as conventional His tags, can be inserted in proteins at the C-terminus or in loops, thus providing high flexibility regarding the site of modification. Finally, the selective and efficient acylation of the therapeutic antibody Rituximab, pure or mixed with other proteins, demonstrates the scope of the Lys-His tag acylation method.

Refined Capsid Structure of Human Adenovirus D26 at 3.4 Å Resolution.

Various adenoviruses are being used as viral vectors for the generation of vaccines against chronic and emerging diseases (e.g., AIDS, COVID-19). Here, we report the improved capsid structure for one of these vectors, human adenovirus D26 (HAdV-D26), at 3.4 Å resolution, by reprocessing the previous cryo-electron microscopy dataset and obtaining a refined model. In addition to overall improvements in the model, the highlights of the structure include (1) locating a segment of the processed peptide of VIII that was previously believed to be released from the mature virions, (2) reorientation of the helical appendage domain (APD) of IIIa situated underneath the vertex region relative to its counterpart observed in the cleavage defective (ts1) mutant of HAdV-C5 that resulted in the loss of interactions between the APD and hexon bases, and (3) the revised conformation of the cleaved N-terminal segments of pre-protein VI (pVIn), located in the hexon cavities, is highly conserved, with notable stacking interactions between the conserved His13 and Phe18 residues. Taken together, the improved model of HAdV-D26 capsid provides a better understanding of protein–protein interactions in HAdV capsids and facilitates the efforts to modify and/or design adenoviral vectors with altered properties. Last but not least, we provide some insights into clotting factors (e.g., FX and PF4) binding to AdV vectors.

Structural polymorphism of coiled-coils from the stalk domain of SARS-CoV-2 spike protein.

Spike trimer plays a key role in SARS‐CoV‐2 infection and vaccine development. It consists of a globular head and a flexible stalk domain that anchors the protein into the viral membrane. While the head domain has been extensively studied, the properties of the adjoining stalk are poorly understood. Here, we characterize the coiled‐coil formation and thermodynamic stability of the stalk domain and its segments. We find that the N‐terminal segment of the stalk does not form coiled‐coils and remains disordered in solution. The C‐terminal stalk segment forms a trimeric coiled‐coil in solution, which becomes significantly stabilized in the context of the full‐length stalk. Its crystal structure reveals a novel antiparallel tetramer coiled‐coil with an unusual combination of a‐d and e‐a‐d hydrophobic core packing. Structural analysis shows that a subset of hydrophobic residues stabilizes different coiled‐coil structures: trimer, tetramer, and heterohexamer, underscoring a highly polymorphic nature of the SARS‐CoV‐2 stalk sequence.

In silico engineering of alternative pore-forming states of VDAC

VDAC (Voltage-Dependent Anion-selective Channel) is the primary metabolite pore in the mitochondrial outer membrane (OM). Atomic structures of VDAC, consistent with a physiological “open” state, consist of 19-strand β-barrels with N-terminal segment folded inside the lumen. However, structures are lacking for VDAC’s partially “closed” states, involving major changes in lumen structure and volume. Likewise, VDAC conformers occur in the OM in which polypeptide segments buried in atomic models are accessible to topological probes.

Declining serum bone turnover markers are associated with the short-term positive change of lumbar spine bone mineral density in postmenopausal women.

Objective:While serum bone turnover markers (BTMs) and bone mineral density (BMD) have been confirmed as useable risk assessment tools for postmenopausal osteoporosis, the associations between BTMs and BMD changes are still ambiguous. The aim of this study was to explore the underlying associations between BTMs and BMD changes in postmenopausal women.Methods:Between January 2015 and October 2020, 135 postmenopausal women were retrospectively enrolled. They were divided into two groups according to lumbar spine (LS) 1-4 BMD change (1 y T-score minus baseline T-score, Group 1 [n = 36] < 0 and Group 2 [n = 99] ≥ 0). The changes of BTMs (N-terminal middle segment osteocalcin [N-MID], propeptide of type I procollagen [P1NP], and β-C-terminal telopeptide of type I collagen [β-CTX]) and their associations with LS 1-4 BMD change were analyzed. The biochemical indices and clinical parameters related with LS 1-4 BMD change were also evaluated.Results:The 1 year N-MID, P1NP, β-CTX and Phosphorus in Group 2 were lower than those in Group 1 (P < 0.05), their changes within 1 year were significantly negatively correlated with LS 1-4 BMD change (R2 = –0.200, P < 0.001; R2 = –0.230, P < 0.001; R2 = –0.186, P < 0.001; R2 = –0.044, P = 0.015; respectively). Except for the Phosphorus change (area under the curve [AUC] = 0.623), the changes of N-MID, P1NP, and β-CTX and their 1 year levels had similar AUC to diagnose the short-term LS 1-4 BMD change (AUC > 0.7 for all, with the AUC of 1 y P1NP being the largest at 0.803). Binary logistic regression analysis showed that the physical activity and drug intervention were the determinant factors for the LS 1-4 BMD change (odds ratio = 6.856, 95% confidence interval: 2.058-22.839, P = 0.002; odds ratio = 5.114, 95% confidence interval: 1.551-16.864, P = 0.007; respectively).Conclusions:Declining N-MID, P1NP, β-CTX, and Phosphorus are associated with the short-term increase of LS 1-4 BMD within 1 year. Physical activity and drug intervention are factors significantly influencing the change of LS 1-4 BMD in postmenopausal women.

Conformational and Solvation Dynamics of an Amyloidogenic Intrinsically Disordered Domain of a Melanosomal Protein.

The conformational plasticity of intrinsically disordered proteins (IDPs) allows them to adopt a range of conformational states that can be important for their biological functions. The driving force for the conformational preference of an IDP emanates from an intricate interplay between chain-chain and chain-solvent interactions. Using ultrafast femtosecond and picosecond time-resolved fluorescence measurements, we characterized the conformational and solvation dynamics around the N- and C-terminal segments of a disordered repeat domain of a melanosomal protein Pmel17 that forms functional amyloid responsible for melanin biosynthesis. Our time-resolved fluorescence anisotropy results revealed slight compaction and slower rotational dynamics around the amyloidogenic C-terminal segment when compared to the proline-rich N-terminal segment of the repeat domain. The compaction of the C-terminal region was also associated with the restrained mobility of hydration water as indicated by our solvation dynamics measurements. Our findings indicate that sequence-dependent chain-solvent interactions govern both the conformational and solvation dynamics that are crucial in directing the conversion of a highly dynamic IDP into an ordered amyloid assembly. Such an interplay of amino acid composition-dependent conformational and solvation dynamics might have important physicochemical consequences in specific water-protein, ion-protein, and protein-protein interactions involved in amyloid formation and phase transitions.

Structural overview of the translocase of the mitochondrial outer membrane complex.

Most mitochondrial proteins are synthesized as precursor proteins (preproteins) in the cytosol and imported into mitochondria. The translocator of the outer membrane (TOM) complex functions as a main entry gate for the import of mitochondrial proteins. The TOM complex is a multi-subunit membrane protein complex composed of a β-barrel channel Tom40 and six single-pass membrane proteins. Recent cryo-EM studies have revealed high-resolution structures of the yeast and human TOM complexes, which enabled us to discuss the mechanism of protein import at an amino-acid residue level. The cryo-EM structures show that two Tom40 β-barrels are surrounded by two sets of small Tom subunits to form a dimeric structure. The intermembrane space (IMS) domains of Tom40, Tom22, and Tom7 form a binding site for presequence-containing preproteins in the middle of the dimer to achieve their efficient transfer of to the downstream translocase, the TIM23 complex. The N-terminal segment of Tom40 spans the channel from the cytosol to the IMS to interact with Tom5 at the periphery of the dimer, where downstream components of presequence-lacking preproteins are recruited. Structure-based biochemical analyses together with crosslinking experiments revealed that each Tom40 channel possesses two distinct paths and exit sites for protein translocation of different sets of mitochondrial preproteins. Here we summarize the current knowledge on the structural features, protein translocation mechanisms, and remaining questions for the TOM complexes, with particular emphasis on their determined cryo-EM structures. This article is an extended version of the Japanese article, Structural basis for protein translocation by the translocase of the outer mitochondrial membrane, published in SEIBUTSU BUTSURI Vol. 60, p. 280-283 (2020).

Multiple binding modes of an N-terminal CCR5-peptide in complex with HIV-1 gp120.

The N-terminal segment of CCR5 contains four tyrosine residues, sulphation of two of which is essential for high-affinity binding to gp120. In the present study, the interactions of gp120YU2 with a 27-residue N-terminal CCR5 peptide sulphated at position Y10 and Y14, i.e. Nt-CCR5, were studied using 13 C-edited-HMQC methyl-NOESY [1 H(13 C)-1 H], combined with transferred NOE NMR spectroscopy. A large number of pairwise interactions were observed between the methyl protons of methionine, threonine, valine and isoleucine residues of gp120, and the aromatic tyrosine-protons of Nt-CCR5. M434, V120 and V200 of gp120 were found to interact with all four tyrosine residues, Y3, sY10, sY14 and Y15. Particularly intriguing was the observation that Y3 and Y15 interact with the same gp120 methyl protons. Such interactions cannot be explained by the single cryo-EM structure of gp120/CD4/CCR5 complex published recently (Nature, 565, 318-323, 2019). Rather, they are consistent with the existence of a dynamic equilibrium involving two or more binding modes of Nt-CCR5 to gp120. These different modes of binding can coexist because the surface of gp120 contains two sites that can optimally interact with a sulphated tyrosine residue and two sites that can interact favorably with a non-sulphated tyrosine residue. Modelling of gp120YU2 complexed with the Nt-CCR5 peptide or with the entire CCR5 receptor provides an explanation for the NMR observations and the existence of these different binding modes of the disordered N-terminus of CCR5. The data presented extend our understanding of the two-step model and suggest a more variable binding mode of Nt-CCR5 with gp120.

WFS1 functions in ER export of vesicular cargo proteins in pancreatic \u03b2-cells

The sorting of soluble secretory proteins from the endoplasmic reticulum (ER) to the Golgi complex is mediated by coat protein complex II (COPII) vesicles and thought to required specific ER membrane cargo-receptor proteins. However, these receptors remain largely unknown. Herein, we show that ER to Golgi transfer of vesicular cargo proteins requires WFS1, an ER-associated membrane protein whose loss of function leads to Wolfram syndrome. Mechanistically, WFS1 directly binds to vesicular cargo proteins including proinsulin via its ER luminal C-terminal segment, whereas pathogenic mutations within this region disrupt the interaction. The specific ER export signal encoded in the cytosolic N-terminal segment of WFS1 is recognized by the COPII subunit SEC24, generating mature COPII vesicles that traffic to the Golgi complex. WFS1 deficiency leads to abnormal accumulation of proinsulin in the ER, impeding the proinsulin processing as well as insulin secretion. This work identifies a vesicular cargo receptor for ER export and suggests that impaired peptide hormone transport underlies diabetes resulting from pathogenic WFS1 mutations.

Abstract 13171: The Role of Novel Brugada Syndrome Gene MAPRE2 in Maintaining Normal Cardiac Electrophysiology

Introduction: Brugada syndrome (BrS) is a significant cause of sudden cardiac death yet only ~20% of patients are found with a loss-of-function variant in SCN5A encoding the cardiac Na + channel, leaving ~80% genetically undiagnosed. A recent genome-wide association study found 12 loci associated with BrS, including one in MAPRE2 encoding microtubule end-binding protein 2 (EB2). Hypothesis: MAPRE2 loss-of-function contributes to BrS and is necessary in maintaining normal cardiac electrophysiology (EP). Methods: Using CRISPR/Cas9, we generated two mapre2 loss-of-function mutants in zebrafish: a full knock-out and a mutant lacking the unique N-terminus of EB2 (delN). Cardiac EP was assessed using optical mapping in larvae, surface ECG in adults, and patch clamping in ventricular myocytes. Cardiac structure was assessed using videomicroscopy, histology, and confocal microscopy. Transcriptional changes were assessed using RNA-seq. Results: No gross changes in cardiac structure were observed with mapre2 loss-of-function. However, in both mutants, voltage mapping showed decreased ventricular conduction velocity and action potential upstroke velocity (V max ) and ECGs showed prolonged QRS, P wave, and corrected QT. Patch clamp of delN ventricular myocytes revealed reduced Na + current density without changes in gating properties consistent with the reduced V max . RNA-seq of larvel hearts suggested disruption of cell adhesion, confirmed by confocal microscopy, whereas Ca 2+ imaging found an increase in Ca 2+ transient amplitude. RNA-seq of adult hearts showed disruption in the Wnt signaling pathway and treatment with GSK3β inhibitor SB217673 rescued mutant ECG abnormalities. Conclusions: Our study supports MAPRE2 as a novel contributing gene in BrS pathogenesis and conduction slowing in general. Beyond its effect on Na + channel function, mapre2 and in particularly its unique N-terminal segment may play a broader role in cellular organization via the microtubule network affecting cellular EP in multiple ways. Our study also implicates for the first time the Wnt signaling pathway in BrS, suggesting not only shared biology with arrhythmogenic cardiomyopathies, but also the possibility of GSK3β modulation as a novel therapeutic approach.

Molecular mechanisms of the influenza fusion peptide: insights from experimental and simulation studies.

A key step in infections by enveloped viruses, such as influenza, is the fusion between the viral envelope and the host cell membrane, which allows the virus to insert its genetic material into the host cell and replicate. The influenza virus fusion process is promoted by hemagglutinin (HA), a glycoprotein that contains three identical monomers composed of two polypeptide chains (HA1 and HA2). Early studies on this protein revealed that HA‐mediated fusion involves the insertion of the HA2 N‐terminal segment into the host membrane and that this segment, known as the fusion peptide, is a key player in the fusion process. This mini‐review highlights the main findings that have been obtained by experimental and computational studies on the HA fusion peptide, which give us a glimpse of its mode of action.

An intrinsically disordered pathological prion variant Y145Stop converts into self-seeding amyloids via liquid–liquid phase separation

Biomolecular condensation via liquid-liquid phase separation of intrinsically disordered proteins/regions (IDPs/IDRs) along with other biomolecules is proposed to control critical cellular functions, whereas aberrant phase transitions are associated with a range of neurodegenerative diseases. Here, we show that a disease-associated stop codon mutation of the prion protein (PrP) at tyrosine 145 (Y145Stop), resulting in a truncated, highly disordered, N-terminal IDR, spontaneously phase-separates into dynamic liquid-like droplets. Phase separation of this highly positively charged N-terminal segment is promoted by the electrostatic screening and a multitude of weak, transient, multivalent, intermolecular interactions. Single-droplet Raman measurements, in conjunction with an array of bioinformatic, spectroscopic, microscopic, and mutagenesis studies, revealed a highly mobile internal organization within the liquid-like condensates. The phase behavior of Y145Stop is modulated by RNA. Lower RNA:protein ratios promote condensation at a low micromolar protein concentration under physiological conditions. At higher concentrations of RNA, phase separation is abolished. Upon aging, these highly dynamic liquid-like droplets gradually transform into ordered, β-rich, amyloid-like aggregates. These aggregates formed via phase transitions display an autocatalytic self-templating characteristic involving the recruitment and binding-induced conformational conversion of monomeric Y145Stop into amyloid fibrils. In contrast to this intrinsically disordered truncated variant, the wild-type full-length PrP exhibits a much lower propensity for both condensation and maturation into amyloids, hinting at a possible protective role of the C-terminal domain. Such an interplay of molecular factors in modulating the protein phase behavior might have much broader implications in cell physiology and disease.

Curly Hair FUE: My Approach Using Classification of Follicle Curvature and Curl

<h3>Abstract</h3> <i>Deinococcus radiodurans</i> is a deep-branching extremophilic bacterium that is remarkably tolerant to numerous environmental stresses, including large doses of ultraviolet radiation and extreme temperatures. It can even survive in outer space for several years. This endurance of <i>D. radiodurans</i> has been partly ascribed to its atypical cell envelope comprising an inner membrane, a large periplasmic space with a thick peptidoglycan (PG) layer, and an outer membrane (OM) covered by a surface layer (S-layer). Despite intense research, molecular principles governing envelope organization and OM stabilization are unclear in <i>D. radiodurans</i> and related bacteria. Here, we report an electron cryomicroscopy (cryo-EM) structure of the abundant <i>D. radiodurans</i> OM protein SlpA, showing how its C-terminal segment forms homotrimers of 30-stranded β-barrels in the OM, whereas its N-terminal segment forms long, homotrimeric coiled coils linking the OM to the PG layer via S-layer homology (SLH) domains. Using the power of structure prediction and sequence-based bioinformatics, we further show that SlpA-like proteins are widespread in deep-branching Gram-negative bacteria, plausibly constituting an ancestral superfamily of OM-PG connectors, important for organizing the cell envelopes of many bacteria. Finally, combining our atomic structures with tomography of cell envelopes, we report a model for the cell surface of <i>D. radiodurans</i>, with implications on understanding the cell surface organization and hyperstability of <i>D. radiodurans</i> and related bacteria. Furthermore, the widespread occurrence of SlpA-like OM-PG connectors in deep-branching bacteria will help in understanding the evolutionary transition between Gram-negative and Gram-positive bacteria.

Synergistic Action of Antimicrobial Lung Proteins against Klebsiella pneumoniae.

As key components of innate immunity, lung antimicrobial proteins play a critical role in warding off invading respiratory pathogens. Lung surfactant protein A (SP-A) exerts synergistic antimicrobial activity with the N-terminal segment of the SP-B proprotein (SP-BN) against Klebsiella pneumoniae K2 in vivo. However, the factors that govern SP-A/SP-BN antimicrobial activity are still unclear. The aim of this study was to identify the mechanisms by which SP-A and SP-BN act synergistically against K. pneumoniae, which is resistant to either protein alone. The effect of these proteins on K. pneumoniae was studied by membrane permeabilization and depolarization assays and transmission electron microscopy. Their effects on model membranes of the outer and inner bacterial membranes were analyzed by differential scanning calorimetry and membrane leakage assays. Our results indicate that the SP-A/SP-BN complex alters the ultrastructure of K. pneumoniae by binding to lipopolysaccharide molecules present in the outer membrane, forming packing defects in the membrane that may favor the translocation of both proteins to the periplasmic space. The SP-A/SP-BN complex depolarized and permeabilized the inner membrane, perhaps through the induction of toroidal pores. We conclude that the synergistic antimicrobial activity of SP-A/SP-BN is based on the capability of this complex, but not either protein alone, to alter the integrity of bacterial membranes.

Targeting the Achilles Heel of FtsZ: The Interdomain Cleft.

Widespread antimicrobial resistance among bacterial pathogens is a serious threat to public health. Thus, identification of new targets and development of new antibacterial agents are urgently needed. Although cell division is a major driver of bacterial colonization and pathogenesis, its targeting with antibacterial compounds is still in its infancy. FtsZ, a bacterial cytoskeletal homolog of eukaryotic tubulin, plays a highly conserved and foundational role in cell division and has been the primary focus of research on small molecule cell division inhibitors. FtsZ contains two drug-binding pockets: the GTP binding site situated at the interface between polymeric subunits, and the inter-domain cleft (IDC), located between the N-terminal and C-terminal segments of the core globular domain of FtsZ. The majority of anti-FtsZ molecules bind to the IDC. Compounds that bind instead to the GTP binding site are much less useful as potential antimicrobial therapeutics because they are often cytotoxic to mammalian cells, due to the high sequence similarity between the GTP binding sites of FtsZ and tubulin. Fortunately, the IDC has much less sequence and structural similarity with tubulin, making it a better potential target for drugs that are less toxic to humans. Over the last decade, a large number of natural and synthetic IDC inhibitors have been identified. Here we outline the molecular structure of IDC in detail and discuss how it has become a crucial target for broad spectrum and species-specific antibacterial agents. We also outline the drugs that bind to the IDC and their modes of action.

Abstract MP240: Site-specific O-Glcnacylation Of Transient Receptor Potential Canonical Type 6 (trpc6) Channel Is Required For Normal Structure And Function

O-GlcNAcylation is a dynamic, reversible posttranslational modification (PTM) that regulates a multitude of biological processes. Fluctuations in O-GlcNAC of various calcium handling proteins impact their functionality in cardiomyocytes. Here, we show for the first time that TRPC6, a nonselective receptor-operated cation channel and mediator of hypertrophy and fibrosis, is constitutively O-GlcNAcylated in the ankyrin repeat domain (AR4), at Ser 14, Thr 70, and Thr 221 within the N-terminal cytoplasmic segment. Of these, only substitution of Thr 221 with alanine (T221A) results in a change of function, notably a hyperactive TRPC6 channel with 5X greater increase in consequent NFAT promoter activity, which is a marker of TRPC6 calcium signaling. Patch-clamp analysis of T221A mutant channels found a 75-80% increased conductance compared to WT. Myocardial injection of T221A in homozygous TRPC6 KO mice by AAV-9 mediated gene transfer results in systolic dysfunction, hypertrophy, and cardiac fibrosis, by loss of OGlcNAc modification at site T221. T221 is highly conserved across species and found in the AR4 domain, which forms the core structure of TRPC6 intracellular domain. Mutating the site in its closest homologs, TRPC3 and TRPC7, also activates channel activity. T221 O-GlcNAcylation also protects the nascent protein from premature proteasomal degradation. Molecular modeling from the crystal structure of human TRPC6 predicts that OGlcNACylation stabilizes electrostatic interactions with the 193-203 loop near AR4, and loop connecting AR4 to the linker helix 1 (LH1) at S199, E200, and E246. Mutating these sites to alanine also increases TRPC6-NFAT signaling similar to what was observed in the T221A mutant. In summary, this study highlights that O-GlcNAcylation of TRPC6 is an important PTM needed to stabilize channel function, and its decline results in gain-of-function related diseases.

Glycogen phosphorylase revisited: extending the resolution of the R- and T-state structures of the free enzyme and in complex with allosteric activators.

The crystal structures of free T-state and R-state glycogen phosphorylase (GP) and of R-state GP in complex with the allosteric activators IMP and AMP are reported at improved resolution. GP is a validated pharmaceutical target for the development of antihyperglycaemic agents, and the reported structures may have a significant impact on structure-based drug-design efforts. Comparisons with previously reported structures at lower resolution reveal the detailed conformation of important structural features in the allosteric transition of GP from the T-state to the R-state. The conformation of the N-terminal segment (residues 7-17), the position of which was not located in previous T-state structures, was revealed to form an α-helix (now termed α0). The conformation of this segment (which contains Ser14, phosphorylation of which leads to the activation of GP) is significantly different between the T-state and the R-state, pointing in opposite directions. In the T-state it is packed between helices α4 and α16 (residues 104-115 and 497-508, respectively), while in the R-state it is packed against helix α1 (residues 22'-38') and towards the loop connecting helices α4' and α5' of the neighbouring subunit. The allosteric binding site where AMP and IMP bind is formed by the ordering of a loop (residues 313-326) which is disordered in the free structure, and adopts a conformation dictated mainly by the type of nucleotide that binds at this site.

Lack of N-Terminal Segment of the Flagellin Protein Results in the Production of a Shortened Polar Flagellum in the Deep-Sea Sedimentary Bacterium Pseudoalteromonas sp. Strain SM9913.

Bacterial polar flagella, comprised of flagellin, are essential for bacterial motility. Pseudoalteromonas sp. strain SM9913 is a bacterium isolated from deep-sea sediments. Unlike other Pseudoalteromonas strains that have a long polar flagellum, strain SM9913 has an abnormally short polar flagellum. Here, we investigated the underlying reason for the short flagellum and found that a single-base mutation was responsible for the altered flagellar assembly. This mutation leads to the fragmentation of the flagellin gene into two genes, PSM_A2281, encoding the core segment and the C-terminal segment, and PSM_A2282, encoding the N-terminal segment, and only gene PSM_A2281 is involved in the production of the short polar flagellum. When a chimeric gene of PSM_A2281 and PSM_A2282 encoding an intact flagellin, A2281::82, was expressed, a long polar flagellum was produced, indicating that the N-terminal segment of flagellin contributes to the production of a polar flagellum of a normal length. Analyses of the simulated structures of A2281 and A2281::82 and that of the flagellar filament assembled with A2281::82 indicate that due to the lack of two α-helices, the core of the flagellar filament assembled with A2281 is incomplete and is likely too weak to support the stability and movement of a long flagellum. This mutation in strain SM9913 had little effect on its growth and only a small effect on its swimming motility, implying that strain SM9913 can live well with this mutation in natural sedimentary environments. This study provides a better understanding of the assembly and production of bacterial flagella. IMPORTANCE Polar flagella, which are essential organelles for bacterial motility, are comprised of multiple flagellin subunits. A flagellin molecule contains an N-terminal segment, a core segment, and a C-terminal segment. The results of this investigation of the deep-sea sedimentary bacterium Pseudoalteromonas sp. strain SM9913 demonstrate that a single-base mutation in the flagellin gene leads to the production of an incomplete flagellin without the N-terminal segment and that the loss of the N-terminal segment of the flagellin protein results in the production of a shortened polar flagellar filament. Our results shed light on the important function of the N-terminal segment of flagellin in the assembly and stability of bacterial flagellar filament.

The structure and flexibility analysis of the Arabidopsis synaptotagmin 1 reveal the basis of its regulation at membrane contact sites.

Non-vesicular lipid transfer at ER and plasma membrane (PM) contact sites (CS) is crucial for the maintenance of membrane lipid homeostasis. Extended synaptotagmins (E-Syts) play a central role in this process as they act as molecular tethers of ER and PM and as lipid transfer proteins between these organelles. E-Syts are proteins constitutively anchored to the ER through an N-terminal hydrophobic segment and bind the PM via a variable number of C-terminal C2 domains. Synaptotagmins (SYTs) are the plant orthologous of E-Syts and regulate the ER-PM communication in response to abiotic stress. Combining different structural and biochemical techniques, we demonstrate that the binding of SYT1 to lipids occurs through a Ca2+-dependent lipid-binding site and by a site for phosphorylated forms of phosphatidylinositol, thus integrating two different molecular signals in response to stress. In addition, we show that SYT1 displays three highly flexible hinge points that provide conformational freedom to facilitate lipid extraction, protein loading, and subsequent transfer between PM and ER.

The disordered regions of the methyltransferase SETD2 govern its function by regulating its proteolysis and phase separation

SETD2 is an important methyltransferase that methylates crucial substrates such as histone H3, tubulin, and STAT1 and also physically interacts with transcription and splicing regulators such as Pol II and various hnRNPs. Of note, SETD2 has a functionally uncharacterized extended N-terminal region, the removal of which leads to its stabilization. How this region regulates SETD2 half-life is unclear. Here we show that SETD2 consists of multiple long disordered regions across its length that cumulatively destabilize the protein by facilitating its proteasomal degradation. SETD2 disordered regions can reduce the half-life of the yeast homolog Set2 in mammalian cells as well as in yeast, demonstrating the importance of intrinsic structural features in regulating protein half-life. In addition to the shortened half-life, by performing fluorescence recovery after photobleaching assay we found that SETD2 forms liquid droplets in vivo, another property associated with proteins that contain disordered regions. The phase-separation behavior of SETD2 is exacerbated upon the removal of its N-terminal segment and results in activator-independent histone H3K36 methylation. Our findings reveal that disordered region-facilitated proteolysis is an important mechanism governing SETD2 function.