Filament structure and subcellular organization of the bacterial intermediate filament-like protein crescentin.

The protein crescentin is required for the crescent shape of the freshwater bacterium Caulobacter crescentus (vibrioides). Crescentin forms a filamentous structure on the inner, concave side of the curved cells. It shares features with eukaryotic intermediate filament (IF) proteins, including the formation of static filaments based on long and parallel coiled coils, the protein's length, structural roles in cell and organelle shape determination and the presence of a coiled coil discontinuity called the "stutter." Here, we have used electron cryomicroscopy (cryo-EM) to determine the structure of the full-length protein and its filament, exploiting a crescentin-specific nanobody. The filament is formed by two strands, related by twofold symmetry, that each consist of two dimers, resulting in an octameric assembly. Crescentin subunits form longitudinal contacts head-to-head and tail-to-tail, making the entire filament non-polar. Using in vivo site-directed cysteine cross-linking, we demonstrated that contacts observed in the in vitro filament structure exist in cells. Electron cryotomography (cryo-ET) of cells expressing crescentin showed filaments on the concave side of the curved cells, close to the inner membrane, where they form a band. When comparing with current models of IF proteins and their filaments, which are also built from parallel coiled coil dimers and lack overall polarity, it emerges that IF proteins form head-to-tail longitudinal contacts in contrast to crescentin and hence several inter-dimer contacts in IFs have no equivalents in crescentin filaments. Our work supports the idea that intermediate filament-like proteins achieve their shared polymerization and mechanical properties through a variety of filament architectures.

What AlphaFold tells us about cohesin's retention on and release from chromosomes.

Cohesin is a trimeric complex containing a pair of SMC proteins (Smc1 and Smc3) whose ATPase domains at the end of long coiled coils (CC) are interconnected by Scc1. During interphase, it organizes chromosomal DNA topology by extruding loops in a manner dependent on Scc1's association with two large hook-shaped proteins called SA (yeast: Scc3) and Nipbl (Scc2). The latter's replacement by Pds5 recruits Wapl, which induces release from chromatin via a process requiring dissociation of Scc1's N-terminal domain (NTD) from Smc3. If blocked by Esco (Eco)-mediated Smc3 acetylation, cohesin containing Pds5 merely maintains pre-existing loops, but a third fate occurs during DNA replication, when Pds5-containing cohesin associates with Sororin and forms structures that hold sister DNAs together. How Wapl induces and Sororin blocks release has hitherto remained mysterious. In the 20 years since their discovery, not a single testable hypothesis has been proposed as to their role. Here, AlphaFold 2 (AF) three-dimensional protein structure predictions lead us to propose formation of a quarternary complex between Wapl, SA, Pds5, and Scc1's NTD, in which the latter is juxtaposed with (and subsequently sequestered by) a highly conserved cleft within Wapl's C-terminal domain. AF also reveals how Scc1's dissociation from Smc3 arises from a distortion of Smc3's CC induced by engagement of SMC ATPase domains, how Esco acetyl transferases are recruited to Smc3 by Pds5, and how Sororin prevents release by binding to the Smc3/Scc1 interface. Our hypotheses explain the phenotypes of numerous existing mutations and are highly testable.

The patterned assembly and stepwise Vps4-mediated disassembly of composite ESCRT-III polymers drives archaeal cell division.

ESCRT-III family proteins form composite polymers that deform and cut membrane tubes in the context of a wide range of cell biological processes across the tree of life. In reconstituted systems, sequential changes in the composition of ESCRT-III polymers induced by the AAA-adenosine triphosphatase Vps4 have been shown to remodel membranes. However, it is not known how composite ESCRT-III polymers are organized and remodeled in space and time in a cellular context. Taking advantage of the relative simplicity of the ESCRT-III-dependent division system in Sulfolobus acidocaldarius, one of the closest experimentally tractable prokaryotic relatives of eukaryotes, we use super-resolution microscopy, electron microscopy, and computational modeling to show how CdvB/CdvB1/CdvB2 proteins form a precisely patterned composite ESCRT-III division ring, which undergoes stepwise Vps4-dependent disassembly and contracts to cut cells into two. These observations lead us to suggest sequential changes in a patterned composite polymer as a general mechanism of ESCRT-III-dependent membrane remodeling.

Efficacy of same-day versus next-day administration of pegfilgrastim for the prevention of chemotherapy-induced febrile neutropenia in breast cancer patients receiving dose-dense doxorubicin and cyclophosphamide: A retrospective multi-site analysis.

INTRODUCTION: Administering pegfilgrastim on the same day as chemotherapy can improve patient satisfaction through convenience and may increase the utilization of cost-effective biosimilars compared to next-day administration, but the effect on clinical outcomes with commonly used breast cancer regimens is unclear. METHODS: This multi-site, retrospective cohort study included breast cancer patients age 18 years or older who received dose-dense doxorubicin and cyclophosphamide (ddAC) and pegfilgrastim between 1 June 2016 and 31 May 2020. Pegfilgrastim was given on the same day as chemotherapy at one site and the day after chemotherapy at the other two sites. The primary endpoint compared the incidence of febrile neutropenia associated with pegfilgrastim administration timing. RESULTS: A total of 360 patients were reviewed (146 same-day administration and 214 next-day administration). In the same-day group 36 patients (24.6%) developed FN compared to 25 patients (11.7%) in the next-day group (p = 0.002). Same-day administration also significantly increased the incidences of additional acute care visits (11.6% vs 2.8%, p = 0.0016), grade ≥ 3 neutropenia (38.4% vs 13.6%, p < 0.0001), chemotherapy dose reductions (21.2% vs 6.1%, p < 0.0001), and antibiotic use (26.7% vs 12.6%, p = 0.001). Same-day administration did not significantly increase the rate of hospitalization (15% vs 11.2%, p = 0.36) and delay of next chemotherapy cycle by ≥1 day (8.2% vs 6.1%, p = 0.57) due to neutropenic complications. CONCLUSIONS: Administering pegfilgrastim on the same day as ddAC led to a significant increase in neutropenic complications. This study confirms the need to administer pegfilgrastim the day after chemotherapy in breast cancer patients receiving ddAC.

Bacterial divisome protein FtsA forms curved antiparallel double filaments when binding to FtsN.

During bacterial cell division, filaments of tubulin-like FtsZ form the Z-ring, which is the cytoplasmic scaffold for divisome assembly. In Escherichia coli, the actin homologue FtsA anchors the Z-ring to the membrane and recruits divisome components, including bitopic FtsN. FtsN regulates the periplasmic peptidoglycan synthase FtsWI. To characterize how FtsA regulates FtsN, we applied electron microscopy to show that E. coli FtsA forms antiparallel double filaments on lipid monolayers when bound to the cytoplasmic tail of FtsN. Using X-ray crystallography, we demonstrate that Vibrio maritimus FtsA crystallizes as an equivalent double filament. We identified an FtsA-FtsN interaction site in the IA-IC interdomain cleft of FtsA using X-ray crystallography and confirmed that FtsA forms double filaments in vivo by site-specific cysteine cross-linking. FtsA-FtsN double filaments reconstituted in or on liposomes prefer negative Gaussian curvature, like those of MreB, the actin-like protein of the elongasome. We propose that curved antiparallel FtsA double filaments together with treadmilling FtsZ filaments organize septal peptidoglycan synthesis in the division plane.

Clamping of DNA shuts the condensin neck gate.

SignificanceDNA needs to be compacted to fit into nuclei and during cell division, when dense chromatids are formed for their mechanical segregation, a process that depends on the protein complex condensin. It forms and enlarges loops in DNA through loop extrusion. Our work resolves the atomic structure of a DNA-bound state of condensin in which ATP has not been hydrolyzed. The DNA is clamped within a compartment that has been reported previously in other structural maintenance of chromosomes (SMC) complexes, including Rad50, cohesin, and MukBEF. With the caveat of important differences, it means that all SMC complexes cycle through at least some similar states and undergo similar conformational changes in their head modules, while hydrolyzing ATP and translocating DNA.

Cryo-EM structure of MukBEF reveals DNA loop entrapment at chromosomal unloading sites.

The ring-like structural maintenance of chromosomes (SMC) complex MukBEF folds the genome of Escherichia coli and related bacteria into large loops, presumably by active DNA loop extrusion. MukBEF activity within the replication terminus macrodomain is suppressed by the sequence-specific unloader MatP. Here, we present the complete atomic structure of MukBEF in complex with MatP and DNA as determined by electron cryomicroscopy (cryo-EM). The complex binds two distinct DNA double helices corresponding to the arms of a plectonemic loop. MatP-bound DNA threads through the MukBEF ring, while the second DNA is clamped by the kleisin MukF, MukE, and the MukB ATPase heads. Combinatorial cysteine cross-linking confirms this topology of DNA loop entrapment in vivo. Our findings illuminate how a class of near-ubiquitous DNA organizers with important roles in genome maintenance interacts with the bacterial chromosome.

Folding of cohesin's coiled coil is important for Scc2/4-induced association with chromosomes.

Cohesin's association with and translocation along chromosomal DNAs depend on an ATP hydrolysis cycle driving the association and subsequent release of DNA. This involves DNA being 'clamped' by Scc2 and ATP-dependent engagement of cohesin's Smc1 and Smc3 head domains. Scc2's replacement by Pds5 abrogates cohesin's ATPase and has an important role in halting DNA loop extrusion. The ATPase domains of all SMC proteins are separated from their hinge dimerisation domains by 50-nm-long coiled coils, which have been observed to zip up along their entire length and fold around an elbow, thereby greatly shortening the distance between hinges and ATPase heads. Whether folding exists in vivo or has any physiological importance is not known. We present here a cryo-EM structure of the apo form of cohesin that reveals the structure of folded and zipped-up coils in unprecedented detail and shows that Scc2 can associate with Smc1's ATPase head even when it is fully disengaged from that of Smc3. Using cysteine-specific crosslinking, we show that cohesin's coiled coils are frequently folded in vivo, including when cohesin holds sister chromatids together. Moreover, we describe a mutation (SMC1D588Y) within Smc1's hinge that alters how Scc2 and Pds5 interact with Smc1's hinge and that enables Scc2 to support loading in the absence of its normal partner Scc4. The mutant phenotype of loading without Scc4 is only explicable if loading depends on an association between Scc2/4 and cohesin's hinge, which in turn requires coiled coil folding.

Cryo-EM structures of holo condensin reveal a subunit flip-flop mechanism.

Complexes containing a pair of structural maintenance of chromosomes (SMC) family proteins are fundamental for the three-dimensional (3D) organization of genomes in all domains of life. The eukaryotic SMC complexes cohesin and condensin are thought to fold interphase and mitotic chromosomes, respectively, into large loop domains, although the underlying molecular mechanisms have remained unknown. We used cryo-EM to investigate the nucleotide-driven reaction cycle of condensin from the budding yeast Saccharomyces cerevisiae. Our structures of the five-subunit condensin holo complex at different functional stages suggest that ATP binding induces the transition of the SMC coiled coils from a folded-rod conformation into a more open architecture. ATP binding simultaneously triggers the exchange of the two HEAT-repeat subunits bound to the SMC ATPase head domains. We propose that these steps result in the interconversion of DNA-binding sites in the catalytic core of condensin, forming the basis of the DNA translocation and loop-extrusion activities.

Cryo-EM structure of the MinCD copolymeric filament from Pseudomonas aeruginosa at 3.1 Å resolution.

Positioning of the division site in many bacterial species relies on the MinCDE system, which prevents the cytokinetic Z-ring from assembling anywhere but the mid-cell, through an oscillatory diffusion-reaction mechanism. MinD dimers bind to membranes and, via their partner MinC, inhibit the polymerization of cell division protein FtsZ into the Z-ring. MinC and MinD form polymeric assemblies in solution and on cell membranes. Here, we report the high-resolution cryo-EM structure of the copolymeric filaments of Pseudomonas aeruginosa MinCD. The filaments consist of three protofilaments made of alternating MinC and MinD dimers. The MinCD protofilaments are almost completely straight and assemble as single protofilaments on lipid membranes, which we also visualized by cryo-EM.

Scc2 Is a Potent Activator of Cohesin's ATPase that Promotes Loading by Binding Scc1 without Pds5.

Cohesin organizes DNA into chromatids, regulates enhancer-promoter interactions, and confers sister chromatid cohesion. Its association with chromosomes is regulated by hook-shaped HEAT repeat proteins that bind Scc1, namely Scc3, Pds5, and Scc2. Unlike Pds5, Scc2 is not a stable cohesin constituent but, as shown here, transiently replaces Pds5. Scc1 mutations that compromise its interaction with Scc2 adversely affect cohesin's ATPase activity and loading. Moreover, Scc2 mutations that alter how the ATPase responds to DNA abolish loading despite cohesin's initial association with loading sites. Lastly, Scc2 mutations that permit loading in the absence of Scc4 increase Scc2's association with chromosomal cohesin and reduce that of Pds5. We suggest that cohesin switches between two states: one with Pds5 bound that is unable to hydrolyze ATP efficiently but is capable of release from chromosomes and another in which Scc2 replaces Pds5 and stimulates ATP hydrolysis necessary for loading and translocation from loading sites.

Structures of actin-like ParM filaments show architecture of plasmid-segregating spindles.

Active segregation of Escherichia coli low-copy-number plasmid R1 involves formation of a bipolar spindle made of left-handed double-helical actin-like ParM filaments. ParR links the filaments with centromeric parC plasmid DNA, while facilitating the addition of subunits to ParM filaments. Growing ParMRC spindles push sister plasmids to the cell poles. Here, using modern electron cryomicroscopy methods, we investigate the structures and arrangements of ParM filaments in vitro and in cells, revealing at near-atomic resolution how subunits and filaments come together to produce the simplest known mitotic machinery. To understand the mechanism of dynamic instability, we determine structures of ParM filaments in different nucleotide states. The structure of filaments bound to the ATP analogue AMPPNP is determined at 4.3 Å resolution and refined. The ParM filament structure shows strong longitudinal interfaces and weaker lateral interactions. Also using electron cryomicroscopy, we reconstruct ParM doublets forming antiparallel spindles. Finally, with whole-cell electron cryotomography, we show that doublets are abundant in bacterial cells containing low-copy-number plasmids with the ParMRC locus, leading to an asynchronous model of R1 plasmid segregation.

MinCD cell division proteins form alternating copolymeric cytomotive filaments.

During bacterial cell division, filaments of the tubulin-like protein FtsZ assemble at midcell to form the cytokinetic Z-ring. Its positioning is regulated by the oscillation of MinCDE proteins. MinC is activated by MinD through an unknown mechanism and prevents Z-ring assembly anywhere but midcell. Here, using X-ray crystallography, electron microscopy and in vivo analyses, we show that MinD activates MinC by forming a new class of alternating copolymeric filaments that show similarity to eukaryotic septin filaments. A non-polymerizing mutation in MinD causes aberrant cell division in Escherichia coli. MinCD copolymers bind to membrane, interact with FtsZ and are disassembled by MinE. Imaging a functional msfGFP-MinC fusion protein in MinE-deleted cells reveals filamentous structures. EM imaging of our reconstitution of the MinCD-FtsZ interaction on liposome surfaces reveals a plausible mechanism for regulation of FtsZ ring assembly by MinCD copolymers.

Closing the cohesin ring: structure and function of its Smc3-kleisin interface.

Through their association with a kleisin subunit (Scc1), cohesin's Smc1 and Smc3 subunits are thought to form tripartite rings that mediate sister chromatid cohesion. Unlike the structure of Smc1/Smc3 and Smc1/Scc1 interfaces, that of Smc3/Scc1 is not known. Disconnection of this interface is thought to release cohesin from chromosomes in a process regulated by acetylation. We show here that the N-terminal domain of yeast Scc1 contains two α helices, forming a four-helix bundle with the coiled coil emerging from Smc3's adenosine triphosphatase head. Mutations affecting this interaction compromise cohesin's association with chromosomes. The interface is far from Smc3 residues, whose acetylation prevents cohesin's dissociation from chromosomes. Cohesin complexes holding chromatids together in vivo do indeed have the configuration of hetero-trimeric rings, and sister DNAs are entrapped within these.

Bacterial actin MreB forms antiparallel double filaments.

Filaments of all actin-like proteins known to date are assembled from pairs of protofilaments that are arranged in a parallel fashion, generating polarity. In this study, we show that the prokaryotic actin homologue MreB forms pairs of protofilaments that adopt an antiparallel arrangement in vitro and in vivo. We provide an atomic view of antiparallel protofilaments of Caulobacter MreB as apparent from crystal structures. We show that a protofilament doublet is essential for MreB's function in cell shape maintenance and demonstrate by in vivo site-specific cross-linking the antiparallel orientation of MreB protofilaments in E. coli. 3D cryo-EM shows that pairs of protofilaments of Caulobacter MreB tightly bind to membranes. Crystal structures of different nucleotide and polymerisation states of Caulobacter MreB reveal conserved conformational changes accompanying antiparallel filament formation. Finally, the antimicrobial agents A22/MP265 are shown to bind close to the bound nucleotide of MreB, presumably preventing nucleotide hydrolysis and destabilising double protofilaments.DOI: http://dx.doi.org/10.7554/eLife.02634.001.

Localized dimerization and nucleoid binding drive gradient formation by the bacterial cell division inhibitor MipZ.

Protein gradients play a central role in the spatial organization of cells, but the mechanisms of their formation are incompletely understood. This study analyzes the determinants responsible for establishing bipolar gradients of the ATPase MipZ, a key regulator of division site placement in Caulobacter crescentus. We have solved the crystal structure of MipZ in different nucleotide states, dissected its ATPase cycle, and investigated its interaction with FtsZ, ParB, and the nucleoid. Our results suggest that the polar ParB complexes locally stimulate the formation of ATP-bound MipZ dimers, which are then retained near the cell poles through association with chromosomal DNA. Due to their intrinsic ATPase activity, dimers eventually dissociate into freely diffusible monomers that undergo spontaneous nucleotide exchange and are recaptured by ParB. These findings clarify the molecular function of a conserved gradient-forming system and reveal mechanistic principles that might be commonly used to sustain protein gradients within cells.

Crystal structures of Bacillus subtilis Lon protease.

Lon ATP-dependent proteases are key components of the protein quality control systems of bacterial cells and eukaryotic organelles. Eubacterial Lon proteases contain an N-terminal domain, an ATPase domain, and a protease domain, all in one polypeptide chain. The N-terminal domain is thought to be involved in substrate recognition, the ATPase domain in substrate unfolding and translocation into the protease chamber, and the protease domain in the hydrolysis of polypeptides into small peptide fragments. Like other AAA+ ATPases and self-compartmentalising proteases, Lon functions as an oligomeric complex, although the subunit stoichiometry is currently unclear. Here, we present crystal structures of truncated versions of Lon protease from Bacillus subtilis (BsLon), which reveal previously unknown architectural features of Lon complexes. Our analytical ultracentrifugation and electron microscopy show different oligomerisation of Lon proteases from two different bacterial species, Aquifex aeolicus and B. subtilis. The structure of BsLon-AP shows a hexameric complex consisting of a small part of the N-terminal domain, the ATPase, and protease domains. The structure shows the approximate arrangement of the three functional domains of Lon. It also reveals a resemblance between the architecture of Lon proteases and the bacterial proteasome-like protease HslUV. Our second structure, BsLon-N, represents the first 209 amino acids of the N-terminal domain of BsLon and consists of a globular domain, similar in structure to the E. coli Lon N-terminal domain, and an additional four-helix bundle, which is part of a predicted coiled-coil region. An unexpected dimeric interaction between BsLon-N monomers reveals the possibility that Lon complexes may be stabilised by coiled-coil interactions between neighbouring N-terminal domains. Together, BsLon-N and BsLon-AP are 36 amino acids short of offering a complete picture of a full-length Lon protease.

Molecular mechanism of sequence-directed DNA loading and translocation by FtsK.

Dimeric circular chromosomes, formed by recombination between monomer sisters, cannot be segregated to daughter cells at cell division. XerCD site-specific recombination at the Escherichia coli dif site converts these dimers to monomers in a reaction that requires the DNA translocase FtsK. Short DNA sequences, KOPS (GGGNAGGG), which are polarized toward dif in the chromosome, direct FtsK translocation. FtsK interacts with KOPS through a C-terminal winged helix domain gamma. The crystal structure of three FtsKgamma domains bound to 8 bp KOPS DNA demonstrates how three gamma domains recognize KOPS. Using covalently linked dimers of FtsK, we infer that three gamma domains per hexamer are sufficient to recognize KOPS and load FtsK and subsequently activate recombination at dif. During translocation, FtsK fails to recognize an inverted KOPS sequence. Therefore, we propose that KOPS act solely as a loading site for FtsK, resulting in a unidirectionally oriented hexameric motor upon DNA.

Bacterial actin: architecture of the ParMRC plasmid DNA partitioning complex.

The R1 plasmid employs ATP-driven polymerisation of the actin-like protein ParM to move newly replicated DNA to opposite poles of a bacterial cell. This process is essential for ensuring accurate segregation of the low-copy number plasmid and is the best characterised example of DNA partitioning in prokaryotes. In vivo, ParM only forms long filaments when capped at both ends by attachment to a centromere-like region parC, through a small DNA-binding protein ParR. Here, we present biochemical and electron microscopy data leading to a model for the mechanism by which ParR-parC complexes bind and stabilise elongating ParM filaments. We propose that the open ring formed by oligomeric ParR dimers with parC DNA wrapped around acts as a rigid clamp, which holds the end of elongating ParM filaments while allowing entry of new ATP-bound monomers. We propose a processive mechanism by which cycles of ATP hydrolysis in polymerising ParM drives movement of ParR-bound parC DNA. Importantly, our model predicts that each pair of plasmids will be driven apart in the cell by just a single double helical ParM filament.

Structural and mutational analysis of the cell division protein FtsQ.

Bacterial cytokinesis requires the divisome, a complex of proteins that co-ordinates the invagination of the cytoplasmic membrane, inward growth of the peptidoglycan layer and the outer membrane. Assembly of the cell division proteins is tightly regulated and the order of appearance at the future division site is well organized. FtsQ is a highly conserved component of the divisome among bacteria that have a cell wall, where it plays a central role in the assembly of early and late cell division proteins. Here, we describe the crystal structure of the major, periplasmic domain of FtsQ from Escherichia coli and Yersinia enterocolitica. The crystal structure reveals two domains; the alpha-domain has a striking similarity to polypeptide transport-associated (POTRA) domains and the C-terminal beta-domain forms an extended beta-sheet overlaid by two, slightly curved alpha-helices. Mutagenesis experiments demonstrate that two functions of FtsQ, localization and recruitment, occur in two separate domains. Proteins that localize FtsQ need the second beta-strand of the POTRA domain and those that are recruited by FtsQ, like FtsL/FtsB, require the surface formed by the tip of the last alpha-helix and the two C-terminal beta-strands. Both domains act together to accomplish the role of FtsQ in linking upstream and downstream cell division proteins within the divisome.