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SARS-CoV-2 is a novel coronavirus responsible for the COVID-19 pandemic. Its high pathogenicity is due to SARS-CoV-2 spike protein (S protein) contacting host-cell receptors. A critical hallmark of COVID-19 is the occurrence of coagulopathies. Here, we report the direct observation of the interactions between S protein and platelets. Live imaging showed that the S protein triggers platelets to deform dynamically, in some cases, leading to their irreversible activation. Strikingly, cellular cryo-electron tomography revealed dense decorations of S protein on the platelet surface, inducing filopodia formation. Hypothesizing that S protein binds to filopodia-inducing integrin receptors, we tested the binding to RGD motif-recognizing platelet integrins and found that S protein recognizes integrin αvβ3. Our results infer that the stochastic activation of platelets is due to weak interactions of S protein with integrin, which can attribute to the pathogenesis of COVID-19 and the occurrence of rare but severe coagulopathies.

Most bacteria replicate and segregate their DNA concomitantly while growing, before cell division takes place. How bacteria synchronize these different cell cycle events to ensure faithful chromosome inheritance by daughter cells is poorly understood. Here, we identify Cell Cycle Regulator protein interacting with FtsZ (CcrZ) as a conserved and essential protein in pneumococci and related Firmicutes such as Bacillus subtilis and Staphylococcus aureus. CcrZ couples cell division with DNA replication by controlling the activity of the master initiator of DNA replication, DnaA. The absence of CcrZ causes mis-timed and reduced initiation of DNA replication, which subsequently results in aberrant cell division. We show that CcrZ from Streptococcus pneumoniae interacts directly with the cytoskeleton protein FtsZ, which places CcrZ in the middle of the newborn cell where the DnaA-bound origin is positioned. This work uncovers a mechanism for control of the bacterial cell cycle in which CcrZ controls DnaA activity to ensure that the chromosome is replicated at the right time during the cell cycle., In this Article, the authors identify CcrZ as a protein that coordinates DNA replication and cell division in Streptococcus pneumoniae and other Firmicutes. CcrZ is localized to the division site by binding directly to the divisome protein FtsZ, and there it activates DnaA, the master initiator of DNA replication, through a still unknown mechanism.

The ParABS system plays an essential role in prokaryotic chromosome segregation. After loading at the parS site on the genome, ParB proteins rapidly redistribute to distances of ~15 kb away from the loading site. It has remained puzzling how this large-distance spreading can occur along DNA that is loaded with hundreds of proteins. Using single-molecule in vitro visualization, we here show that, unexpectedly, ParB can load onto DNA independently and distantly of parS, whereby loaded ParB molecules are themselves able to recruit additional ParB proteins from bulk. Strikingly, this recruitment can occur in-cis but also in-trans whereby, at low tensions within the DNA, newly recruited ParB can bypass roadblocks as it gets loaded to spatially proximal but genomically distant DNA regions. The data are supported by Molecular Dynamics simulations which also show that cooperative ParB-ParB recruitment enhances spreading. ParS-independent recruitment explains how ParB can cover substantial genomic distance during chromosome segregation which is vital for the bacterial cell cycle.

Three-component ParABS systems are widely distributed factors for plasmid partitioning and chromosome segregation in bacteria. ParB protein acts as an adaptor between the 16 bp centromeric parS DNA sequences and the DNA segregation ATPase ParA. It accumulates at high concentrations at and near a parS site by assembling a partition complex. ParB dimers form a DNA sliding clamp whose closure at parS requires CTP binding. The mechanism underlying ParB loading and the role of CTP hydrolysis however remain unclear. We show that CTP hydrolysis is dispensable for Smc recruitment to parS sites in Bacillus subtilis but is essential for chromosome segregation by ParABS in the absence of Smc. Our results suggest that CTP hydrolysis contributes to partition complex assembly via two mechanisms. It recycles off-target ParB clamps to allow for new attempts at parS targeting and it limits the extent of spreading from parS by promoting DNA unloading. We also propose a model for how parS DNA catalyzes ParB clamp closure involving a steric clash between ParB protomers binding to opposing parS half sites.

Eukaryotic cells employ three SMC complexes to control DNA folding and topology. The Smc5/6 complex plays roles in DNA repair and in preventing the accumulation of deleterious DNA junctions. To elucidate how specific features of Smc5/6 govern these functions, we reconstituted the yeast holo-complex. We found that the Nse5/6 sub-complex strongly inhibited the Smc5/6 ATPase by preventing productive ATP binding. This inhibition was relieved by plasmid DNA binding but not by short linear DNA, while opposing effects were observed without Nse5/6. We uncovered two binding sites for Nse5/6 on Smc5/6, based on an Nse5/6 crystal structure and cross-linking mass spectrometry data. One binding site is located at the Smc5/6 arms and one at the heads, the latter likely exerting inhibitory effects on ATP hydrolysis. Cysteine cross-linking demonstrated that the interaction with Nse5/6 anchored the ATPase domains in a non-productive state, which was destabilized by ATP and DNA. Under similar conditions, the Nse4/3/1 module detached from the ATPase. Altogether, we show how DNA substrate selection is modulated by direct inhibition of the Smc5/6 ATPase by Nse5/6.

Multi-subunit SMC ATPases control chromosome superstructure apparently by catalyzing a DNA-loop-extrusion reaction. SMC proteins harbor an ABC-type ATPase "head" and a "hinge" dimerization domain connected by a coiled coil "arm." Two arms in a SMC dimer can co-align, thereby forming a rod-shaped particle. Upon ATP binding, SMC heads engage, and arms are thought to separate. Here, we study the shape of Bacillus subtilis Smc-ScpAB by electron-spin resonance spectroscopy. Arm separation is readily detected proximal to the heads in the absence of ligands, and separation near the hinge largely depends on ATP and DNA. Artificial blockage of arm opening eliminates DNA stimulation of ATP hydrolysis but does not prevent basal ATPase activity. We report an arm contact as being important for controlling the transformations. Point mutations at this arm interface eliminated Smc function. We propose that partially open, intermediary conformations provide directionality to SMC DNA translocation by (un)binding suitable DNA substrates., Cell Reports, 35 (4), ISSN:2666-3864, ISSN:2211-1247

Correct bioriented attachment of sister chromatids to the mitotic spindle is essential for chromosome segregation. In budding yeast, the conserved protein shugoshin (Sgo1) contributes to biorientation by recruiting the protein phosphatase PP2A-Rts1 and the condensin complex to centromeres. Using peptide prints, we identified a Serine-Rich Motif (SRM) of Sgo1 that mediates the interaction with condensin and is essential for centromeric condensin recruitment and the establishment of biorientation. We show that the interaction is regulated via phosphorylation within the SRM and we determined the phospho-sites using mass spectrometry. Analysis of the phosphomimic and phosphoresistant mutants revealed that SRM phosphorylation disrupts the shugoshin–condensin interaction. We present evidence that Mps1, a central kinase in the spindle assembly checkpoint, directly phosphorylates Sgo1 within the SRM to regulate the interaction with condensin and thereby condensin localization to centromeres. Our findings identify novel mechanisms that control shugoshin activity at the centromere in budding yeast., Author summary Proper chromosome segregation in eukaryotes is ensured through correct attachment of the spindle microtubules to the centromeric chromosomal regions. The attachment is mediated via the multimolecular proteinaceous complex called the kinetochore. This enables the establishment of bioirentation, when each sister chromatid is attached to microtubules emanating from opposite spindle poles. Shugoshin (Sgo1) is a conserved centromeric protein that facilitates biorientation through its interactions with the protein phosphatase PP2A-Rts1, chromosome passenger complex and centromeric condensin. Here, we identified a serine-rich motif that is required for the interaction of shugoshin with the condensin complex. We show that loss of this region impairs condensin enrichment at the centromere, chromosome biorientation, segregation as well as the function of the chromosome passenger complex in the error correction. Moreover, the interaction is phosphoregulated, as phosphorylation of the serine-rich motif on Sgo1 disrupts its interaction with condensin. Finally, we show that the conserved spindle assembly checkpoint kinase Mps1 is responsible for this phosphorylation. Our findings uncover novel regulatory mechanisms that facilitate proper chromosome segregation.

The Rad50-Mre11 nuclease complex plays a vital role in DNA repair in all domains of life. It recognizes and processes DNA double-strand breaks. Rad50 proteins fold into an extended structure with a ~20-60 nm long coiled coil connecting a globular ABC ATPase domain with a zinc hook dimerization domain. A published structure of an archaeal Rad50 zinc hook shows coiled coils pointing away from each other. Here we present the crystal structure of an alternate conformation displaying co-aligned coiled coils. Archaeal Rad50 may thus switch between rod-shaped and ring-like conformations as recently proposed for a bacterial homolog.

Most bacteria replicate and segregate their DNA concomitantly while growing, before cell division takes place. How bacteria synchronize these different cell cycle events to ensure faithful chromosome inheritance is poorly understood. Here, we identified a conserved and essential protein in pneumococci and related Firmicutes named CcrZ (for Cell Cycle Regulator protein interacting with FtsZ) that couples cell division with DNA replication by controlling the activity of the master initiator of DNA replication, DnaA. The absence of CcrZ causes mis-timed and reduced initiation of DNA replication, which subsequently results in aberrant cell division. We show that CcrZ from Streptococcus pneumoniae directly interacts with the cytoskeleton protein FtsZ to place it in the middle of the newborn cell where the DnaA-bound origin is positioned. Together, this work uncovers a new mechanism for the control of the bacterial cell cycle in which CcrZ controls DnaA activity to ensure that the chromosome is replicated at the right time during the cell cycle.

Eukaryotic structural maintenance of chromosomes (SMC)-kleisin complexes form large, ring-shaped assemblies that promote accurate chromosome segregation. Their asymmetric structural core comprises SMC heterodimers that associate with both ends of a kleisin subunit. However, prokaryotic condensin Smc-ScpAB is composed of symmetric Smc homodimers associated with the kleisin ScpA in a postulated symmetrical manner. Here, we demonstrate that Smc molecules have two distinct binding sites for ScpA. The N terminus of ScpA binds the Smc coiled coil, whereas the C terminus binds the Smc ATPase domain. We show that in Bacillus subtilis cells, an Smc dimer is bridged by a single ScpAB to generate asymmetric tripartite rings analogous to eukaryotic SMC complexes. We define a molecular mechanism that ensures asymmetric assembly, and we conclude that the basic architecture of SMC-kleisin rings evolved before the emergence of eukaryotes.

Summary SMC condensin complexes are central modulators of chromosome superstructure in all branches of life. Their SMC subunits form a long intramolecular coiled coil, which connects a constitutive “hinge” dimerization domain with an ATP-regulated “head” dimerization module. Here, we address the structural arrangement of the long coiled coils in SMC complexes. We unequivocally show that prokaryotic Smc-ScpAB, eukaryotic condensin, and possibly also cohesin form rod-like structures, with their coiled coils being closely juxtaposed and accurately anchored to the hinge. Upon ATP-induced binding of DNA to the hinge, however, Smc switches to a more open configuration. Our data suggest that a long-distance structural transition is transmitted from the Smc head domains to regulate Smc-ScpAB’s association with DNA. These findings uncover a conserved architectural theme in SMC complexes, provide a mechanistic basis for Smc’s dynamic engagement with chromosomes, and offer a molecular explanation for defects in Cornelia de Lange syndrome., Graphical Abstract, Highlights • Prokaryotic Smc-ScpAB complexes form rod-like structures • Binding of ATP and DNA induces a rod-to-ring transition in prokaryotic condensin • The condensin hinge is rigidly anchored to its coiled coil • The rod-like conformation is a conserved feature of SMC protein dimers, Soh et al. show that the rod-like conformation is a conserved architectural scheme of SMC complexes. Upon ATP-induced binding to DNA, the juxtaposed coiled coils of prokaryotic Smc-ScpAB adopt an open conformation to expose a DNA binding site at the inner surface of the hinge domain.

An outer membrane protein BP26/OMP28 of Brucella, BP26, is identified as a major immunodominant antigen and widely used as a diagnostic marker and for vaccination against Brucellosis. BP26 belongs to the family of proteins that contains a SIMPL (signaling molecule that associates with the mouse pelle-like kinase) domain, whose structure and function have been unknown. Here, we present the crystal structure of BP26 revealing that 16 BP26 molecules form a novel channel-like assembly as also shown by electron microscopy analysis. Eight BP26 molecules forming a ring structure contain a hole at the center of the octamer, and another octamer interacts with each other to form a channel having a large internal cavity. BP26 is found to be structurally similar to a bacteriophage protein involved in infection, implicating that BP26 might function during Brucella infection. In addition, the BP26 structure suggests that the protein functions as a multimeric channel-like form and provides a canonical model for the SIMPL domains.

CTP hydrolysis organizes chromosomes The bacterial DNA parS centromere recruits the ParB protein to the bacterial chromosome. Soh et al. found that the widespread family of ParB proteins not only bind DNA but also bind and hydrolyze cytidine triphosphate (CTP) (see the Perspective by Funnell). ParB CTP hydrolysis is stimulated by parS and regulates the spreading of ParB protein to the parS flanking regions, which is crucial for organizing the bacterial chromosome. The cytidine triphosphatase domain is conserved in a large variety of protein sequences, suggesting its potential roles in other cellular processes. Science , this issue p. 1129 ; see also p. 1072