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Maltodextrin glucosidase (MalZ) is a key enzyme in the maltose utilization pathway in Escherichia coli that liberates glucose from the reducing end of the short malto-oligosaccharides. Unlike other enzymes in the GH13_21 subfamily, the hydrolytic activity of MalZ is limited to maltodextrin rather than long starch substrates, forming various transglycosylation products in α-1,3, α-1,4 or α-1,6 linkages. The mechanism for the substrate binding and hydrolysis of this enzyme is not well understood yet. Here, we present the dimeric crystal structure of MalZ, with the N-domain generating a unique substrate binding groove. The N-domain bears CBM34 architecture and forms a part of the active site in the catalytic domain of the adjacent molecule. The groove found between the N-domain and catalytic domain from the adjacent molecule, shapes active sites suitable for short malto-oligosaccharides, but hinders long stretches of oligosaccharides. The conserved residue of E44 protrudes at subsite +2, elucidating the hydrolysis pattern of the substrate by the glucose unit from the reducing end. The structural analysis provides a molecular basis for the substrate specificity and the enzymatic property, and has potential industrial application for protein engineering.

Adenosine diphosphate (ADP) ribosylation is a reversible posttranslational modification involved in the regulation of numerous cellular processes. Prototype ADP ribosyltransferases (ARTs) from many pathogenic bacteria are known to function as toxins, while other bacterial ARTs have just recently emerged. Recent studies have shown that bacteria also possess enzymes that function as poly-ADP ribose (ADPr) glycohydrolases (PARGs), which reverse poly-ADP ribosylation. However, how bacteria manipulate host target proteins by coordinated reactions of ARTs and ADPr hydrolases (ARHs) remains elusive. The intracellular bacterial pathogen Legionella pneumophila, the causative agent of Legionnaires’ disease, transports a large array of effector proteins via the Dot/Icm type IV secretion system to host cells. The effector proteins, which mostly function as enzymes, modulate host cellular processes for the bacteria’s benefit. In this study, we identified a pair of L. pneumophila effector proteins, Lpg0080 and Lpg0081, which function as an ART and an ARH, respectively. The two proteins were shown to coordinately modulate mitochondrial ADP/adenosine triphosphate (ATP) translocases (ANTs) by their enzymatic activities to conjugate ADPr to, and remove it from, a key arginine residue. The crystal structures of Lpg0081 and the Lpg0081:ADPr complex indicated that Lpg0081 is a macroD-type ARH with a noncanonical macrodomain, whose folding topology is strikingly distinct from that of the canonical macrodomain that is ubiquitously found in eukaryotic PARGs and ARHs. Our results illustrate that L. pneumophila has acquired an effector pair that coordinately manipulate mitochondrial activity via reversible chemical modification of ANTs.

The Legionella pneumophila Dot/Icm type IVB secretion system (T4BSS) is extremely versatile, translocating ~300 effector proteins into host cells. This specialized secretion system employs the Dot/Icm type IVB coupling protein (T4CP) complex, which includes IcmS, IcmW and LvgA, that are known to selectively assist the export of a subclass of effectors. Herein, the crystal structure of a four-subunit T4CP subcomplex bound to the effector protein VpdB reveals an interaction between LvgA and a linear motif in the C-terminus of VpdB. The same binding interface of LvgA also interacts with the C-terminal region of three additional effectors, SidH, SetA and PieA. Mutational analyses identified a FxxxLxxxK binding motif that is shared by VpdB and SidH, but not by SetA and PieA, showing that LvgA recognizes more than one type of binding motif. Together, this work provides a structural basis for how the Dot/Icm T4CP complex recognizes effectors, and highlights the multiple substrate-binding specificities of its adaptor subunit., The Legionella pneumophila Dot/Icm type IVB secretion system (T4BSS) translocates effector proteins into host cells, and the recognition of these effectors is mediated by the Dot/Icm type IV coupling protein (T4CP) complex. Here, the authors present the crystal structure of a four-subunit containing T4CP subcomplex bound to the effector protein VpdB, and identify a FxxxLxxxK binding motif that is present in a subset of the effectors and which is recognized by the T4CP adaptor subunit LvgA.

A marine phytoplankton dinoflagellate, Alexandrium sp. is known to cause worldwide harmful algal blooms, resulting in paralytic shellfish poisoning. In this study, we isolated a novel compound secreted by the marine bacterium Pseudoruegeria sp. M32A2M, and showed that it displays algicidal activity against A. catenella (group I). The molecular structure of the compound was analyzed by using

The marine dinoflagellate Alexandrium is associated with harmful algal blooms (HABs) worldwide, causing paralytic shellfish poisoning (PSP) in humans. We found that the marine bacterium Pseudoruegeria sp. M32A2M exhibits algicidal activity against Alexandrium catenella (Group I), inhibiting its motility and consequently inducing cell disruption after 24 h of co-culture. To understand the communication between the two organisms, we investigated the time-course cellular responses through genome-wide transcriptome analysis. Functional analysis of differentially expressed genes revealed that the core reactions of the photosystem in A. catenella were inhibited within 2 h, eventually downregulating the entire pathways of oxidative phosphorylation and carbon fixation, as well as associated metabolic pathways. Conversely, Pseudoruegeria upregulated its glycolysis, tricarboxylic acid cycle, and oxidative phosphorylation pathways. Also, the transporters for nutrients such as C3/C4 carbohydrates and peptides were highly upregulated, leading to the speculation that nutrients released by disrupted A. catenella cells affect the central metabolism of Pseudoruegeria. In addition, we analyzed the secondary metabolite-synthesizing clusters of Pseudoruegeria that were upregulated by co-culture, suggesting their potential roles in algicidal activity. Our time-course transcriptome analysis elucidates how A. catenella is affected by algicidal bacteria and how these bacteria obtain functional benefits through metabolic pathways.

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.

Uracil DNA glycosylases (UDGs) are important DNA repair enzymes that excise uracil from DNA, yielding an abasic site. Recently, UdgX, an unconventional UDG with extremely tight binding to DNA containing uracil, was discovered. The structure of UdgX from Mycobacterium smegmatis in complex with DNA shows an overall similarity to that of family 4 UDGs except for a protruding loop at the entrance of the uracil-binding pocket. Surprisingly, H109 in the loop was found to make a covalent bond to the abasic site to form a stable intermediate, while the excised uracil remained in the pocket of the active site. H109 functions as a nucleophile to attack the oxocarbenium ion, substituting for the catalytic water molecule found in other UDGs. To our knowledge, this change from a catalytic water attack to a direct nucleophilic attack by the histidine residue is unprecedented. UdgX utilizes a unique mechanism of protecting cytotoxic abasic sites from exposure to the cellular environment.

Naturally occurring protein switches have been repurposed for the development of biosensors and reporters for cellular and clinical applications1. However, the number of such switches is limited, and reengineering them is challenging. Here we show that a general class of protein-based biosensors can be created by inverting the flow of information through de novo designed protein switches in which the binding of a peptide key triggers biological outputs of interest2. The designed sensors are modular molecular devices with a closed dark state and an open luminescent state; analyte binding drives the switch from the closed to the open state. Because the sensor is based on the thermodynamic coupling of analyte binding to sensor activation, only one target binding domain is required, which simplifies sensor design and allows direct readout in solution. We create biosensors that can sensitively detect the anti-apoptosis protein BCL-2, the IgG1 Fc domain, the HER2 receptor, and Botulinum neurotoxin B, as well as biosensors for cardiac troponin I and an anti-hepatitis B virus antibody with the high sensitivity required to detect these molecules clinically. Given the need for diagnostic tools to track the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)3, we used the approach to design sensors for the SARS-CoV-2 spike protein and antibodies against the membrane and nucleocapsid proteins. The former, which incorporates a de novo designed spike receptor binding domain (RBD) binder4, has a limit of detection of 15 pM and a luminescence signal 50-fold higher than the background level. The modularity and sensitivity of the platform should enable the rapid construction of sensors for a wide range of analytes, and highlights the power of de novo protein design to create multi-state protein systems with new and useful functions. A modular de novo designed biosensor platform consisting of a cage and key molecule is developed, and used to create sensors for seven distinct proteins including the spike protein from SARS-CoV-2 and anti-SARS antibodies.

Contrary to the general concept, the archaeal Smc-based complex appears to lack the kite subunit ScpB that is essential in the bacterial Smc–ScpAB complex that mediates chromosome organization., SMC complexes play a central role in chromosome organization in all domains of life. The bacterial Smc–ScpAB complex is a three-subunit complex composed of Smc, ScpA and ScpB. ScpA bridges the two ATPase domains of the Smc homodimer, while ScpB, which belongs to the kite family of proteins, interacts with ScpA. The three subunits are known to be equally important for the function of Smc–ScpAB in bacteria. From crystallographic and biochemical studies, evidence is provided that six archaeal ScpA proteins are unable to interact with the only putative ScpB found in these species. Structure-based sequence alignment reveals that these archaeal ScpAs lack the ScpB-binding segment that is commonly present in the middle of bacterial ScpA sequences, which is thus responsible for their inability to interact with ScpB. ScpA proteins lacking the ScpB-binding segment are found to prevail in archaea. Moreover, two archaeal ScpA proteins with a longer middle region also failed to bind their putative ScpB partner. Furthermore, all or most species belonging to five out of 14 euryarchaeotal orders contain Smc and ScpA but not a detectable ScpB homologue. These data support the notion that archaeal Smc-based complexes generally function as a two-subunit complex composed of only Smc and ScpA.

Multifunctional autoprocessing repeats-in-toxin (MARTX) toxins are secreted by Gram-negative bacteria and function as primary virulence-promoting macromolecules that deliver multiple cytopathic and cytotoxic effector domains into the host cytoplasm. Among these effectors, Ras/Rap1-specific endopeptidase (RRSP) catalyzes the sequence-specific cleavage of the Switch I region of the cellular substrates Ras and Rap1 that are crucial for host innate immune defenses during infection. To dissect the molecular basis underpinning RRSP-mediated substrate inactivation, we determined the crystal structure of an RRSP from the sepsis-causing bacterial pathogen Vibrio vulnificus (VvRRSP). Structural and biochemical analyses revealed that VvRRSP is a metal-independent TIKI family endopeptidase composed of an N-terminal membrane-localization and substrate-recruitment domain (N lobe) connected via an inter-lobe linker to the C-terminal active site–coordinating core β-sheet–containing domain (C lobe). Structure-based mutagenesis identified the 2His/2Glu catalytic residues in the core catalytic domain that are shared with other TIKI family enzymes and that are essential for Ras processing. In vitro KRas cleavage assays disclosed that deleting the N lobe in VvRRSP causes complete loss of enzymatic activity. Endogenous Ras cleavage assays combined with confocal microscopy analysis of HEK293T cells indicated that the N lobe functions both in membrane localization via the first α-helix and in substrate assimilation by altering the functional conformation of the C lobe to facilitate recruitment of cellular substrates. Collectively, these results indicate that RRSP is a critical virulence factor that robustly inactivates Ras and Rap1 and augments the pathogenicity of invading bacteria via the combined effects of its N and C lobes.

Protein tyrosine phosphatases (PTPs) originating from eukaryotes or bacteria have been under intensive structural and biochemical investigation, whereas archaeal PTP proteins have not been investigated extensively; therefore, they are poorly understood. Here, we present the crystal structures of Tk-PTP derived from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1, in both the active and inactive forms. Tk-PTP adopts a common dual-specificity phosphatase (DUSP) fold, but it undergoes an atypical temperature-dependent conformational change in its P-loop and α4−α5 loop regions, switching between the inactive and active forms. Through comprehensive analyses of Tk-PTP, including additional structural determination of the G95A mutant form, enzymatic activity assays, and structural comparison with the other archaeal PTP, it was revealed that the presence of the GG motif in the P-loop is necessary but not sufficient for the structural flexibility of Tk-PTP. It was also proven that Tk-PTP contains dual general acid/base residues unlike most of the other DUSP proteins, and that both the residues are critical in its phosphatase activity. This work provides the basis for expanding our understanding of the previously uncharacterized PTP proteins from archaea, the third domain of living organisms.

SummaryThe CRISPR-Cas system is the RNA-guided immune defense mechanism in bacteria and archaea. Csm1 belongs to the Cas10 family, which is the common signature protein of the type III system. Csm1 is the largest subunit of the Csm interference complex in the type III-A subtype, which targets foreign DNA or RNA. Here, we report crystallographic and biochemical analyses of Thermococcus onnurineus Csm1, revealing a five-domain organization and single-stranded DNA (ssDNA)-specific nuclease activity associated with the N-terminal HD domain. This domain folds into permuted secondary structures in comparison with the HD domain of Cas3 and contains all the catalytically important residues. It exhibited both endo- and exonuclease activities in an Ni2+ or Mn2+-dependent manner. The narrow width of the active-site cleft appears to restrict the substrate specificity to ssDNA and thus to prevent Csm1 from cleaving double-stranded chromosomal DNA. These data suggest that Csm1 may function in DNA interference by the Csm effector complex.

In meiotic DNA recombination, the Hop2−Mnd1 complex promotes Dmc1-mediated single-stranded DNA (ssDNA) invasion into homologous chromosomes to form a synaptic complex by a yet-unclear mechanism. Here, the crystal structure of Hop2−Mnd1 reveals that it forms a curved rod-like structure consisting of three leucine zippers and two kinked junctions. One end of the rod is linked to two juxtaposed winged-helix domains, and the other end is capped by extra α-helices to form a helical bundle-like structure. Deletion analysis shows that the helical bundle-like structure is sufficient for interacting with the Dmc1-ssDNA nucleofilament, and molecular modeling suggests that the curved rod could be accommodated into the helical groove of the nucleofilament. Remarkably, the winged-helix domains are juxtaposed at fixed relative orientation, and their binding to DNA is likely to perturb the base pairing according to molecular simulations. These findings allow us to propose a model explaining how Hop2−Mnd1 juxtaposes Dmc1-bound ssDNA with distorted recipient double-stranded DNA and thus facilitates strand invasion.

Polo-like kinase 4 (Plk4) is a key regulator of centriole duplication, an event critical for the maintenance of genomic integrity. Here we showed that Plk4 relocalizes from the inner Cep192 ring to the outer Cep152 ring as newly recruited Cep152 assembles around the Cep192-encircled daughter centriole. Crystal structure analyses revealed that Cep192 - and Cep152-derived peptides bind the cryptic polo box (CPB) of Plk4 in opposite orientations and in a mutually exclusive manner. The Cep152-peptide bound to the CPB markedly better than the Cep192-peptide and effectively snatched the CPB away from a preformed CPB–Cep192-peptide complex. A cancer-associated Cep152 mutation impairing the Plk4 interaction induced defects in procentriole assembly and chromosome segregation. Thus, Plk4 is intricately regulated in time and space through ordered interactions with two distinct scaffolds, Cep192 and Cep152, and a failure in this process may lead to human cancer.

SummaryRobust immune responses are essential for eliminating pathogens but must be metered to avoid prolonged immune activation and potential host damage. Upon recognition of microbial DNA, the cytosolic DNA sensor cyclic GMP-AMP (cGAMP) synthetase (cGAS) produces the second messenger cGAMP to initiate the stimulator of interferon genes (STING) pathway and subsequent interferon (IFN) production. We report that the direct interaction between cGAS and the Beclin-1 autophagy protein not only suppresses cGAMP synthesis to halt IFN production upon double-stranded DNA (dsDNA) stimulation or herpes simplex virus-1 infection, but also enhances autophagy-mediated degradation of cytosolic pathogen DNA to prevent excessive cGAS activation and persistent immune stimulation. Specifically, this interaction releases Rubicon, a negative autophagy regulator, from the Beclin-1 complex, activating phosphatidylinositol 3-kinase class III activity and thereby inducing autophagy to remove cytosolic pathogen DNA. Thus, the cGAS-Beclin-1 interaction shapes innate immune responses by regulating both cGAMP production and autophagy, resulting in well-balanced antimicrobial immune responses.

Human papillomaviruses (HPVs) are causative agents of various diseases associated with cellular hyperproliferation, including cervical cancer, one of the most prevalent tumors in women. E7 is one of the two HPV-encoded oncoproteins and directs recruitment and subsequent degradation of tumor-suppressive proteins such as retinoblastoma protein (pRb) via its LxCxE motif. E7 also triggers tumorigenesis in a pRb-independent pathway through its C-terminal domain, which has yet been largely undetermined, with a lack of structural information in a complex form with a host protein. Herein, we present the crystal structure of the E7 C-terminal domain of HPV18 belonging to the high-risk HPV genotypes bound to the catalytic domain of human nonreceptor-type protein tyrosine phosphatase 14 (PTPN14). They interact directly and potently with each other, with a dissociation constant of 18.2 nM. Ensuing structural analysis revealed the molecular basis of the PTPN14-binding specificity of E7 over other protein tyrosine phosphatases and also led to the identification of PTPN21 as a direct interacting partner of E7. Disruption of HPV18 E7 binding to PTPN14 by structure-based mutagenesis impaired E7’s ability to promote keratinocyte proliferation and migration. Likewise, E7 binding-defective PTPN14 was resistant for degradation via proteasome, and it was much more effective than wild-type PTPN14 in attenuating the activity of downstream effectors of Hippo signaling and negatively regulating cell proliferation, migration, and invasion when examined in HPV18-positive HeLa cells. These results therefore demonstrated the significance and therapeutic potential of the intermolecular interaction between HPV E7 and host PTPN14 in HPV-mediated cell transformation and tumorigenesis., Human papillomaviruses cause various diseases associated with cellular hyperproliferation, including cervical cancer. Structural, biochemical, and cellular analyses reveal the molecular basis and significance of the intermolecular interaction between the E7 protein of human papillomavirus 18 and the human tumor suppressor protein PTPN14.

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.

The CRISPR-Cas system is an adaptive and heritable immune response that destroys invading foreign nucleic acids. The effector complex of the Type III CRISPR-Cas system targets RNA and DNA in a transcription-coupled manner, but the exact mechanism of DNA targeting by this complex remains elusive. In this study, an effector Csm holocomplex derived from Thermococcus onnurineus is reconstituted with a minimalistic combination of Csm1121334151, and shows RNA targeting and RNA-activated single-stranded DNA (ssDNA) targeting activities. Unexpectedly, in the absence of an RNA transcript, it cleaves ssDNA containing a sequence complementary to the bound crRNA guide region in a manner dependent on the HD domain of the Csm1 subunit. This nuclease activity is blocked by a repeat tag found in the host CRISPR loci. The specific cleavage of ssDNA without a target RNA suggests a novel ssDNA targeting mechanism of the Type III system, which could facilitate the efficient and complete degradation of foreign nucleic acids.

A hypothetical protein TON_0340 of a Thermococcus species is a protein conserved in a variety of organisms including human. Herein, we present four different crystal structures of TON_0340, leading to the identification of an active-site cavity harboring a metal-binding site composed of six invariant aspartate and glutamate residues that coordinate one to three metal ions. Biochemical and mutational analyses involving many phosphorous compounds show that TON_0340 is a Mn2+-dependent phosphatase. Mg2+ binds to TON_0340 less tightly and activates the phosphatase activity less efficiently than Mn2+. Whereas Ca2+ and Zn2+ are able to bind to the protein, they are unable to activate its enzymatic activity. Since the active-site cavity is small and largely composed of nearly invariant stretches of 11 or 13 amino acids, the physiological substrates of TON_0340 and its homologues are likely to be a small and the same molecule. The Mn2+-bound TON_0340 structure provides a canonical model for the ubiquitously present TON_0340 homologues and lays a strong foundation for the elucidation of their substrate and biological function.

Protrudin is a FYVE (Fab 1, YOTB, Vac 1, and EEA1) domain-containing protein involved in transport of neuronal cargoes and implicated in the onset of hereditary spastic paraplegia. Our image-based screening of the lipid binding domain library revealed novel plasma membrane localization of the FYVE domain of protrudin unlike canonical FYVE domains that are localized to early endosomes. The membrane binding study by surface plasmon resonance analysis showed that this FYVE domain preferentially binds phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)), phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P(2)), and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) unlike canonical FYVE domains that specifically bind phosphatidylinositol 3-phosphate (PtdIns(3)P). Furthermore, we found that these phosphoinositides (PtdInsP) differentially regulate shuttling of protrudin between endosomes and plasma membrane via its FYVE domain. Protrudin mutants with reduced PtdInsP-binding affinity failed to promote neurite outgrowth in primary cultured hippocampal neurons. These results suggest that novel PtdInsP selectivity of the protrudin-FYVE domain is critical for its cellular localization and its role in neurite outgrowth.

The surface of proteins can be charged with zinc ions and the anomalous signals from these zinc ions can be used for structure determination of proteins., Zinc is a suitable metal for anomalous dispersion phasing methods in protein crystallography. Structure determination using zinc anomalous scattering has been almost exclusively limited to proteins with intrinsically bound zinc(s). Here, it is reported that multiple zinc ions can easily be charged onto the surface of proteins with no intrinsic zinc-binding site by using zinc-containing solutions. Zn derivatization of protein surfaces appears to be a largely unnoticed but promising method of protein structure determination.

Staphylothermus marinus maltogenic amylase (SMMA) is a novel extreme thermophile maltogenic amylase with an optimal temperature of 100 °C, which hydrolyzes α-(1-4)-glycosyl linkages in cyclodextrins and in linear malto-oligosaccharides. This enzyme has a long N-terminal extension that is conserved among archaic hyperthermophilic amylases but is not found in other hydrolyzing enzymes from the glycoside hydrolase 13 family. The SMMA crystal structure revealed that the N-terminal extension forms an N' domain that is similar to carbohydrate-binding module 48, with the strand-loop-strand region forming a part of the substrate binding pocket with several aromatic residues, including Phe-95, Phe-96, and Tyr-99. A structural comparison with conventional cyclodextrin-hydrolyzing enzymes revealed a striking resemblance between the SMMA N' domain position and the dimeric N domain position in bacterial enzymes. This result suggests that extremophilic archaea that live at high temperatures may have adopted a novel domain arrangement that combines all of the substrate binding components within a monomeric subunit. The SMMA structure provides a molecular basis for the functional properties that are unique to hyperthermophile maltogenic amylases from archaea and that distinguish SMMA from moderate thermophilic or mesophilic bacterial enzymes.

GDP-bound prenylated Rabs, sequestered by GDI (GDP dissociation inhibitor) in the cytosol, are delivered to destined sub-cellular compartment and subsequently activated by GEFs (guanine nucleotide exchange factors) catalysing GDP-to-GTP exchange. The dissociation of GDI from Rabs is believed to require a GDF (GDI displacement factor). Only two RabGDFs, human PRA-1 and Legionella pneumophila SidM/DrrA, have been identified so far and the molecular mechanism of GDF is elusive. Here, we present the structure of a SidM/DrrA fragment possessing dual GEF and GDF activity in complex with Rab1. SidM/DrrA reconfigures the Switch regions of the GTPase domain of Rab1, as eukaryotic GEFs do toward cognate Rabs. Structure-based mutational analyses show that the surface of SidM/DrrA, catalysing nucleotide exchange, is involved in GDI1 displacement from prenylated Rab1:GDP. In comparison with an eukaryotic GEF TRAPP I, this bacterial GEF/GDF exhibits high binding affinity for Rab1 with GDP retained at the active site, which appears as the key feature for the GDF activity of the protein.

Condensin complexes are the key mediators of chromosome condensation. The MukB–MukE–MukF complex is a bacterial condensin, in which the MukB subunit forms a V-shaped dimeric structure with two ATPase head domains. MukE and MukF together form a tight complex, which binds to the MukB head via the C-terminal winged-helix domain (C-WHD) of MukF. One of the two bound C-WHDs of MukF is forced to detach from two ATP-bound, engaged MukB heads, and this detachment reaction depends on the MukF flexible linker preceding the C-WHD. Whereas MukB is known to focally localize at particular positions in cells by an unknown mechanism, mukE- or mukF-null mutation causes MukB to become dispersed in cells. Here, we report that mutations in MukF causing a defect in the detachment reaction interfere with the focal localization of MukB, and that the dispersed distribution of MukB in cells correlates directly with defects in cell growth and division. The data strongly suggest that the MukB–MukE–MukF condensin forms huge clusters through the ATP-dependent detachment reaction, and this cluster formation is critical for chromosome condensation by this machinery. We also show that the MukF flexible linker is involved in the dimerization and ATPase activity of the MukB head. Structured digital abstract • MINT-7216106: mukBhd (uniprotkb:P22523), mukF (uniprotkb:P60293) and mukE (uniprotkb:P22524) physically interact (MI:0915) by blue native page (MI:0276)

To elucidate the relationship between the substrate size and geometric shape of the catalytic site of Thermus maltogenic amylase, Gly50, Asp109, and Val431, located at the interface of the dimer, were replaced with bulky amino acids. The k(cat)/K(m) value of the mutant for amylose increased significantly, whereas that for amylopectin decreased as compared to that of the wild-type enzyme. Thus, the substituted bulky amino acid residues modified the shape of the catalytic site, such that the ability of the enzyme to distinguish between small and large molecules like amylose and amylopectin was enhanced.

Recognition of lysine-type peptidoglycan by peptidoglycan recognition protein (PGRP)-SA provokes the activation of the Toll and prophenoloxidase pathways. Here we reveal that a soluble fragment of lysine-type peptidoglycan, a long glycan chain with short stem peptides, is a potent activator of the Drosophila Toll pathway and the prophenoloxidase activation cascade in the beetle Tenebrio molitor . Using this peptidoglycan fragment, we present biochemical evidence that clustering of PGRP-SA molecules on the peptidoglycan is required for the activation of the prophenoloxidase cascade. We subsequently highlight that the lysozyme-mediated partial digestion of highly cross-linked lysine-type peptidoglycan dramatically increases the binding of PGRP-SA, presumably by inducing clustering of PGRP-SA, which then recruits the Gram-negative bacteria-binding protein 1 homologue and a modular serine protease containing low-density lipoprotein and complement control protein domains. The crucial role of lysozyme in the prophenoloxidase activation cascade is further confirmed in vivo by using a lysozyme inhibitor. Taken together, we propose a model whereby lysozyme presents a processed form of lysine-type peptidoglycan for clustering of PGRP-SA that recruits Gram-negative bacteria-binding protein 1 and the modular serine protease, which leads to the activation of both the Toll and prophenoloxidase pathways.

Ultraviolet (UV)-induced DNA damage, a major risk factor for skin cancers, is primarily repaired by nucleotide excision repair (NER). UV-radiation Resistance Associated Gene (UVRAG) is a tumor suppressor involved in autophagy. It was initially isolated as a cDNA partially complementing UV sensitivity in Xeroderma Pigmentosum (XP), but this was not explored further. Here we show that UVRAG plays an integral role in UV-induced DNA damage repair. It localizes to photolesions and associates with DDB1 to promote the assembly and activity of the DDB2-DDB1-Cul4A–Roc1 (CRL4DDB2) ubiquitin-ligase complex, leading to efficient XPC recruitment and global-genomic NER. UVRAG depletion decreased substrate handover to XPC and conferred UV-damage hypersensitivity. We confirmed the importance of UVRAG for UV-damage tolerance using a Drosophila model. Furthermore, increased UV-signature mutations in melanoma correlate with reduced expression of UVRAG. Our results identify UVRAG as a regulator of CRL4DDB2-mediated NER and suggest that its expression levels may influence melanoma predisposition.

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.

Kaposi's sarcoma-associated herpesvirus (KSHV) evades host defenses through tight suppression of autophagy by targeting each step of its signal transduction: by viral Bcl-2 (vBcl-2) in vesicle nucleation, by viral FLIP (vFLIP) in vesicle elongation, and by K7 in vesicle maturation. By exploring the roles of KSHV autophagy-modulating genes, we found, surprisingly, that vBcl-2 is essential for KSHV lytic replication, whereas vFLIP and K7 are dispensable. Knocking out vBcl-2 from the KSHV genome resulted in decreased lytic gene expression at the mRNA and protein levels, a lower viral DNA copy number, and, consequently, a dramatic reduction in the amount of progeny infectious viruses, as also described in the accompanying article ( A. Gelgor, I. Kalt, S. Bergson, K. F. Brulois, J. U. Jung, and R. Sarid , J Virol 89:5298–5307, 2015). More importantly, the antiapoptotic and antiautophagic functions of vBcl-2 were not required for KSHV lytic replication. Using a comprehensive mutagenesis analysis, we identified that glutamic acid 14 (E 14 ) of vBcl-2 is critical for KSHV lytic replication. Mutating E 14 to alanine totally blocked KSHV lytic replication but showed little or no effect on the antiapoptotic and antiautophagic functions of vBcl-2. Our study indicates that vBcl-2 harbors at least three important and genetically separable functions to modulate both cellular signaling and the virus life cycle. IMPORTANCE The present study shows for the first time that vBcl-2 is essential for KSHV lytic replication. Removal of the vBcl-2 gene results in a lower level of KSHV lytic gene expression, impaired viral DNA replication, and consequently, a dramatic reduction in the level of progeny production. More importantly, the role of vBcl-2 in KSHV lytic replication is genetically separated from its antiapoptotic and antiautophagic functions, suggesting that the KSHV Bcl-2 carries a novel function in viral lytic replication.