Structural basis for plasmid restriction by SMC JET nuclease.

DNA loop-extruding SMC complexes play crucial roles in chromosome folding and DNA immunity. Prokaryotic SMC Wadjet (JET) complexes limit the spread of plasmids through DNA cleavage, yet the mechanisms for plasmid recognition are unresolved. We show that artificial DNA circularization renders linear DNA susceptible to JET nuclease cleavage. Unlike free DNA, JET cleaves immobilized plasmid DNA at a specific site, the plasmid-anchoring point, showing that the anchor hinders DNA extrusion but not DNA cleavage. Structures of plasmid-bound JetABC reveal two presumably stalled SMC motor units that are drastically rearranged from the resting state, together entrapping a U-shaped DNA segment, which is further converted to kinked V-shaped cleavage substrate by JetD nuclease binding. Our findings uncover mechanical bending of residual unextruded DNA as molecular signature for plasmid recognition and non-self DNA elimination. We moreover elucidate key elements of SMC loop extrusion, including the motor direction and the structure of a DNA-holding state.

Proteasome-mediated processing of Def1, a critical step in the cellular response to transcription stress.

DNA damage triggers polyubiquitylation and degradation of the largest subunit of RNA polymerase II (RNAPII), a "mechanism of last resort" employed during transcription stress. In yeast, this process is dependent on Def1 through a previously unresolved mechanism. Here, we report that Def1 becomes activated through ubiquitylation- and proteasome-dependent processing. Def1 processing results in the removal of a domain promoting cytoplasmic localization, resulting in nuclear accumulation of the clipped protein. Nuclear Def1 then binds RNAPII, utilizing a ubiquitin-binding domain to recruit the Elongin-Cullin E3 ligase complex via a ubiquitin-homology domain in the Ela1 protein. This facilitates polyubiquitylation of Rpb1, triggering its proteasome-mediated degradation. Together, these results outline the multistep mechanism of Rpb1 polyubiquitylation triggered by transcription stress and uncover the key role played by Def1 as a facilitator of Elongin-Cullin ubiquitin ligase function.

Biochemically validated structural model of the 15-subunit intraflagellar transport complex IFT-B.

Cilia are ubiquitous eukaryotic organelles impotant for cellular motility, signaling, and sensory reception. Cilium formation requires intraflagellar transport of structural and signaling components and involves 22 different proteins organized into intraflagellar transport (IFT) complexes IFT-A and IFT-B that are transported by molecular motors. The IFT-B complex constitutes the backbone of polymeric IFT trains carrying cargo between the cilium and the cell body. Currently, high-resolution structures are only available for smaller IFT-B subcomplexes leaving > 50% structurally uncharacterized. Here, we used Alphafold to structurally model the 15-subunit IFT-B complex. The model was validated using cross-linking/mass-spectrometry data on reconstituted IFT-B complexes, X-ray scattering in solution, diffraction from crystals as well as site-directed mutagenesis and protein-binding assays. The IFT-B structure reveals an elongated and highly flexible complex consistent with cryo-electron tomographic reconstructions of IFT trains. The IFT-B complex organizes into IFT-B1 and IFT-B2 parts with binding sites for ciliary cargo and the inactive IFT dynein motor, respectively. Interestingly, our results are consistent with two different binding sites for IFT81/74 on IFT88/70/52/46 suggesting the possibility of different structural architectures for the IFT-B1 complex. Our data present a structural framework to understand IFT-B complex assembly, function, and ciliopathy variants.

Nse5/6 inhibits the Smc5/6 ATPase and modulates DNA substrate binding.

Eukaryotic cells employ three SMC (structural maintenance of chromosomes) 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.

IFT proteins interact with HSET to promote supernumerary centrosome clustering in mitosis.

Centrosome amplification is a hallmark of cancer, and centrosome clustering is essential for cancer cell survival. The mitotic kinesin HSET is an essential contributor to this process. Recent studies have highlighted novel functions for intraflagellar transport (IFT) proteins in regulating motors and mitotic processes. Here, using siRNA knock-down of various IFT proteins or AID-inducible degradation of endogenous IFT88 in combination with small-molecule inhibition of HSET, we show that IFT proteins together with HSET are required for efficient centrosome clustering. We identify a direct interaction between the kinesin HSET and IFT proteins, and we define how IFT proteins contribute to clustering dynamics during mitosis using high-resolution live imaging of centrosomes. Finally, we demonstrate the requirement of IFT88 for efficient centrosome clustering in a variety of cancer cell lines naturally harboring supernumerary centrosomes and its importance for cancer cell proliferation. Overall, our data unravel a novel role for the IFT machinery in centrosome clustering during mitosis in cells harboring supernumerary centrosomes.

Intraflagellar transport proteins 172, 80, 57, 54, 38, and 20 form a stable tubulin-binding IFT-B2 complex.

Intraflagellar transport (IFT) relies on the IFT complex and is required for ciliogenesis. The IFT-B complex consists of 9-10 stably associated core subunits and six "peripheral" subunits that were shown to dissociate from the core structure at moderate salt concentration. We purified the six "peripheral"IFT-B subunits of Chlamydomonas reinhardtiias recombinant proteins and show that they form a stable complex independently of the IFT-B core. We suggest a nomenclature of IFT-B1 (core) and IFT-B2 (peripheral) for the two IFT-B subcomplexes. We demonstrate that IFT88, together with the N-terminal domain of IFT52, is necessary to bridge the interaction between IFT-B1 and B2. The crystal structure of IFT52N reveals highly conserved residues critical for IFT-B1/IFT-B2 complex formation. Furthermore, we show that of the three IFT-B2 subunits containing a calponin homology (CH) domain (IFT38, 54, and 57), only IFT54 binds αß-tubulin as a potential IFT cargo, whereas the CH domains of IFT38 and IFT57 mediate the interaction with IFT80 and IFT172, respectively. Crystal structures of IFT54 CH domains reveal that tubulin binding is mediated by basic surface-exposed residues.

Intraflagellar transport protein IFT52 recruits IFT46 to the basal body and flagella.

Cilia are microtubule-based organelles and perform motile, sensing and signaling functions. The assembly and maintenance of cilia depend on intraflagellar transport (IFT). Besides ciliary localization, most IFT proteins accumulate at basal bodies. However, little is known about the molecular mechanism of basal body targeting of IFT proteins. We first identified the possible basal body-targeting sequence in IFT46 by expressing IFT46 truncation constructs in an ift46-1 mutant. The C-terminal sequence between residues 246-321, termed BBTS3, was sufficient to target YFP to basal bodies in the ift46-1 strain. Interestingly, BBTS3 is also responsible for the ciliary targeting of IFT46. BBTS3::YFP moves bidirectionally in flagella and interacts with other IFT complex B (IFT-B) proteins. Using IFT and motor mutants, we show that the basal body localization of IFT46 depends on IFT52, but not on IFT81, IFT88, IFT122, FLA10 or DHC1b. IFT52 interacts with IFT46 through residues L285 and L286 of IFT46 and recruits it to basal bodies. Ectopic expression of the C-terminal domain of IFT52 in the nucleus resulted in accumulation of IFT46 in nuclei. These data suggest that IFT52 and IFT46 can preassemble as a complex in the cytoplasm, which is then targeted to basal bodies.

Synthesis, Characterization, and Copper(II) Chelates of 1,11-Dithia-4,8-diazacyclotetradecane.

Synthesis of 1,11-dithia-4,8-diazacyclotetradecane (L1), a constitutional isomer of the macrocyclic [14]aneN2S2 series, is accompanied with reaction and method optimization. Chelation of L1 with copper(II) provided assessment of lattice packing, ring contortion, and evidence of conformational fluxionality in solution through two unique crystal structures: L1Cu(ClO4)2 and [(L1Cu)2µ-Cl](ClO4)3. Multiple synthetic approaches are presented, supplemented with reaction methodology and reagent screening to access [14]aneN2S2 L1. Reductive alkylation of bis-tosyl-cystamine was integrated into the synthetic route, eliminating the use and isolation of volatile thiols and streamlining the synthetic scale-up. Late-stage cleavage of protecting sulfonamides was addressed using reductive N-S cleavage to furnish macrocyclic freebase L1.

Structural basis of outer dynein arm intraflagellar transport by the transport adaptor protein ODA16 and the intraflagellar transport protein IFT46.

Motile cilia are found on unicellular organisms such as the green alga Chlamydomonas reinhardtii, on sperm cells, and on cells that line the trachea and fallopian tubes in mammals. The motility of cilia relies on a number of large protein complexes including the force-generating outer dynein arms (ODAs). The transport of ODAs into cilia has been previously shown to require the transport adaptor ODA16, as well as the intraflagellar transport (IFT) protein IFT46, but the molecular mechanism by which ODAs are recognized and transported into motile cilia is still unclear. Here, we determined the high-resolution crystal structure of C. reinhardtii ODA16 (CrODA16) and mapped the binding to IFT46 and ODAs. The CrODA16 structure revealed a small 80-residue N-terminal domain and a C-terminal 8-bladed ß-propeller domain that are both required for the association with the N-terminal 147 residues of IFT46. The dissociation constant of the IFT46-ODA16 complex was 200 nm, demonstrating that CrODA16 associates with the IFT complex with an affinity comparable with that of the individual IFT subunits. Furthermore, we show, using ODAs extracted from the axonemes of C. reinhardtii, that the C-terminal ß-propeller but not the N-terminal domain of CrODA16 is required for the interaction with ODAs. These data allowed us to present an architectural model for ODA16-mediated IFT of ODAs.

Lone-Pair-Induced Topicity Observed in Macrobicyclic Tetra-thia Lactams and Cryptands: Synthesis, Spectral Identification, and Computational Assessment.

The synthesis of a rigid macrobicyclic N,S lactam L1 and a topologically favored in/in N,S cryptand L2 are reported with X-ray structure analysis, dynamic correlation NMR spectroscopy, and computational analysis. Lactam L1 exhibits two distinct rotameric conformations (plus their enantiomeric counterparts) at 25 °C, as confirmed via NMR spectroscopy and computational analysis. Coalescence of the resonances of L1 was observed at 115 °C, allowing for complete nuclei to frequency correlation. Combining computational investigations with experimental data, topological equilibria and relative energies/strain relating to the perturbation of the pore were determined. Due to the increased conformational strain of the N2S2 template, the nitrogen lone pairs in L2 elicit a unique transannular interaction, resulting in a thermodynamically favored in/in nephroidal racemate. The combination of preferred topology, steric relief, and electronic localization of L2 induces a chiral environment imparted through the amine with a computed inversion barrier of 10.3 kcal mol-1.

Reversal of RNA polymerase II ubiquitylation by the ubiquitin protease Ubp3.

The final outcome of protein polyubiquitylation is often proteasome-mediated proteolysis, meaning that "proofreading" of ubiquitylation by ubiquitin proteases (UBPs) is crucial. Transcriptional arrest can trigger ubiquitin-mediated proteolysis of RNA polymerase II (RNAPII) so a UBP reversing RNAPII ubiquitylation might be expected. Here, we show that Ubp3 deubiquitylates RNAPII in yeast. Genetic characterization of ubp3 cells is consistent with a role in elongation, and Ubp3 can be purified with RNAPII, Def1, and the elongation factors Spt5 and TFIIF. This Ubp3 complex deubiquitylates both mono- and polyubiquitylated RNAPII in vitro, and ubp3 cells have elevated levels of ubiquitylated RNAPII in vivo. Moreover, RNAPII is degraded faster in a ubp3 mutant after UV irradiation. Problems posed by damage-arrested RNAPII are thought to be resolved either by removing the damage or degrading the polymerase. In agreement with this, cells with compromised DNA repair are better equipped to survive UV damage when UPB3 is deleted.

IFT81, encoding an IFT-B core protein, as a very rare cause of a ciliopathy phenotype.

BACKGROUND: Bidirectional intraflagellar transport (IFT) consists of two major protein complexes, IFT-A and IFT-B. In contrast to the IFT-B complex, all components of IFT-A have recently been linked to human ciliopathies when defective. We therefore hypothesised that mutations in additional IFT-B encoding genes can be found in patients with multisystemic ciliopathies. METHODS: We screened 1628 individuals with reno-ocular ciliopathies by targeted next-generation sequencing of ciliary candidate genes, including all IFT-B encoding genes. RESULTS: Consequently, we identified a homozygous mutation in IFT81 affecting an obligatory donor splice site in an individual with nephronophthisis and polydactyly. Further, we detected a loss-of-stop mutation with extension of the deduced protein by 10 amino acids in an individual with neuronal ceroid lipofuscinosis-1. This proband presented with retinal dystrophy and brain lesions including cerebellar atrophy, a phenotype to which the IFT81 variant might contribute. Cultured fibroblasts of this latter affected individual showed a significant decrease in ciliated cell abundance compared with controls and increased expression of the transcription factor GLI2 suggesting deranged sonic hedgehog signalling. CONCLUSIONS: This work describes identification of mutations of IFT81 in individuals with symptoms consistent with the clinical spectrum of ciliopathies. It might represent the rare case of a core IFT-B complex protein found associated with human disease. Our data further suggest that defects in the IFT-B core are an exceedingly rare finding, probably due to its indispensable role for ciliary assembly in development.

Complex Reconstitution from Individual Protein Modules.

Cellular function relies on protein complexes that work as nano-machines. The structure and function of protein complexes is an outcome of the specific combination of protein subunits, or modules, within the complex. A major focus of molecular biology is thus to understand how protein subunits assemble to form complexes with distinct biological function. To this end, in vitro reconstitution of complexes from individual subunits to study their assembly, structure and activity is of central importance. With purified individual subunits and sub-modules at hand one can systematically dissect the hierarchical assembly of larger complexes using direct protein-protein interaction assays. Furthermore, activity assays can be carried out with individual subunits or smaller sub-complexes and compared to those of the fully assembled complex to precisely map functional sites and provide a molecular basis for in vivo observations. In this chapter we review methods for protein complex assembly from individual subunits and provide examples of advantages and potential pitfalls to this approach.

Crystal structure of the intraflagellar transport complex 25/27.

The cilium is an important organelle that is found on many eukaryotic cells, where it serves essential functions in motility, sensory reception and signalling. Intraflagellar transport (IFT) is a vital process for the formation and maintenance of cilia. We have determined the crystal structure of Chlamydomonas reinhardtii IFT25/27, an IFT sub-complex, at 2.6 Å resolution. IFT25 and IFT27 interact via a conserved interface that we verify biochemically using structure-guided mutagenesis. IFT27 displays the fold of Rab-like small guanosine triphosphate hydrolases (GTPases), binds GTP and GDP with micromolar affinity and has very low intrinsic GTPase activity, suggesting that it likely requires a GTPase-activating protein (GAP) for robust GTP turnover. A patch of conserved surface residues contributed by both IFT25 and IFT27 is found adjacent to the GTP-binding site and could mediate the binding to other IFT proteins as well as to a potential GAP. These results provide the first step towards a high-resolution structural understanding of the IFT complex.

Atomic resolution structure of human α-tubulin acetyltransferase bound to acetyl-CoA.

Acetylation of lysine residues is an important posttranslational modification found in all domains of life. α-Tubulin is specifically acetylated on lysine 40, a modification that serves to stabilize microtubules of axons and cilia. Whereas histone acetyltransferases have been extensively studied, there is no structural and mechanistic information available on α-tubulin acetyltransferases. Here, we present the structure of the human α-tubulin acetyltransferase catalytic domain bound to its cosubstrate acetyl-CoA at 1.05 Å resolution. Compared with other lysine acetyltransferases of known structure, α-tubulin acetyltransferase displays a relatively well-conserved cosubstrate binding pocket but is unique in its active site and putative α-tubulin binding site. Using acetylation assays with structure-guided mutants, we map residues important for acetyl-CoA binding, substrate binding, and catalysis. This analysis reveals a basic patch implicated in substrate binding and a conserved glutamine residue required for catalysis, demonstrating that the family of α-tubulin acetyltransferases uses a reaction mechanism different from other lysine acetyltransferases characterized to date.

A conserved antigen induces respiratory Th17-mediated broad serotype protection against pneumococcal superinfection.

Several vaccines targeting bacterial pathogens show reduced efficacy upon concurrent viral infection, indicating that a new vaccinology approach is required. To identify antigens for the human pathogen Streptococcus pneumoniae that are effective following influenza infection, we performed CRISPRi-seq in a murine model of superinfection and identified the conserved lafB gene as crucial for virulence. We show that LafB is a membrane-associated, intracellular protein that catalyzes the formation of galactosyl-glucosyl-diacylglycerol, a glycolipid important for cell wall homeostasis. Respiratory vaccination with recombinant LafB, in contrast to subcutaneous vaccination, was highly protective against S. pneumoniae serotypes 2, 15A, and 24F in a murine model. In contrast to standard capsule-based vaccines, protection did not require LafB-specific antibodies but was dependent on airway CD4+ T helper 17 cells. Healthy human individuals can elicit LafB-specific immune responses, indicating LafB antigenicity in humans. Collectively, these findings present a universal pneumococcal vaccine antigen that remains effective following influenza infection.

Crystal structure of the invertebrate bifunctional purine biosynthesis enzyme PAICS at 2.8 Å resolution.

Two important steps of the de novo purine biosynthesis pathway are catalyzed by the 5-aminoimidazole ribonucleotide carboxylase and the 4-(N-succinylcarboxamide)-5-aminoimidazole ribonucleotide synthetase enzymes. In most eukaryotic organisms, these two activities are present in the bifunctional enzyme complex known as PAICS. We have determined the 2.8-Å resolution crystal structure of the 350-kDa invertebrate PAICS from insect cells (Trichoplusia ni) using single-wavelength anomalous dispersion methods. Comparison of insect PAICS to human and prokaryotic homologs provides insights into substrate binding and reveals a highly conserved enzymatic framework across divergent species.

Architecture and function of IFT complex proteins in ciliogenesis.

Cilia and flagella (interchangeable terms) are evolutionarily conserved organelles found on many different types of eukaryotic cells where they fulfill important functions in motility, sensory reception and signaling. The process of Intraflagellar Transport (IFT) is of central importance for both the assembly and maintenance of cilia, as it delivers building blocks from their site of synthesis in the cell body to the ciliary assembly site at the tip of the cilium. A key player in this process is the multi-subunit IFT-complex, which acts as an adapter between the motor proteins required for movement and the ciliary cargo proteins. Since the discovery of IFT more than 15 years ago, considerable effort has gone into the purification and characterization of the IFT complex proteins. Even though this has led to very interesting findings and has greatly improved our knowledge of the IFT process, we still know very little about the overall architecture of the IFT complex and the specific functions of the various subunits. In this review we will give an update on the knowledge of the structure and function of individual IFT proteins, and the way these proteins interact to form the complex that facilitates IFT.

DNA segment capture by Smc5/6 holocomplexes.

Three distinct structural maintenance of chromosomes (SMC) complexes facilitate chromosome folding and segregation in eukaryotes, presumably by DNA loop extrusion. How SMCs interact with DNA to extrude loops is not well understood. Among the SMC complexes, Smc5/6 has dedicated roles in DNA repair and preventing a buildup of aberrant DNA junctions. In the present study, we describe the reconstitution of ATP-dependent DNA loading by yeast Smc5/6 rings. Loading strictly requires the Nse5/6 subcomplex which opens the kleisin neck gate. We show that plasmid molecules are topologically entrapped in the kleisin and two SMC subcompartments, but not in the full SMC compartment. This is explained by the SMC compartment holding a looped DNA segment and by kleisin locking it in place when passing between the two flanks of the loop for neck-gate closure. Related segment capture events may provide the power stroke in subsequent DNA extrusion steps, possibly also in other SMC complexes, thus providing a unifying principle for DNA loading and extrusion.

Biochemical mapping of interactions within the intraflagellar transport (IFT) B core complex: IFT52 binds directly to four other IFT-B subunits.

Cilia and flagella are complex structures emanating from the surface of most eukaroytic cells and serve important functions including motility, signaling, and sensory reception. A process called intraflagellar transport (IFT) is of central importance to ciliary assembly and maintenance. The IFT complex is required for this transport and consists of two distinct multisubunit subcomplexes, IFT-A and IFT-B. Despite the importance of the IFT complex, little is known about its overall architecture. This paper presents a biochemical dissection of the molecular interactions within the IFT-B core complex. Two stable subcomplexes consisting of IFT88/70/52/46 and IFT81/74/27/25 were recombinantly co-expressed and purified. We identify a novel interaction between IFT70/52 and map the interaction domains between IFT52 and the other subunits within the IFT88/70/52/46 complex. Additionally, we show that IFT52 binds directly to the IFT81/74/27/25 complex, indicating that it could mediate the interaction between the two subcomplexes. Our data lead to an improved architectural map for the IFT-B core complex with new interactions as well as domain resolution mapping for several subunits.