ATPase Motor Research Articles

Tad pilus-mediated twitching motility is essential for DNA uptake and survival of Liberibacters.

Axenically cultured Liberibacter crescens (Lcr) is a closely related surrogate for uncultured plant pathogenic species of the genus Liberibacter, including 'Candidatus L. asiaticus' (CLas) and 'Ca. L. solanacearum' (CLso). All Liberibacters encode a completely conserved gene repertoire for both flagella and Tad (Tight Adherence) pili and all are missing genes critical for nucleotide biosynthesis. Both flagellar swimming and Tad pilus-mediated twitching motility in Lcr were demonstrated for the first time. A role for Tad pili in the uptake of extracellular dsDNA for food in Liberibacters was suspected because both twitching and DNA uptake are impossible without repetitive pilus extension and retraction, and no genes encoding other pilus assemblages or mechanisms for DNA uptake were predicted to be even partially present in any of the 35 fully sequenced Liberibacter genomes. Insertional mutations of the Lcr Tad pilus genes cpaA, cpaB, cpaE, cpaF and tadC all displayed such severely reduced growth and viability that none could be complemented. A mutation affecting cpaF (motor ATPase) was further characterized and the strain displayed concomitant loss of twitching, viability and reduced periplasmic uptake of extracellular dsDNA. Mutations of comEC, encoding the inner membrane competence channel, had no effect on either motility or growth but completely abolished natural transformation in Lcr. The comEC mutation was restored by complementation using comEC from Lcr but not from CLas strain psy62 or CLso strain RS100, indicating that unlike Lcr, these pathogens were not naturally competent for transformation. This report provides the first evidence that the Liberibacter Tad pili are dynamic and essential for both motility and DNA uptake, thus extending their role beyond surface adherence.

SecA - a multidomain and multitask bacterial export protein.

Most bacterial secretory proteins destined to the extracytoplasmic space are secreted posttranslationally by the Sec translocase. SecA, a key component of the Sec system, is the ATPase motor protein, directly responsible for transferring the preprotein across the cytoplasmic membrane. SecA is a large protein, composed of several domains, capable of binding client preproteins and a variety of partners, including the SecYEG inner membrane channel complex, membrane phospholipids and ribosomes. SecA-mediated translocation can be divided into two major steps: (1) targeting of the preproteins to the membrane translocation apparatus and (2) transport across the membrane through the SecYEG channel. In this review we present current knowledge regarding SecA structure and function of this protein in both translocation steps. The most recent model of the SecA-dependent preprotein mechanical translocation across the bacterial cytoplasmic membrane is described. A possibility of targeting SecA with inhibitory compounds as a strategy to combat pathogenic bacteria will be discussed as well.

1H, 15N and 13C resonance assignments of the C-terminal domain of PulL, a component of the Klebsiella oxytoca type II secretion system.

Type II secretion systems (T2SS) allow Gram-negative bacteria to transport toxins and enzymes from the periplasm to the external milieu, and are thus important for the pathogenicity of bacteria. To drive secretion, T2SS assemble filaments called pseudopili closely related to bacterial type IV pili. These filaments are non-covalent polymers of proteins that are assembled by an inner membrane complex called the assembly platform connected to a cytoplasmic ATPase motor. In the Klebsiella oxytoca T2SS, the PulL protein from the assembly platform is essential for pseudopilus assembly and protein secretion. However, its role in these processes is not well understood. To decipher the molecular basis of PulL function, we used solution NMR to study its structure and interactions with other components of the machinery. Here as a first step, we report the 1H, 15N and 13C backbone and side-chain chemical shift assignments of the C-terminal periplasmic domain of PulL and its secondary structure based on NMR data.

Autophosphorylation of the KaiC-like protein ArlH inhibits oligomerization and interaction with ArlI, the motor ATPase of the archaellum.

Motile archaea are propelled by the archaellum, whose motor complex consists of the membrane protein ArlJ, the ATPase ArlI, and the ATP-binding protein ArlH. Despite its essential function and the existence of structural and biochemical data on ArlH, the role of ArlH in archaellum assembly and function remains elusive. ArlH is a structural homolog of KaiC, the central component of the cyanobacterial circadian clock. Since autophosphorylation and dephosphorylation of KaiC are central properties for the function of KaiC, we asked whether autophosphorylation is also a property of ArlH proteins. We observed that both ArlH from the euryarchaeon Pyrococcus furiosus (PfArlH) and from the crenarchaeon Sulfolobus acidocaldarius (SaArlH) have autophosphorylation activity. Using a combination of single-molecule fluorescence measurements and biochemical assays, we show that autophosphorylation of ArlH is closely linked to its oligomeric state when bound to hexameric ArlI. These experiments also strongly suggest that ArlH is a hexamer in its ArlI-bound state. Mutagenesis of the putative catalytic residue (Glu-57 in SaArlH) in ArlH results in a reduced autophosphorylation activity and abolished archaellation and motility in S. acidocaldarius, indicating that optimum phosphorylation activity of ArlH is essential for archaellation and motility.

Structural basis of ALC1/CHD1L autoinhibition and the mechanism of activation by the nucleosome

Chromatin remodeler ALC1 (amplification in liver cancer 1) is crucial for repairing damaged DNA. It is autoinhibited and activated by nucleosomal epitopes. However, the mechanisms by which ALC1 is regulated remain unclear. Here we report the crystal structure of human ALC1 and the cryoEM structure bound to the nucleosome. The structure shows the macro domain of ALC1 binds to lobe 2 of the ATPase motor, sequestering two elements for nucleosome recognition, explaining the autoinhibition mechanism of the enzyme. The H4 tail competes with the macro domain for lobe 2-binding, explaining the requirement for this nucleosomal epitope for ALC1 activation. A dual-arginine-anchor motif of ALC1 recognizes the acidic pocket of the nucleosome, which is critical for chromatin remodeling in vitro. Together, our findings illustrate the structures of ALC1 and shed light on its regulation mechanisms, paving the way for the discovery of drugs targeting ALC1 for the treatment of cancer.

Abstract 2063: TLK1 phosphorylates RAD54 to promote homology driven DSB repair

Abstract Background: Homologous recombination repair (HRR) is a faithful mechanism of maintaining genomic integrity in cells. The regulation of this pathway is critical particularly to guard against tumorigenesis due to chromosomal rearrangements. RAD54L, the DNA motor ATPase, performs as the key player in HRR whose regulation in higher eukaryotes is still unclear. Studies have shown post-translational modification of RAD54L imparting specific functions toward the progression of HRR. Tousled-like kinase homolog1 (TLK1), a Ser/Thr Kinase, is known to regulate DNA damage response through NEK1-ATR axis. But its role in the mechanism in DSB repair is still unknown. We recently uncovered the interaction between TLK1 and RAD54L through an invitro proteomic array of TLK1 interacting partners. The crystal structure of RAD54L reveals distinct features about the N-terminal domain (NTD) and the C-terminal domain (CTD) of the protein. The unstructured NTD (1-90) is important for interaction with its physiological partner protein RAD51 while the structured CTD (660-747) (Zn-finger-like motif) contacts the DNA backbone in the donor template. Therefore, it is interesting to study the regulation of RAD54 function by TLK1. Material and methods: We employed immunoprecipitation and pull-down assays to show that TLK1 interacts with RAD54L in cells. We performed an in-vitro kinase assay using purified recombinant TLK1 to phosphorylate purified human RAD54L followed by mass spectrometry (MS). We use DR-GFP assay, standard technique to measure HR efficiency in HeLa cell line to understand the effect of TLK1 and mutation on RAD54 at the novel sites. Results: We find that upon irradiation (10Gy), TLK1 interaction with RAD54L increases during the initial 30mins post DNA damage, while the interaction decreases over time (4-12hrs). MS analyses reveal three novel sites of phosphorylation on RAD54L at T41, T59, and T700. We hypothesize that phosphorylation of RAD54L at both NTD and CTD by TLK1 promotes HRR through RAD51-nucleofilament interaction and formation of D-loop and DNA translocase activity. HRR activity in cells can be monitored through the DR-GFP assay which results in GFP positive cells following efficient homologous recombination event after site-specific cleavage with SceI. Our results with TLK1 specific inhibitor (J54) and parallel knockdown using shRNA against TLK1 confirm that inhibition or depletion of TLK1 suppresses HRR activity in cells. We have performed site-directed mutagenesis from T to D at RAD54L which mimics the phosphorylated state of RAD54L and generated HeLa -RAD54L mutant cell line variants with DR-GFP integrated cassette in RAD54L genetically deleted background (CRISPR/Cas9) - from Dr. Wiese lab). We plan to use these as a tool to monitor the HRR activity in hyper-phosphorylated RAD54L state. Conclusion: We expect that RAD54L T>D mutants will show higher gene conversion than WT Rad54-expressing cells. Citation Format: Ishita Ghosh, Youngho Kwon, Jing Chen, Platon Selemenakis, Claudia Wiese, Patrick Sung, Arrigo DeBenedetti. TLK1 phosphorylates RAD54 to promote homology driven DSB repair [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2063.

A two-hybrid system reveals previously uncharacterized protein\u2013protein interactions within the Helicobacter pylori NIF iron\u2013sulfur maturation system

Iron–sulfur (Fe–S) proteins play essential roles in all living organisms. The gastric pathogen Helicobacter pylori relies exclusively on the NIF system for biosynthesis and delivery of Fe–S clusters. Previously characterized components include two essential proteins, NifS (cysteine desulfurase) and NifU (scaffold protein), and a dispensable Fe–S carrier, Nfu. Among 38 proteins previously predicted to coordinate Fe–S clusters, two proteins, HP0207 (a member of the Nbp35/ApbC ATPase family) and HP0277 (previously annotated as FdxA, a member of the YfhL ferredoxin-like family) were further studied, using a bacterial two-hybrid system approach to identify protein–protein interactions. ApbC was found to interact with 30 proteins, including itself, NifS, NifU, Nfu and FdxA, and alteration of the conserved ATPase motif in ApbC resulted in a significant (50%) decrease in the number of protein interactions, suggesting the ATpase activity is needed for some ApbC-target protein interactions. FdxA was shown to interact with 21 proteins, including itself, NifS, ApbC and Nfu, however no interactions between NifU and FdxA were detected. By use of cross-linking studies, a 51-kDa ApbC-Nfu heterodimer complex was identified. Attempts to generate apbC chromosomal deletion mutants in H. pylori were unsuccessful, therefore indirectly suggesting the hp0207 gene is essential. In contrast, mutants in the fdxA gene were obtained, albeit only in one parental strain (26695). Taken together, these results suggest both ApbC and FdxA are important players in the H. pylori NIF maturation system.

Mud binds the kinesin-14 Ncd in Drosophila

Maintenance of proper mitotic spindle structure is necessary for error-free chromosome segregation and cell division. Spindle assembly is controlled by force-generating kinesin motors that contribute to its geometry and bipolarity, and balancing motor-dependent forces between opposing kinesins is critical to the integrity of this process. Non-claret dysjunctional (Ncd), a Drosophila kinesin-14 member, crosslinks and slides microtubule minus-ends to focus spindle poles and sustain bipolarity. However, mechanisms that regulate Ncd activity during mitosis are underappreciated. Here, we identify Mushroom body defect (Mud), the fly ortholog of human NuMA, as a direct Ncd binding partner. We demonstrate this interaction involves a short coiled-coil domain within Mud (MudCC) binding the N-terminal, non-motor microtubule-binding domain of Ncd (NcdnMBD). We further show that the C-terminal ATPase motor domain of Ncd (NcdCTm) directly interacts with NcdnMBD as well. Mud binding competes against this self-association and also increases NcdnMBD microtubule binding in vitro. Our results describe an interaction between two spindle-associated proteins and suggest a potentially new mode of minus-end motor protein regulation at mitotic spindle poles.

A viral genome packaging motor transitions between cyclic and helical symmetry to translocate dsDNA.

Molecular segregation and biopolymer manipulation require the action of molecular motors to do work by applying directional forces to macromolecules. The additional strand conserved E (ASCE) ring motors are an ancient family of molecular motors responsible for diverse biological polymer manipulation tasks. Viruses use ASCE segregation motors to package their genomes into their protein capsids and provide accessible experimental systems due to their relative simplicity. We show by cryo-EM-focused image reconstruction that ASCE ATPases in viral double-stranded DNA (dsDNA) packaging motors adopt helical symmetry complementary to their dsDNA substrates. Together with previous data, our results suggest that these motors cycle between helical and planar configurations, providing a possible mechanism for directional translocation of DNA. Similar changes in quaternary structure have been observed for proteasome and helicase motors, suggesting an ancient and common mechanism of force generation that has been adapted for specific tasks over the course of evolution.

Ubiquitin binding modulates the conformational dynamics and substrate degradation of the 26S proteasome

The 26S proteasome is an ATP-dependent molecular machine that degrades ubiquitin-tagged proteins in several well-coordinated steps, including ubiquitin binding, substrate engagement, mechanical unfolding, de-ubiquitination, translocation, and proteolytic cleavage. Recently developed single-molecule FRET measurements, relying on the labeling of proteasome-incorporated unnatural amino acids, allow us to follow individual substrate proteins through the central channel and dissect the kinetics and coordination of processing steps. Mutational studies thereby reveal new details about the mechanisms of substrate engagement by the AAA+ ATPase motor and the contributions of individual ATPase subunits depending on their position in the hexameric spiral-staircase arrangement of the motor. Our FRET-based measurements with double-labeled proteasomes provide exciting insights into the conformational dynamics of the proteasomal regulatory particle and how ubiquitin-chain binding to certain receptor subunits influences the switching between substrate-free and substrate-processing states. Through this regulation of the proteasome conformational dynamics, ubiquitin chains accelerate substrate engagement by the AAA+ motor and potentiate mechanical unfolding, leading to faster and more efficient degradation. These effects depend on the length and linkage type of ubiquitin chains, indicating how the “ubiquitin code” may be utilized by the proteasome to prioritize substrates and fine-tune degradation in a complex cellular environment.

Role of Chromodomain-Helicase-DNA-Binding Protein 4 (CHD4) in Breast Cancer.

Chromodomain-helicase-DNA-binding protein 4 (CHD4) is an epigenetic regulator identified as an oncogenic element that may provide a novel therapeutic target for the treatment of breast cancer (BC). CHD4—the core component of the nucleosome remodeling and deacetylase (NuRD) complex—may be mutated in patients with this disease. However, information on CHD4 mutants that might allow their use as biomarkers of therapeutic success and prognosis is lacking. The present work examines mutations in CHD4 reported in patients with breast cancer and included in public databases and attempts to identify their roles in its development. The databases revealed 81 point mutations across different types of breast cancer (19 of which also appeared in endometrial, intestinal, nervous system, kidney, and lymphoid organ cancers). 71.6% of the detected mutations were missense mutations, 13.6% were silent, and 6.2% nonsense. Over 50% affected conserved residues of the ATPase motor (ATPase and helicase domains), and domains of unknown function in the C-terminal region. Thirty one mutations were classified in the databases as either ‘deleterious’, ‘probably/possibly damaging’ or as ‘high/medium pathogenic’; another five nonsense and one splice-site variant were predicted to produce potentially harmful truncated proteins. Eight of the 81 mutations were categorized as putative driver mutations and have been found in other cancer types. Some mutations seem to influence ATPase and DNA translocation activities (R1162W), while others may alter protein stability (R877Q/H, R975H) or disrupt DNA binding and protein activity (R572*, X34_splice) suggesting CHD4 function may be affected. In vivo tumorigenecity studies in endometrial cancer have revealed R975H and R1162W as mutations that lead to CHD4 loss-of-function. Our study provides insight into the molecular mechanism whereby CHD4, and some of its mutants could play a role in breast cancer and suggest important implications for the biological comprehension and prognosis of breast cancer, identifying CHD4 as a novel therapeutic target for BC patients.

Multifaceted Function of Myosin-18, an Unconventional Class of the Myosin Superfamily.

Myosin is a diverse superfamily of motor proteins responsible for actin-based motility and contractility in eukaryotic cells. Myosin-18 family, including myosin-18A and myosin-18B, belongs to an unconventional class of myosin, which lacks ATPase motor activity, and the investigations on their functions and molecular mechanisms in vertebrate development and diseases have just been initiated in recent years. Myosin-18A is ubiquitously expressed in mammalian cells, whereas myosin-18B shows strong enrichment in striated muscles. Myosin-18 family is important for cell motility, sarcomere formation, and mechanosensing, mostly by interacting with other cytoskeletal proteins and cellular apparatus. Myosin-18A participates in several intracellular transport processes, such as Golgi trafficking, and has multiple roles in focal adhesions, stress fibers, and lamellipodia formation. Myosin-18B, on the other hand, participates in actomyosin alignment and sarcomere assembly, thus relating to cell migration and muscle contractility. Mutations of either Myo18a or Myo18b cause cardiac developmental defects in mouse, emphasizing their crucial role in muscle development and cardiac diseases. In this review, we revisit the discovery history of myosin-18s and summarize the evolving understanding of the molecular functions of myosin-18A and myosin-18B, with an emphasis on their separate yet closely related functions in cell motility and contraction. Moreover, we discuss the diseases tightly associated with myosin-18s, especially cardiovascular defects and cancer, as well as highlight the unanswered questions and potential future research perspectives on myosin-18s.

Comparative proteomic analysis of kinesin-8B deficient Plasmodium berghei during gametogenesis

Plasmodium blood stages, responsible for human to vector transmission, termed gametocytes, are the precursor cells that develop into gametes in the mosquito. Male gametogenesis works as a bottleneck for the parasite life cycle, where, during a peculiar and rapid exflagellation, a male gametocyte produces 8 intracellular axonemes that generate by budding 8 motile gametes. Understanding the molecular mechanisms of gametogenesis is key to design strategies for controlling malaria transmission. In the rodent P. berghei, the microtubule-based motor kinesin-8B (PbKIN8B) is essential for flagellum assembly during male gametogenesis and its gene disruption impacts on completion of the parasitic life cycle. In efforts to improve our knowledge about male gametogenesis, we performed an iTRAQ-based quantitative proteomic comparison of P. berghei mutants with disrupted kinesin-8B gene (ΔPbkin8B) and wild type parasites. During the 15 min of gametogenesis, ΔPbkin8B parasites exhibited important motor protein dysregulation that suggests an essential role of PbKIN8B for the correct interaction or integration of axonemal proteins within the growing axoneme. The energy metabolism of ΔPbkin8B mutants was further affected, as well as the response to stress proteins, protein synthesis, as well as chromatin organisation and DNA processes, although endomitoses seemed to occur. SignificanceMalaria continues to be a global scourge, mainly in subtropical and tropical areas. The disease is caused by parasites from the Plasmodium genus. Plasmodium life cycle alternates between female Anopheles mosquitoes and vertebrate hosts through bites. Gametocytes are the parasite blood forms responsible for transmission from vertebrates to vectors. Inside the mosquito midgut, after stimulation, male and female gametocytes transform into gametes resulting in fertilization. During male gametogenesis, one gametocyte generates eight intracytoplasmic axonemes that generate, by budding, flagellated motile gametes involving a process termed exflagellation. Sexual development has a central role in ensuring malaria transmission. However, molecular data on male gametogenesis and particularly on intracytoplasmic axoneme assembly are still lacking. Since rodent malaria parasites permit the combination of in vivo and in vitro experiments and reverse genetic studies, our group investigated the molecular events in rodent P. berghei gametogenesis. The P. berghei motor ATPase kinesin-8B is proposed as an important component for male gametogenesis. We generated Pbkin8B gene-disrupted gametocytes (ΔPbkin8B) that were morphologically similar to the wild- type (WT) parasites. However, in mutants, male gametogenesis is impaired, male gametocytes are disabled in their ability to assemble axonemes and to exflagellate to release gametes, reducing fertilization drastically. Using a comparative quantitative proteomic analysis, we associated the nonfunctional axoneme of the mutants with the abnormal differential expression of proteins essential to axoneme organisation and stability. We also observed a differential dysregulation of proteins involved in protein biosynthesis and degradation, chromatin organisation and DNA processes in ΔPbkin8B parasites, although DNA condensation, mitotic spindle formation and endomitoses seem to occur. This is the first functional proteomic study of a kinesin gene-disrupted Plasmodium parasite providing new insights into Plasmodium male gametogenesis.

Autoinhibitory elements of the Chd1 remodeler block initiation of twist defects by destabilizing the ATPase motor on the nucleosome

Chromatin remodelers are ATP (adenosine triphosphate)-powered motors that reposition nucleosomes throughout eukaryotic chromosomes. Remodelers possess autoinhibitory elements that control the direction of nucleosome sliding, but underlying mechanisms of inhibition have been unclear. Here, we show that autoinhibitory elements of the yeast Chd1 remodeler block nucleosome sliding by preventing initiation of twist defects. We show that two autoinhibitory elements-the chromodomains and bridge-reinforce each other to block sliding when the DNA-binding domain is not bound to entry-side DNA. Our data support a model where the chromodomains and bridge target nucleotide-free and ADP-bound states of the ATPase motor, favoring a partially disengaged state of the ATPase motor on the nucleosome. By bypassing distortions of nucleosomal DNA prior to ATP binding, we propose that autoinhibitory elements uncouple the ATP binding/hydrolysis cycle from DNA translocation around the histone core.

Rhythmic adenosine triphosphate release from the cyanobacterial circadian clock protein KaiC revealed by real-time monitoring of bioluminescence using firefly luciferase.

The cyanobacterial circadian clock is composed of three clock proteins, KaiA, KaiB and KaiC. This KaiABC clock system can be reconstituted in vitro in the presence of adenosine triphosphate (ATP) and Mg2+ , and shows circadian rhythms in the phosphorylation level and ATPase activity of KaiC. Previously, we found that ATP regulates a complex formation between KaiB and KaiC, and KaiC releases ATP from KaiC itself (PLoS One, 8, 2013, e80200). In this study, we examined whether the ATP release from KaiC shows any rhythms in vitro. We monitored the release of ATP from wild-type and ATPase motif mutants of KaiC as a bioluminescence in real time using a firefly luciferase assay in vitro and obtained the following results: (a) ATP release from KaiC oscillated even without KaiA and KaiB although period of the oscillation was not 24hr; (b) ATP was mainly released from the N-terminal domain of KaiC; and (c) the ATP release was enhanced and suppressed by KaiB and KaiA, respectively. These results suggest that KaiC can generate basal oscillation as a core clock without KaiA and KaiB, whereas these two proteins contribute to adjusting and stabilizing the oscillation.

Biophysics of Chromatin Remodeling.

As primary carriers of epigenetic information and gatekeepers of genomic DNA, nucleosomes are essential for proper growth and development of all eukaryotic cells. Although they are intrinsically dynamic, nucleosomes are actively reorganized by ATP-dependent chromatin remodelers. Chromatin remodelers contain helicase-like ATPase motor domains that can translocate along DNA, and a long-standing question in the field is how this activity is used to reposition or slide nucleosomes. In addition to ratcheting along DNA like their helicase ancestors, remodeler ATPases appear to dictate specific alternating geometries of the DNA duplex, providing an unexpected means for moving DNA past the histone core. Emerging evidence supports twist-based mechanisms for ATP-driven repositioning of nucleosomes along DNA. In this review, we discuss core experimental findings and ideas that have shaped the view of how nucleosome sliding may be achieved.

The ATPase mechanism of myosin 15, the molecular motor mutated in DFNB3 human deafness

Cochlear hair cells each possess an exquisite bundle of actin-based stereocilia that detect sound. Unconventional myosin 15 (MYO15) traffics and delivers critical molecules required for stereocilia development and thus is essential for building the mechanosensory hair bundle. Mutations in the human MYO15A gene interfere with stereocilia trafficking and cause hereditary hearing loss, DFNB3, but the impact of these mutations is not known, as MYO15 itself is poorly characterized. To learn more, we performed a kinetic study of the ATPase motor domain to characterize its mechanochemical cycle. Using the baculovirus–Sf9 system, we purified a recombinant minimal motor domain (S1) by coexpressing the mouse MYO15 ATPase, essential and regulatory light chains that bind its IQ domains, and UNC45 and HSP90A chaperones required for correct folding of the ATPase. MYO15 purified with either UNC45A or UNC45B coexpression had similar ATPase activities (kcat = ∼ 6 s−1 at 20 °C). Using stopped-flow and quenched-flow transient kinetic analyses, we measured the major rate constants describing the ATPase cycle, including ATP, ADP, and actin binding; hydrolysis; and phosphate release. Actin-attached ADP release was the slowest measured transition (∼12 s−1 at 20 °C), although this did not rate-limit the ATPase cycle. The kinetic analysis shows the MYO15 motor domain has a moderate duty ratio (∼0.5) and weak thermodynamic coupling between ADP and actin binding. These findings are consistent with MYO15 being kinetically adapted for processive motility when oligomerized. Our kinetic characterization enables future studies into how deafness-causing mutations affect MYO15 and disrupt stereocilia trafficking necessary for hearing.

In vivo localization and oligomerization of PixD and PixE for controlling phototaxis in the cyanobacterium Synechocystis sp. PCC 6803.

Phototaxis is a phenomenon where cyanobacteria move toward a light source. Previous studies have shown that the blue-light-using-flavin (BLUF)-type photoreceptor PixD and the response regulator-like protein PixE control the phototaxis in the cyanobacterium Synechocystis sp. PCC 6803. The pixD-null mutant moves away from light, whereas WT, pixE mutant, and pixD pixE double mutant move toward the light. This indicates that PixE functions downstream of PixD and influences the direction of movement. However, it is still unclear how the light signal received by PixD is transmitted to PixE, and then subsequently transmitted to the type IV pili motor mechanism. Here, we investigated intracellular localization and oligomerization of PixD and PixE to elucidate mechanisms of phototaxis regulation. Blue-native PAGE analysis, coupled with western blotting, indicated that most PixD exist as a dimer in soluble fractions, whereas PixE localized in ~250 kDa and ~450 kDa protein complexes in membrane fractions. When blue-native PAGE was performed after illuminating the membrane fractions with blue light, PixE levels in the ~250 kDa and ~450 kDa complexes were reduced and increased, respectively. These results suggest that PixE, localized in the ~450 kDa complex, controls activity of the motor ATPase PilB1 to regulate pilus motility.

Inhibition of potato leafroll virus multiplication and systemic translocation by siRNA constructs against putative ATPase fold of movement protein

Viruses cause many severe plant diseases, resulting in immense losses of crop yield worldwide. Therefore, developing novel approaches to control plant viruses is crucial to meet the demands of a growing world population. Recently, RNA interference (RNAi) has been widely used to develop virus-resistant plants. Once genome replication and assembly of virion particles is completed inside the host plant, mature virions or sometimes naked viral genomes spread cell-to-cell through plasmodesmata by interacting with the virus-encoded movement protein (MP). We used the RNAi approach to suppress MP gene expression, which in turn prevented potato leafroll virus (PLRV) systemic infection in Solanum tuberosum cv. Khufri Ashoka. Potato plants agroinfiltrated with MP siRNA constructs exhibited no rolling symptoms upon PLRV infection, indicating that the silencing of MP gene expression is an efficient method for generating PLRV-resistant potato plants. Further, we identified novel ATPase motifs in MP that may be involved in DNA binding and translocation through plasmodesmata. We also showed that the ATPase activity of MP was stimulated in the presence of DNA/RNA. Overall, our findings provide a robust technology to generate PLRV-resistant potato plants, which can be extended to other species. Moreover, this approach also contributes to the study of genome translocation mechanisms of plant viruses.

Role of SpoIVA ATPase Motifs during Clostridioides difficile Sporulation.

The nosocomial pathogen Clostridioides difficile is a spore-forming obligate anaerobe that depends on its aerotolerant spore form to transmit infections. Functional spore formation depends on the assembly of a proteinaceous layer known as the coat around the developing spore. In C. difficile, coat assembly depends on the conserved spore protein SpoIVA and the clostridial-organism-specific spore protein SipL, which directly interact. Mutations that disrupt their interaction cause the coat to mislocalize and impair spore formation. In Bacillus subtilis, SpoIVA is an ATPase that uses ATP hydrolysis to drive its polymerization around the forespore. Loss of SpoIVA ATPase activity impairs B. subtilis SpoIVA encasement of the forespore and activates a quality control mechanism that eliminates these defective cells. Since this mechanism is lacking in C. difficile, we tested whether mutations in the C. difficile SpoIVA ATPase motifs impact functional spore formation. Disrupting C. difficile SpoIVA ATPase motifs resulted in phenotypes that were typically >104-fold less severe than the equivalent mutations in B. subtilis Interestingly, mutation of ATPase motif residues predicted to abrogate SpoIVA binding to ATP decreased the SpoIVA-SipL interaction, whereas mutation of ATPase motif residues predicted to disrupt ATP hydrolysis but maintain ATP binding enhanced the SpoIVA-SipL interaction. When a sipL mutation known to reduce binding to SpoIVA was combined with a spoIVA mutation predicted to prevent SpoIVA binding to ATP, spore formation was severely exacerbated. Since this phenotype is allele specific, our data imply that SipL recognizes the ATP-bound form of SpoIVA and highlight the importance of this interaction for functional C. difficile spore formation.IMPORTANCE The major pathogen Clostridioides difficile depends on its spore form to transmit disease. However, the mechanism by which C. difficile assembles spores remains poorly characterized. We previously showed that binding between the spore morphogenetic proteins SpoIVA and SipL regulates assembly of the protective coat layer around the forespore. In this study, we determined that mutations in the C. difficile SpoIVA ATPase motifs result in relatively minor defects in spore formation, in contrast with Bacillus subtilis Nevertheless, our data suggest that SipL preferentially recognizes the ATP-bound form of SpoIVA and identify a specific residue in the SipL C-terminal LysM domain that is critical for recognizing the ATP-bound form of SpoIVA. These findings advance our understanding of how SpoIVA-SipL interactions regulate C. difficile spore assembly.