Higher-order SPOP assembly reveals a basis for cancer mutant dysregulation.

The speckle-type POZ protein (SPOP) functions in the Cullin3-RING ubiquitin ligase (CRL3) as a receptor for the recognition of substrates involved in cell growth, survival, and signaling. SPOP mutations have been attributed to the development of many types of cancers, including prostate and endometrial cancers. Prostate cancer mutations localize in the substrate-binding site of the substrate recognition (MATH) domain and reduce or prevent binding. However, most endometrial cancer mutations are dispersed in seemingly inconspicuous solvent-exposed regions of SPOP, offering no clear basis for their cancer-causing and peculiar gain-of-function properties. Herein, we present the first structure of SPOP in its oligomeric form, uncovering several new interfaces important for SPOP self-assembly and normal function. Given that many previously unaccounted-for cancer mutations are localized in these newly identified interfaces, we uncover molecular mechanisms underlying dysregulation of SPOP function, with effects ranging from gross structural changes to enhanced self-association, and heightened stability and activity.

Activation and closed-state inactivation mechanisms of the human voltage-gated KV4 channel complexes.

The voltage-gated ion channel activity depends on both activation (transition from the resting state to the open state) and inactivation. Inactivation is a self-restraint mechanism to limit ion conduction and is as crucial to membrane excitability as activation. Inactivation can occur when the channel is open or closed. Although open-state inactivation is well understood, the molecular basis of closed-state inactivation has remained elusive. We report cryo-EM structures of human KV4.2 channel complexes in inactivated, open, and closed states. Closed-state inactivation of KV4 involves an unprecedented symmetry breakdown for pore closure by only two of the four S4-S5 linkers, distinct from known mechanisms of open-state inactivation. We further capture KV4 in a putative resting state, revealing how voltage sensor movements control the pore. Moreover, our structures provide insights regarding channel modulation by KChIP2 and DPP6 auxiliary subunits. Our findings elucidate mechanisms of closed-state inactivation and voltage-dependent activation of the KV4 channel.

The Lipid Kinase PI5P4Kß Is an Intracellular GTP Sensor for Metabolism and Tumorigenesis.

While cellular GTP concentration dramatically changes in response to an organism's cellular status, whether it serves as a metabolic cue for biological signaling remains elusive due to the lack of molecular identification of GTP sensors. Here we report that PI5P4Kß, a phosphoinositide kinase that regulates PI(5)P levels, detects GTP concentration and converts them into lipid second messenger signaling. Biochemical analyses show that PI5P4Kß preferentially utilizes GTP, rather than ATP, for PI(5)P phosphorylation, and its activity reflects changes in direct proportion to the physiological GTP concentration. Structural and biological analyses reveal that the GTP-sensing activity of PI5P4Kß is critical for metabolic adaptation and tumorigenesis. These results demonstrate that PI5P4Kß is the missing GTP sensor and that GTP concentration functions as a metabolic cue via PI5P4Kß. The critical role of the GTP-sensing activity of PI5P4Kß in cancer signifies this lipid kinase as a cancer therapeutic target.

[Improving the Pap Smear Screening Rate for Cervical Cancer in a Health Management Center: A Quality Improvement Program].

BACKGROUND & PROBLEMS: Early detection tests are highly effective in helping adult women prevent the onset of cervical cancer. However, the cervical Pap smear screening rate in a health management center was only 54.3% in 2020. PURPOSE: This project was developed to improve the Pap smear screening rate for cervical cancer in a health management center. RESOLUTION: The strategies developed included revising the health examination lists, developing an online appointment booking system, designing a patient decision aid, creating a standardized simulation moulage for education, and rechecking patient's National Health Insurance cards. RESULTS: After implementation of these strategies, the Pap smear screening rate for cervical cancer rose from 54.3% to 81.2%. The screening rate at the health management center in 2022 reached 96.6%. CONCLUSIONS: Shared decision-making can elucidate the comprehensive options available to clients and support them in considering their options and achieving informed choices regarding Pap smear preferences.

A class-A GPCR solubilized under high hydrostatic pressure retains its ligand binding ability.

The effect of high hydrostatic pressure (HHP) on the solubilization of a class-A G protein-coupled receptor, the silkmoth pheromone biosynthesis-activating neuropeptide receptor (PBANR), was investigated. PBANR was expressed in expresSF+ insect cells as a C-terminal fusion protein with EGFP. The membrane fraction was subjected to HHP treatment (200MPa) at room temperature for 1-16h in the presence of 0-2.0% (w/v) n-dodecyl-ß-D-maltopyranoside (DDM). The solubilization yield of PBANR-EGFP in the presence of 0.6% (w/v) DDM increased to ~1.5-fold after 1h HHP treatment. Fluorescence-detection size-exclusion chromatography demonstrated that the PBANR-EGFP ligand binding ability was retained after HHP-mediated solubilization. The PBANR-EGFP solubilized with 1.0% DDM under HHP at room temperature for 6h retained ligand binding ability, whereas solubilization in the absence of HHP treatment resulted in denaturation.

Structural basis of human NOX5 activation.

NADPH oxidase 5 (NOX5) catalyzes the production of superoxide free radicals and regulates physiological processes from sperm motility to cardiac rhythm. Overexpression of NOX5 leads to cancers, diabetes, and cardiovascular diseases. NOX5 is activated by intracellular calcium signaling, but the underlying molecular mechanism of which - in particular, how calcium triggers electron transfer from NADPH to FAD - is still unclear. Here we capture motions of full-length human NOX5 upon calcium binding using single-particle cryogenic electron microscopy (cryo-EM). By combining biochemistry, mutagenesis analyses, and molecular dynamics (MD) simulations, we decode the molecular basis of NOX5 activation and electron transfer. We find that calcium binding to the EF-hand domain increases NADPH dynamics, permitting electron transfer between NADPH and FAD and superoxide production. Our structural findings also uncover a zinc-binding motif that is important for NOX5 stability and enzymatic activity, revealing modulation mechanisms of reactive oxygen species (ROS) production.

Crystal structure and DNA-binding mode of Klebsiella pneumoniae primosomal PriB protein.

PriB is a primosomal DNA replication protein required for the re-initiation of replication in bacteria. In this study, we investigated the gene expression of PriB in Klebsiella pneumoniae (KpPriB) and characterized the gene product through crystal structural and functional analyses. Quantitative polymerase chain reaction analysis (Q-PCR) indicated that the 104-aa priB was expressed in K. pneumoniae with a C(T) value of 22.4. The crystal structure of KpPriB (Protein Data Bank entry: 4APV) determined at a resolution of 2.1 Å was similar to that of Escherichia coli PriB (EcPriB). KpPriB formed a single complex with single-stranded DNA (ssDNA) of different lengths, suggesting a highly cooperative process. Structure-based mutational analysis revealed that substitution at K18, F42, R44, W47, K82, K84, or K89 but not R34 in KpPriB had a significant effect on both ssDNA and double-stranded DNA (dsDNA) binding. Based on these findings, the known ssDNA interaction sites of PriB were expanded to include R44 and F42, thus allowing nucleic acids to wrap around the whole PriB protein.

Communication network within the essential AAA-ATPase Rix7 drives ribosome assembly.

Rix7 is an essential AAA+ ATPase that functions during the early stages of ribosome biogenesis. Rix7 is composed of three domains including an N-terminal domain (NTD) and two AAA+ domains (D1 and D2) that assemble into an asymmetric stacked hexamer. It was recently established that Rix7 is a presumed protein translocase that removes substrates from preribosomes by translocating them through its central pore. However, how the different domains of Rix7 coordinate their activities within the overall hexameric structure was unknown. We captured cryo-electron microscopy (EM) structures of single and double Walker B variants of full length Rix7. The disordered NTD was not visible in the cryo-EM reconstructions, but cross-linking mass spectrometry revealed that the NTD can associate with the central channel in vitro. Deletion of the disordered NTD enabled us to obtain a structure of the Rix7 hexamer to 2.9 Å resolution, providing high resolution details of critical motifs involved in substrate translocation and interdomain communication. This structure coupled with cell-based assays established that the linker connecting the D1 and D2 domains as well as the pore loops lining the central channel are essential for formation of the large ribosomal subunit. Together, our work shows that Rix7 utilizes a complex communication network to drive ribosome biogenesis.

The GTP responsiveness of PI5P4Kß evolved from a compromised trade-off between activity and specificity.

Unlike most kinases, phosphatidylinositol 5-phosphate 4-kinase ß (PI5P4Kß) utilizes GTP as a physiological phosphate donor and regulates cell growth under stress (i.e., GTP-dependent stress resilience). However, the genesis and evolution of its GTP responsiveness remain unknown. Here, we reveal that PI5P4Kß has acquired GTP preference by generating a short dual-nucleotide-recognizing motif called the guanine efficient association (GEA) motif. Comparison of nucleobase recognition with 660 kinases and 128 G proteins has uncovered that most kinases and PI5P4Kß use their main-chain atoms for adenine recognition, while the side-chain atoms are required for guanine recognition. Mutational analysis of the GEA motif revealed that the acquisition of GTP reactivity is accompanied by an extended activity toward inosine triphosphate (ITP) and xanthosine triphosphate (XTP). Along with the evolutionary analysis data that point to strong negative selection of the GEA motif, these results suggest that the GTP responsiveness of PI5P4Kß has evolved from a compromised trade-off between activity and specificity, underpinning the development of the GTP-dependent stress resilience.

The crystal structure of a replicative hexameric helicase DnaC and its complex with single-stranded DNA.

DNA helicases are motor proteins that play essential roles in DNA replication, repair and recombination. In the replicative hexameric helicase, the fundamental reaction is the unwinding of duplex DNA; however, our understanding of this function remains vague due to insufficient structural information. Here, we report two crystal structures of the DnaB-family replicative helicase from Geobacillus kaustophilus HTA426 (GkDnaC) in the apo-form and bound to single-stranded DNA (ssDNA). The GkDnaC-ssDNA complex structure reveals that three symmetrical basic grooves on the interior surface of the hexamer individually encircle ssDNA. The ssDNA-binding pockets in this structure are directed toward the N-terminal domain collar of the hexameric ring, thus orienting the ssDNA toward the DnaG primase to facilitate the synthesis of short RNA primers. These findings provide insight into the mechanism of ssDNA binding and provide a working model to establish a novel mechanism for DNA translocation at the replication fork.

Shaping the Nascent Ribosome: AAA-ATPases in Eukaryotic Ribosome Biogenesis.

AAA-ATPases are molecular engines evolutionarily optimized for the remodeling of proteins and macromolecular assemblies. Three AAA-ATPases are currently known to be involved in the remodeling of the eukaryotic ribosome, a megadalton range ribonucleoprotein complex responsible for the translation of mRNAs into proteins. The correct assembly of the ribosome is performed by a plethora of additional and transiently acting pre-ribosome maturation factors that act in a timely and spatially orchestrated manner. Minimal disorder of the assembly cascade prohibits the formation of functional ribosomes and results in defects in proliferation and growth. Rix7, Rea1, and Drg1, which are well conserved across eukaryotes, are involved in different maturation steps of pre-60S ribosomal particles. These AAA-ATPases provide energy for the efficient removal of specific assembly factors from pre-60S particles after they have fulfilled their function in the maturation cascade. Recent structural and functional insights have provided the first glimpse into the molecular mechanism of target recognition and remodeling by Rix7, Rea1, and Drg1. Here we summarize current knowledge on the AAA-ATPases involved in eukaryotic ribosome biogenesis. We highlight the latest insights into their mechanism of mechano-chemical complex remodeling driven by advanced cryo-EM structures and the use of highly specific AAA inhibitors.

IT'S 2 for the price of 1: Multifaceted ITS2 processing machines in RNA and DNA maintenance.

Cells utilize sophisticated RNA processing machines to ensure the quality of RNA. Many RNA processing machines have been further implicated in regulating the DNA damage response signifying a strong link between RNA processing and genome maintenance. One of the most intricate and highly regulated RNA processing pathways is the processing of the precursor ribosomal RNA (pre-rRNA), which is paramount for the production of ribosomes. Removal of the Internal Transcribed Spacer 2 (ITS2), located between the 5.8S and 25S rRNA, is one of the most complex steps of ribosome assembly. Processing of the ITS2 is initiated by the newly discovered endoribonuclease Las1, which cleaves at the C2 site within the ITS2, generating products that are further processed by the polynucleotide kinase Grc3, the 5'→3' exonuclease Rat1, and the 3'→5' RNA exosome complex. In addition to their defined roles in ITS2 processing, these critical cellular machines participate in other stages of ribosome assembly, turnover of numerous cellular RNAs, and genome maintenance. Here we summarize recent work defining the molecular mechanisms of ITS2 processing by these essential RNA processing machines and highlight their emerging roles in transcription termination, heterochromatin function, telomere maintenance, and DNA repair.

Cryo-EM structure of the essential ribosome assembly AAA-ATPase Rix7.

Rix7 is an essential type II AAA-ATPase required for the formation of the large ribosomal subunit. Rix7 has been proposed to utilize the power of ATP hydrolysis to drive the removal of assembly factors from pre-60S particles, but the mechanism of release is unknown. Rix7's mammalian homolog, NVL2 has been linked to cancer and mental illness disorders, highlighting the need to understand the molecular mechanisms of this essential machine. Here we report the cryo-EM reconstruction of the tandem AAA domains of Rix7 which form an asymmetric stacked homohexameric ring. We trapped Rix7 with a polypeptide in the central channel, revealing Rix7's role as a molecular unfoldase. The structure establishes that type II AAA-ATPases lacking the aromatic-hydrophobic motif within the first AAA domain can engage a substrate throughout the entire central channel. The structure also reveals that Rix7 contains unique post-α7 insertions within both AAA domains important for Rix7 function.

Combining X-Ray Crystallography with Small Angle X-Ray Scattering to Model Unstructured Regions of Nsa1 from S. Cerevisiae.

Determination of the full-length structure of ribosome assembly factor Nsa1 from Saccharomyces cerevisiae (S. cerevisiae) is challenging because of the disordered and protease labile C-terminus of the protein. This manuscript describes the methods to purify recombinant Nsa1 from S. cerevisiae for structural analysis by both X-ray crystallography and SAXS. X-ray crystallography was utilized to solve the structure of the well-ordered N-terminal WD40 domain of Nsa1, and then SAXS was used to resolve the structure of the C-terminus of Nsa1 in solution. Solution scattering data was collected from full-length Nsa1 in solution. The theoretical scattering amplitudes were calculated from the high-resolution crystal structure of the WD40 domain, and then a combination of rigid body and ab initio modeling revealed the C-terminus of Nsa1. Through this hybrid approach the quaternary structure of the entire protein was reconstructed. The methods presented here should be generally applicable for the hybrid structural determination of other proteins composed of a mix of structured and unstructured domains.

Structural Analysis Reveals Features of Ribosome Assembly Factor Nsa1/WDR74 Important for Localization and Interaction with Rix7/NVL2.

Ribosome assembly is a complex process that requires hundreds of essential assembly factors, including Rix7 (NVL2 in mammals) and Nsa1 (WDR74 in mammals). Rix7 is a type II double ring, AAA-ATPase, which is closely related to the well-known Cdc48/p97. Previous studies in Saccharomyces cerevisiae suggest that Rix7 mediates the release of Nsa1 from nucleolar pre-60S particles; however, the underlying mechanisms of this release are unknown. Through multiple structural analyses we show that S. cerevisiae Nsa1 is composed of an N-terminal seven-bladed WD40 domain followed by a lysine-rich C terminus that extends away from the WD40 domain and is required for nucleolar localization. Co-immunoprecipitation assays with the mammalian homologs identified a well-conserved interface within WDR74 that is important for its association with NVL2. We further show that WDR74 associates with the D1 AAA domain of NVL2, which represents a novel mode of binding of a substrate with a type II AAA-ATPase.

Structural reverse genetics study of the PI5P4Kß-nucleotide complexes reveals the presence of the GTP bioenergetic system in mammalian cells.

Reverse genetic analysis can connect a gene and its protein counterpart to a biological function(s) by knockout or knockdown of the specific gene. However, when a protein has multiple biochemical activities, the conventional genetics strategy is incapable of distinguishing which biochemical activity of the protein is critical for the particular biological function(s). Here, we propose a structural reverse genetics strategy to overcome this problem. In a structural reverse genetics study, multiple biochemical activities of a protein are segregated by mapping those activities to a structural element(s) in the atomic resolution tertiary structure. Based on the structural mapping, a mutant lacking one biochemical activity of interest can be produced with the other activities kept intact. Expression of the mutant by knockin or ectopic expression in the knockout strain along with the following analysis can connect the single biochemical activity of interest to a biological function. Using the structural reverse genetics strategy, we have dissected the newly identified GTP-dependent activity of a lipid kinase PI5P4Kß from its ATP-dependent activity. The GTP-insensitive mutant has demonstrated the existence of the GTP bioenergetic sensor system in mammalian cells and its critical role in tumorigenesis. As structural reverse genetics can identify in vivo significance of individual biochemical activity, it is a powerful approach to reveal hidden biological functions, which could be a novel pharmacological target for therapeutic intervention. Given the recent expansion of choices in structural biological methods and advances in genome editing technologies, the time is ripe for structural reverse genetics strategies.

Mutations altering the interplay between GkDnaC helicase and DNA reveal an insight into helicase unwinding.

Replicative helicases are essential molecular machines that utilize energy derived from NTP hydrolysis to move along nucleic acids and to unwind double-stranded DNA (dsDNA). Our earlier crystal structure of the hexameric helicase from Geobacillus kaustophilus HTA426 (GkDnaC) in complex with single-stranded DNA (ssDNA) suggested several key residues responsible for DNA binding that likely play a role in DNA translocation during the unwinding process. Here, we demonstrated that the unwinding activities of mutants with substitutions at these key residues in GkDnaC are 2-4-fold higher than that of wild-type protein. We also observed the faster unwinding velocities in these mutants using single-molecule experiments. A partial loss in the interaction of helicase with ssDNA leads to an enhancement in helicase efficiency, while their ATPase activities remain unchanged. In strong contrast, adding accessory proteins (DnaG or DnaI) to GkDnaC helicase alters the ATPase, unwinding efficiency and the unwinding velocity of the helicase. It suggests that the unwinding velocity of helicase could be modulated by two different pathways, the efficiency of ATP hydrolysis or protein-DNA interaction.

Molecular interplay between the replicative helicase DnaC and its loader protein DnaI from Geobacillus kaustophilus.

Helicase loading factors are thought to transfer the hexameric ring-shaped helicases onto the replication fork during DNA replication. However, the mechanism of helicase transfer onto DNA remains unclear. In Bacillus subtilis, the protein DnaI, which belongs to the AAA+ family of ATPases, is responsible for delivering the hexameric helicase DnaC onto DNA. Here we investigated the interaction between DnaC and DnaI from Geobacillus kaustophilus HTA426 (GkDnaC and GkDnaI, respectively) and determined that GkDnaI forms a stable complex with GkDnaC with an apparent stoichiometry of GkDnaC(6)-GkDnaI(6) in the absence of ATP. Surface plasmon resonance analysis indicated that GkDnaI facilitates loading of GkDnaC onto single-stranded DNA (ssDNA) and supports complex formation with ssDNA in the presence of ATP. Additionally, the GkDnaI C-terminal AAA+ domain alone could bind ssDNA, and binding was modulated by nucleotides. We also determined the crystal structure of the C-terminal AAA+ domain of GkDnaI in complex with ADP at 2.5 A resolution. The structure not only delineates the binding of ADP in the expected Walker A and B motifs but also reveals a positively charged region that may be involved in ssDNA binding. These findings provide insight into the mechanism of replicative helicase loading onto ssDNA.