Search

Protein Ser/Thr phosphatase PP1 is always associated with one or two regulatory subunits or RIPPOs. One of the earliest evolved RIPPOs is PPP1R2, also known as Inhibitor-2. Since its discovery nearly 5 decades ago, PPP1R2 has been variously described as an inhibitor, activator or (metal) chaperone of PP1, but it is still unknown how PPP1R2 affects the function of PP1 in intact cells. Here, using specific research tools, we demonstrate that PPP1R2 stabilises a subgroup of PP1 holoenzymes, exemplified by PP1:RepoMan, thereby promoting the dephosphorylation of their substrates. Mechanistically, the recruitment of PPP1R2 disrupts an inhibitory, fuzzy interaction between the C-terminal tail and catalytic domain of PP1, and generates an additional C-terminal RepoMan-interaction site. The resulting holoenzyme is further stabilized by a direct PPP1R2:RepoMan interaction, which renders it refractory to competitive disruption by RIPPOs that do not interact with PPP1R2. Our data demonstrate that PPP1R2 modulates the function of PP1 by altering the balance between holoenzymes through stabilisation of specific subunit interactions., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

In eukaryotes, the leading strand DNA is synthesized by Polε and the lagging strand by Polδ. These replicative polymerases have higher processivity when paired with the DNA clamp PCNA. While the structure of the yeast Polε catalytic domain has been determined, how Polε interacts with PCNA is unknown in any eukaryote, human or yeast. Here we report two cryo-EM structures of human Polε-PCNA-DNA complex, one in an incoming nucleotide bound state and the other in a nucleotide exchange state. The structures reveal an unexpected three-point interface between the Polε catalytic domain and PCNA, with the conserved PIP (PCNA interacting peptide)-motif, the unique P-domain, and the thumb domain each interacting with a different protomer of the PCNA trimer. We propose that the multi-point interface prevents other PIP-containing factors from recruiting to PCNA while PCNA functions with Polε. Comparison of the two states reveals that the finger domain pivots around the [4Fe-4S] cluster-containing tip of the P-domain to regulate nucleotide exchange and incoming nucleotide binding., (© 2024. The Author(s).)

Herpes simplex virus 1 (HSV-1), a double-stranded DNA virus, replicates using seven essential proteins encoded by its genome. Among these, the UL30 DNA polymerase, complexed with the UL42 processivity factor, orchestrates leading and lagging strand replication of the 152 kb viral genome. UL30 polymerase is a prime target for antiviral therapy, and resistance to current drugs can arise in immunocompromised individuals. Using electron cryo-microscopy (cryo-EM), we unveil the dynamic changes of the UL30/UL42 complex with DNA in three distinct states. First, a pre-translocation state with an open fingers domain ready for nucleotide incorporation. Second, a halted elongation state where the fingers close, trapping dATP in the dNTP pocket. Third, a DNA-editing state involving significant conformational changes to allow DNA realignment for exonuclease activity. Additionally, the flexible UL30 C-terminal domain interacts with UL42, forming an extended positively charged surface binding to DNA, thereby enhancing processive synthesis. These findings highlight substantial structural shifts in the polymerase and its DNA interactions during replication, offering insights for future antiviral drug development., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The calcium calmodulin protein kinase II (CaMKII) is a multi-subunit ring assembly with a central hub formed by the association domains. There is evidence for hub polymorphism between and within CaMKII isoforms, but the link between polymorphism and subunit exchange has not been resolved. Here, we present near-atomic resolution cryogenic electron microscopy (cryo-EM) structures revealing that hubs from the α and β isoforms, either standalone or within an β holoenzyme, coexist as 12 and 14 subunit assemblies. Single-molecule fluorescence microscopy of Venus-tagged holoenzymes detects intermediate assemblies and progressive dimer loss due to intrinsic holoenzyme lability, and holoenzyme disassembly into dimers upon mutagenesis of a conserved inter-domain contact. Molecular dynamics (MD) simulations show the flexibility of 4-subunit precursors, extracted in-silico from the β hub polymorphs, encompassing the curvature of both polymorphs. The MD explains how an open hub structure also obtained from the β holoenzyme sample could be created by dimer loss and analysis of its cryo-EM dataset reveals how the gap could open further. An assembly model, considering dimer concentration dependence and strain differences between polymorphs, proposes a mechanism for intrinsic hub lability to fine-tune the stoichiometry of αβ heterooligomers for their dynamic localization within synapses in neurons., (© 2024. The Author(s).)

The year 2022 was marked by the mpox outbreak caused by the human monkeypox virus (MPXV), which is approximately 98% identical to the vaccinia virus (VACV) at the sequence level with regard to the proteins involved in DNA replication. We present the production in the baculovirus-insect cell system of the VACV DNA polymerase holoenzyme, which consists of the E9 polymerase in combination with its co-factor, the A20-D4 heterodimer. This led to the 3.8 Å cryo-electron microscopy (cryo-EM) structure of the DNA-free form of the holoenzyme. The model of the holoenzyme was constructed from high-resolution structures of the components of the complex and the A20 structure predicted by AlphaFold 2. The structures do not change in the context of the holoenzyme compared to the previously determined crystal and NMR structures, but the E9 thumb domain became disordered. The E9-A20-D4 structure shows the same compact arrangement with D4 folded back on E9 as observed for the recently solved MPXV holoenzyme structures in the presence and the absence of bound DNA. A conserved interface between E9 and D4 is formed by a cluster of hydrophobic residues. Small-angle X-ray scattering data show that other, more open conformations of E9-A20-D4 without the E9-D4 contact exist in solution using the flexibility of two hinge regions in A20. Biolayer interferometry (BLI) showed that the E9-D4 interaction is indeed weak and transient in the absence of DNA although it is very important, as it has not been possible to obtain viable viruses carrying mutations of key residues within the E9-D4 interface., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Burmeister et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The binding of calcium/calmodulin (CAM) to calcium/calmodulin-dependent protein kinase II (CaMKII) initiates an ATP-driven cascade that triggers CaMKII autophosphorylation. The autophosphorylation in turn increases the CaMKII affinity for CAM. Here, we studied the ATP dependence of CAM association with the actin-binding CaMKIIβ isoform using single-molecule total internal reflection fluorescence microscopy. Rhodamine-CAM associations/dissociations to surface-immobilized Venus-CaMKIIβ were resolved with 0.5 s resolution from video records, batch-processed with a custom algorithm. CAM occupancy was determined simultaneously with spot-photobleaching measurement of CaMKII holoenzyme stoichiometry. We show the ATP-dependent increase of the CAM association requires dimer formation for both the α and β isoforms. The study of mutant β holoenzymes revealed that the ATP-dependent increase in CAM affinity results in two distinct states. The phosphorylation-defective (T287.306-307A) holoenzyme resides only in the low-affinity state. CAM association is further reduced in the T287A holoenzyme relative to T287.306-307A. In the absence of ATP, the affinity of CAM for the T287.306-307A mutant and the wild-type monomer are comparable. The affinity of the ATP-binding impaired (K43R) mutant is even weaker. In ATP, the K43R holoenzyme resides in the low-affinity state. The phosphomimetic mutant (T287D) resides only in a 1000-fold higher-affinity state, with mean CAM occupancy of more than half of the 14-mer holoenzyme stoichiometry in picomolar CAM. ATP promotes T287D holoenzyme disassembly but does not elevate CAM occupancy. Single Poisson distributions characterized the ATP-dependent CAM occupancy of mutant holoenzymes. In contrast, the CAM occupancy of the wild-type population had a two-state distribution with both low- and high-affinity states represented. The low-affinity state was the dominant state, a result different from published in vitro assays. Differences in assay conditions can alter the balance between activating and inhibitory autophosphorylation. Bound ATP could be sufficient for CaMKII structural function, while antagonistic autophosphorylations may tune CaMKII kinase-regulated action-potential frequency decoding in vivo., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The serine/threonine protein kinase CK2 is implicated in the regulation of fundamental processes in eukaryotic cells. CK2 consists of two catalytic α or α' isoforms and two regulatory CK2β subunits. These three proteins exist in a free form, bound to other cellular proteins, as tetrameric holoenzymes composed of CK2α 2 /β 2 , CK2αα'/β 2 , or CK2α' 2 /β 2 as well as in higher molecular forms of the tetramers. The catalytic domains of CK2α and CK2α' share a 90% identity. As CK2α contains a unique C-terminal sequence. Both proteins function as protein kinases. These properties raised the question of whether both isoforms are just backups of each other or whether they are regulated differently and may then function in an isoform-specific manner. The present review provides observations that the regulation of both CK2α isoforms is partly different concerning the subcellular localization, post-translational modifications, and aggregation. Up to now, there are only a few isoform-specific cellular binding partners. The expression of both CK2α isoforms seems to vary in different cell lines, in tissues, in the cell cycle, and with differentiation. There are different reports about the expression and the functions of the CK2α isoforms in tumor cells and tissues. In many cases, a cell-type-specific expression and function is known, which raises the question about cell-specific regulators of both isoforms. Another future challenge is the identification or design of CK2α'-specific inhibitors.

Transposable elements are genomic parasites that expand within and spread between genomes 1 . PIWI proteins control transposon activity, notably in the germline 2,3 . These proteins recognize their targets through small RNA co-factors named PIWI-interacting RNAs (piRNAs), making piRNA biogenesis a key specificity-determining step in this crucial genome immunity system. Although the processing of piRNA precursors is an essential step in this process, many of the molecular details remain unclear. Here, we identify an endoribonuclease, precursor of 21U RNA 5'-end cleavage holoenzyme (PUCH), that initiates piRNA processing in the nematode Caenorhabditis elegans. Genetic and biochemical studies show that PUCH, a trimer of Schlafen-like-domain proteins (SLFL proteins), executes 5'-end piRNA precursor cleavage. PUCH-mediated processing strictly requires a 7-methyl-G cap (m 7 G-cap) and a uracil at position three. We also demonstrate how PUCH interacts with PETISCO, a complex that binds to piRNA precursors 4 , and that this interaction enhances piRNA production in vivo. The identification of PUCH concludes the search for the 5'-end piRNA biogenesis factor in C. elegans and uncovers a type of RNA endonuclease formed by three SLFL proteins. Mammalian Schlafen (SLFN) genes have been associated with immunity 5 , exposing a molecular link between immune responses in mammals and deeply conserved RNA-based mechanisms that control transposable elements., (© 2023. The Author(s).)

CRISPR-associated transposons (CAST) are programmable mobile genetic elements that insert large DNA cargos using an RNA-guided mechanism 1-3 . CAST elements contain multiple conserved proteins: a CRISPR effector (Cas12k or Cascade), a AAA+ regulator (TnsC), a transposase (TnsA-TnsB) and a target-site-associated factor (TniQ). These components are thought to cooperatively integrate DNA via formation of a multisubunit transposition integration complex (transpososome). Here we reconstituted the approximately 1 MDa type V-K CAST transpososome from Scytonema hofmannii (ShCAST) and determined its structure using single-particle cryo-electon microscopy. The architecture of this transpososome reveals modular association between the components. Cas12k forms a complex with ribosomal subunit S15 and TniQ, stabilizing formation of a full R-loop. TnsC has dedicated interaction interfaces with TniQ and TnsB. Of note, we observe TnsC-TnsB interactions at the C-terminal face of TnsC, which contribute to the stimulation of ATPase activity. Although the TnsC oligomeric assembly deviates slightly from the helical configuration found in isolation, the TnsC-bound target DNA conformation differs markedly in the transpososome. As a consequence, TnsC makes new protein-DNA interactions throughout the transpososome that are important for transposition activity. Finally, we identify two distinct transpososome populations that differ in their DNA contacts near TniQ. This suggests that associations with the CRISPR effector can be flexible. This ShCAST transpososome structure enhances our understanding of CAST transposition systems and suggests ways to improve CAST transposition for precision genome-editing applications., (© 2022. The Author(s).)

Telomeres are the physical ends of linear chromosomes. They are composed of short repeating sequences (such as TTGGGG in the G-strand for Tetrahymena thermophila) of double-stranded DNA with a single-strand 3' overhang of the G-strand and, in humans, the six shelterin proteins: TPP1, POT1, TRF1, TRF2, RAP1 and TIN2 1,2 . TPP1 and POT1 associate with the 3' overhang, with POT1 binding the G-strand 3 and TPP1 (in complex with TIN2 4 ) recruiting telomerase via interaction with telomerase reverse transcriptase 5 (TERT). The telomere DNA ends are replicated and maintained by telomerase 6 , for the G-strand, and subsequently DNA polymerase α-primase 7,8 (PolαPrim), for the C-strand 9 . PolαPrim activity is stimulated by the heterotrimeric complex CTC1-STN1-TEN1 10-12 (CST), but the structural basis of the recruitment of PolαPrim and CST to telomere ends remains unknown. Here we report cryo-electron microscopy (cryo-EM) structures of Tetrahymena CST in the context of the telomerase holoenzyme, in both the absence and the presence of PolαPrim, and of PolαPrim alone. Tetrahymena Ctc1 binds telomerase subunit p50, a TPP1 orthologue, on a flexible Ctc1 binding motif revealed by cryo-EM and NMR spectroscopy. The PolαPrim polymerase subunit POLA1 binds Ctc1 and Stn1, and its interface with Ctc1 forms an entry port for G-strand DNA to the POLA1 active site. We thus provide a snapshot of four key components that are required for telomeric DNA synthesis in a single active complex-telomerase-core ribonucleoprotein, p50, CST and PolαPrim-that provides insights into the recruitment of CST and PolαPrim and the handoff between G-strand and C-strand synthesis., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Methanobactins (Mbns) are a family of copper-binding peptides involved in copper uptake by methanotrophs, and are potential therapeutic agents for treating diseases characterized by disordered copper accumulation. Mbns are produced via modification of MbnA precursor peptides at cysteine residues catalyzed by the core biosynthetic machinery containing MbnB, an iron-dependent enzyme, and MbnC. However, mechanistic details underlying the catalysis of the MbnBC holoenzyme remain unclear. Here, we present crystal structures of MbnABC complexes from two distinct species, revealing that the leader peptide of the substrate MbnA binds MbnC for recruitment of the MbnBC holoenzyme, while the core peptide of MbnA resides in the catalytic cavity created by the MbnB-MbnC interaction which harbors a unique tri-iron cluster. Ligation of the substrate sulfhydryl group to the tri-iron center achieves a dioxygen-dependent reaction for oxazolone-thioamide installation. Structural analysis of the MbnABC complexes together with functional investigation of MbnB variants identified a conserved catalytic aspartate residue as a general base required for MbnBC-mediated MbnA modification. Together, our study reveals the similar architecture and function of MbnBC complexes from different species, demonstrating an evolutionarily conserved catalytic mechanism of the MbnBC holoenzymes., (© 2022. The Author(s).)

The vacuolar ATPase (V-ATPase) is a rotary motor proton pump that is regulated by an assembly equilibrium between active holoenzyme and autoinhibited V 1 -ATPase and V o proton channel subcomplexes. Here, we report cryo-EM structures of yeast V-ATPase assembled in vitro from lipid nanodisc reconstituted V o and mutant V 1 . Our analysis identified holoenzymes in three active rotary states, indicating that binding of V 1 to V o provides sufficient free energy to overcome V o autoinhibition. Moreover, the structures suggest that the unequal spacing of V o 's proton-carrying glutamic acid residues serves to alleviate the symmetry mismatch between V 1 and V o motors, a notion that is supported by mutagenesis experiments. We also uncover a structure of free V 1 bound to Oxr1, a conserved but poorly characterized factor involved in the oxidative stress response. Biochemical experiments show that Oxr1 inhibits V 1 -ATPase and causes disassembly of the holoenzyme, suggesting that Oxr1 plays a direct role in V-ATPase regulation., (© 2021 The Authors Published under the terms of the CC BY NC ND 4.0 license.)

Single-particle cryogenic electron microscopy (cryo-EM) has become a standard technique for determining protein structures at atomic resolution 1-3 . However, cryo-EM studies of protein-free RNA are in their early days. The Tetrahymena thermophila group I self-splicing intron was the first ribozyme to be discovered and has been a prominent model system for the study of RNA catalysis and structure-function relationships 4 , but its full structure remains unknown. Here we report cryo-EM structures of the full-length Tetrahymena ribozyme in substrate-free and bound states at a resolution of 3.1 Å. Newly resolved peripheral regions form two coaxially stacked helices; these are interconnected by two kissing loop pseudoknots that wrap around the catalytic core and include two previously unforeseen (to our knowledge) tertiary interactions. The global architecture is nearly identical in both states; only the internal guide sequence and guanosine binding site undergo a large conformational change and a localized shift, respectively, upon binding of RNA substrates. These results provide a long-sought structural view of a paradigmatic RNA enzyme and signal a new era for the cryo-EM-based study of structure-function relationships in ribozymes., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Telomerase adds telomeric repeats at chromosome ends to compensate for the telomere loss that is caused by incomplete genome end replication 1 . In humans, telomerase is upregulated during embryogenesis and in cancers, and mutations that compromise the function of telomerase result in disease 2 . A previous structure of human telomerase at a resolution of 8 Å revealed a vertebrate-specific composition and architecture 3 , comprising a catalytic core that is flexibly tethered to an H and ACA (hereafter, H/ACA) box ribonucleoprotein (RNP) lobe by telomerase RNA. High-resolution structural information is necessary to develop treatments that can effectively modulate telomerase activity as a therapeutic approach against cancers and disease. Here we used cryo-electron microscopy to determine the structure of human telomerase holoenzyme bound to telomeric DNA at sub-4 Å resolution, which reveals crucial DNA- and RNA-binding interfaces in the active site of telomerase as well as the locations of mutations that alter telomerase activity. We identified a histone H2A-H2B dimer within the holoenzyme that was bound to an essential telomerase RNA motif, which suggests a role for histones in the folding and function of telomerase RNA. Furthermore, this structure of a eukaryotic H/ACA RNP reveals the molecular recognition of conserved RNA and protein motifs, as well as interactions that are crucial for understanding the molecular pathology of many mutations that cause disease. Our findings provide the structural details of the assembly and active site of human telomerase, which paves the way for the development of therapeutic agents that target this enzyme.

Ribosomal RNA (rRNA) is most highly expressed in rapidly growing bacteria and is drastically downregulated under stress conditions by the global transcriptional regulator DksA and the alarmone ppGpp. Here, we determined cryo-electron microscopy structures of the Escherichia coli RNA polymerase (RNAP) σ 70 holoenzyme during rRNA promoter recognition with and without DksA/ppGpp. RNAP contacts the UP element using dimerized α subunit carboxyl-terminal domains and scrunches the template DNA with the σ finger and β' lid to select the transcription start site favorable for rapid promoter escape. Promoter binding induces conformational change of σ domain 2 that opens a gate for DNA loading and ejects σ 1.1 from the RNAP cleft to facilitate open complex formation. DksA/ppGpp binding also opens the DNA loading gate, which is not coupled to σ 1.1 ejection and impedes open complex formation. These results provide a molecular basis for the exceptionally active rRNA transcription and its vulnerability to DksA/ppGpp.

All cellular genetic information is transcribed into RNA by multisubunit RNA polymerases (RNAPs). The basal transcription initiation factors of cellular RNAPs stimulate the initial RNA synthesis via poorly understood mechanisms. Here, we explored the mechanism employed by the bacterial factor σ in promoter-independent initial transcription. We found that the RNAP holoenzyme lacking the promoter-binding domain σ4 is ineffective in de novo transcription initiation and displays high propensity to pausing upon extension of RNAs 3 to 7 nucleotides in length. The nucleotide at the RNA 3' end determines the pause lifetime. The σ4 domain stabilizes short RNA:DNA hybrids and suppresses pausing by stimulating RNAP active-center translocation. The antipausing activity of σ4 is modulated by its interaction with the β subunit flap domain and by the σ remodeling factors AsiA and RbpA. Our results suggest that the presence of σ4 within the RNA exit channel compensates for the intrinsic instability of short RNA:DNA hybrids by increasing RNAP processivity, thus favoring productive transcription initiation. This "RNAP boosting" activity of the initiation factor is shaped by the thermodynamics of RNA:DNA interactions and thus, should be relevant for any factor-dependent RNAP., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The nonreceptor protein tyrosine kinase Fyn and protein Ser/Thr phosphatase 2A (PP2A) are major multifunctional signaling molecules. Deregulation of Fyn and altered PP2A methylation are implicated in cancer and Alzheimer's disease (AD). Here, we tested the hypothesis that the methylation state of PP2A catalytic subunit, which influences PP2A subunit composition and substrate specificity, can affect Fyn regulation and function. Using Neuro-2a (N2a) neuroblastoma cell models, we first show that methylated PP2A holoenzymes containing the Bα subunit coimmunoprecipitate and copurify with Fyn in membrane rafts. PP2A methylation status regulates Fyn distribution and Fyn-dependent neuritogenesis, likely in part by affecting actin dynamics. A methylation-incompetent PP2A mutant fails to interact with Fyn. It perturbs the normal partitioning of Fyn and amyloid precursor protein (APP) in membrane microdomains, which governs Fyn function and APP processing. This correlates with enhanced amyloidogenic cleavage of APP, a hallmark of AD pathogenesis. Conversely, enhanced PP2A methylation promotes the nonamyloidogenic cleavage of APP in a Fyn-dependent manner. Disturbances in one-carbon metabolic pathways that control cellular methylation are associated with AD and cancer. Notably, they induce a parallel loss of membrane-associated methylated PP2A and Fyn enzymes in N2a cells and acute mouse brain slices. One-carbon metabolism also modulates Fyn-dependent process outgrowth in N2a cells. Thus, our findings identify a novel methylation-dependent PP2A/Fyn signaling module. They highlight the underestimated importance of cross talks between essential metabolic pathways and signaling scaffolds that are involved in normal cell homeostasis and currently being targeted for cancer and AD treatment., Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Protein phosphorylation is a post-translational modification essential for the control of the activity of most enzymes in the cell. This protein modification results from a fine-tuned balance between kinases and phosphatases. PP2A is one of the major serine/threonine phosphatases that is involved in the control of a myriad of different signaling cascades. This enzyme, often misregulated in cancer, is considered a tumor suppressor. In this review, we will focus on PP2A-B55, a particular holoenzyme of the family of the PP2A phosphatases whose specific role in cancer development and progression has only recently been highlighted. The discovery of the Greatwall (Gwl)/Arpp19-ENSA cascade, a new pathway specifically controlling PP2A-B55 activity, has been shown to be frequently altered in cancer. Herein, we will review the current knowledge about the mechanisms controlling the formation and the regulation of the activity of this phosphatase and its misregulation in cancer.

PRKACA and PRKACB code for two catalytic subunits (Cα and Cβ) of cAMP-dependent protein kinase (PKA), a pleiotropic holoenzyme that regulates numerous fundamental biological processes such as metabolism, development, memory, and immune response. We report seven unrelated individuals presenting with a multiple congenital malformation syndrome in whom we identified heterozygous germline or mosaic missense variants in PRKACA or PRKACB. Three affected individuals were found with the same PRKACA variant, and the other four had different PRKACB mutations. In most cases, the mutations arose de novo, and two individuals had offspring with the same condition. Nearly all affected individuals and their affected offspring shared an atrioventricular septal defect or a common atrium along with postaxial polydactyly. Additional features included skeletal abnormalities and ectodermal defects of variable severity in five individuals, cognitive deficit in two individuals, and various unusual tumors in one individual. We investigated the structural and functional consequences of the variants identified in PRKACA and PRKACB through the use of several computational and experimental approaches, and we found that they lead to PKA holoenzymes which are more sensitive to activation by cAMP than are the wild-type proteins. Furthermore, expression of PRKACA or PRKACB variants detected in the affected individuals inhibited hedgehog signaling in NIH 3T3 fibroblasts, thereby providing an underlying mechanism for the developmental defects observed in these cases. Our findings highlight the importance of both Cα and Cβ subunits of PKA during human development., (Copyright © 2020 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

PPP-family phosphatases such as PP1 have little intrinsic specificity. Cofactors can target PP1 to substrates or subcellular locations, but it remains unclear how they might confer sequence-specificity on PP1. The cytoskeletal regulator Phactr1 is a neuronally enriched PP1 cofactor that is controlled by G-actin. Structural analysis showed that Phactr1 binding remodels PP1's hydrophobic groove, creating a new composite surface adjacent to the catalytic site. Using phosphoproteomics, we identified mouse fibroblast and neuronal Phactr1/PP1 substrates, which include cytoskeletal components and regulators. We determined high-resolution structures of Phactr1/PP1 bound to the dephosphorylated forms of its substrates IRSp53 and spectrin αII. Inversion of the phosphate in these holoenzyme-product complexes supports the proposed PPP-family catalytic mechanism. Substrate sequences C-terminal to the dephosphorylation site make intimate contacts with the composite Phactr1/PP1 surface, which are required for efficient dephosphorylation. Sequence specificity explains why Phactr1/PP1 exhibits orders-of-magnitude enhanced reactivity towards its substrates, compared to apo-PP1 or other PP1 holoenzymes., Competing Interests: RF, MP, AB, RL, NO, HF, AS, NE, SU, SM, RT No competing interests declared, (© 2020, Fedoryshchak et al.)

SARS-CoV-2 is the causative agent of the 2019-2020 pandemic. The SARS-CoV-2 genome is replicated and transcribed by the RNA-dependent RNA polymerase holoenzyme (subunits nsp7/nsp8 2 /nsp12) along with a cast of accessory factors. One of these factors is the nsp13 helicase. Both the holo-RdRp and nsp13 are essential for viral replication and are targets for treating the disease COVID-19. Here we present cryoelectron microscopic structures of the SARS-CoV-2 holo-RdRp with an RNA template product in complex with two molecules of the nsp13 helicase. The Nidovirales order-specific N-terminal domains of each nsp13 interact with the N-terminal extension of each copy of nsp8. One nsp13 also contacts the nsp12 thumb. The structure places the nucleic acid-binding ATPase domains of the helicase directly in front of the replicating-transcribing holo-RdRp, constraining models for nsp13 function. We also observe ADP-Mg 2+ bound in the nsp12 N-terminal nidovirus RdRp-associated nucleotidyltransferase domain, detailing a new pocket for anti-viral therapy development., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The phosphatases PP1 and PP2A are responsible for the majority of dephosphorylation reactions on phosphoserine (pSer) and phosphothreonine (pThr), and are involved in virtually all cellular processes and numerous diseases. The catalytic subunits exist in cells in form of holoenzymes, which impart substrate specificity. The contribution of the catalytic subunits to the recognition of substrates is unclear. By developing a phosphopeptide library approach and a phosphoproteomic assay, we demonstrate that the specificity of PP1 and PP2A holoenzymes towards pThr and of PP1 for basic motifs adjacent to the phosphorylation site are due to intrinsic properties of the catalytic subunits. Thus, we dissect this amino acid specificity of the catalytic subunits from the contribution of regulatory proteins. Furthermore, our approach enables discovering a role for PP1 as regulator of the GRB-associated-binding protein 2 (GAB2)/14-3-3 complex. Beyond this, we expect that this approach is broadly applicable to detect enzyme-substrate recognition preferences.

Recent studies of bacterial DNA replication have led to a picture of the replisome as an entity that freely exchanges DNA polymerases and displays intermittent coupling between the helicase and polymerase(s). Challenging the textbook model of the polymerase holoenzyme acting as a stable complex coordinating the replisome, these observations suggest a role of the helicase as the central organizing hub. We show here that the molecular origin of this newly found plasticity lies in the 500-fold increase in strength of the interaction between the polymerase holoenzyme and the replicative helicase upon association of the primase with the replisome. By combining in vitro ensemble-averaged and single-molecule assays, we demonstrate that this conformational switch operates during replication and promotes recruitment of multiple holoenzymes at the fork. Our observations provide a molecular mechanism for polymerase exchange and offer a revised model for the replication reaction that emphasizes its stochasticity., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Most eukaryotic mRNAs harbor a characteristic 5' m7GpppN cap that promotes pre-mRNA splicing, mRNA nucleocytoplasmic transport and translation while also protecting mRNAs from exonucleolytic attacks. mRNA caps are eliminated by Dcp2 during mRNA decay, allowing 5'-3' exonucleases to degrade mRNA bodies. However, the Dcp2 decapping enzyme is poorly active on its own and requires binding to stable or transient protein partners to sever the cap of target mRNAs. Here, we analyse the role of one of these partners, the yeast Pby1 factor, which is known to co-localize into P-bodies together with decapping factors. We report that Pby1 uses its C-terminal domain to directly bind to the decapping enzyme. We solved the structure of this Pby1 domain alone and bound to the Dcp1-Dcp2-Edc3 decapping complex. Structure-based mutant analyses reveal that Pby1 binding to the decapping enzyme is required for its recruitment into P-bodies. Moreover, Pby1 binding to the decapping enzyme stimulates growth in conditions in which decapping activation is compromised. Our results point towards a direct connection of Pby1 with decapping and P-body formation, both stemming from its interaction with the Dcp1-Dcp2 holoenzyme., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) is a Ser/Thr kinase necessary for long-term memory formation and other Ca 2+ -dependent signaling cascades such as fertilization. Here, we investigated the stability of CaMKIIα using a combination of differential scanning calorimetry (DSC), X-ray crystallography, and mass photometry (MP). The kinase domain has a low thermal stability (apparent T m = 36°C), which is slightly stabilized by ATP/MgCl 2 binding (apparent T m = 40°C) and significantly stabilized by regulatory segment binding (apparent T m = 60°C). We crystallized the kinase domain of CaMKII bound to p-coumaric acid in the active site. This structure reveals solvent-exposed hydrophobic residues in the substrate-binding pocket, which are normally buried in the autoinhibited structure when the regulatory segment is present. This likely accounts for the large stabilization that we observe in DSC measurements comparing the kinase alone with the kinase plus regulatory segment. The hub domain alone is extremely stable (apparent T m  ~ 90°C), and the holoenzyme structure has multiple unfolding transitions ranging from ~60°C to 100°C. Using MP, we compared a CaMKIIα holoenzyme with different variable linker regions and determined that the dissociation of both these holoenzymes occurs at a higher concentration (is less stable) compared with the hub domain alone. We conclude that within the context of the holoenzyme structure, the kinase domain is stabilized, whereas the hub domain is destabilized. These data support a model where domains within the holoenzyme interact., (© 2020 The Protein Society.)

Cholesterol is an essential component of mammalian cell membranes, constituting up to 50% of plasma membrane lipids. By contrast, it accounts for only 5% of lipids in the endoplasmic reticulum (ER) 1 . The ER enzyme sterol O-acyltransferase 1 (also named acyl-coenzyme A:cholesterol acyltransferase, ACAT1) transfers a long-chain fatty acid to cholesterol to form cholesteryl esters that coalesce into cytosolic lipid droplets. Under conditions of cholesterol overload, ACAT1 maintains the low cholesterol concentration of the ER and thereby has an essential role in cholesterol homeostasis 2,3 . ACAT1 has also been implicated in Alzheimer's disease 4 , atherosclerosis 5 and cancers 6 . Here we report a cryo-electron microscopy structure of human ACAT1 in complex with nevanimibe 7 , an inhibitor that is in clinical trials for the treatment of congenital adrenal hyperplasia. The ACAT1 holoenzyme is a tetramer that consists of two homodimers. Each monomer contains nine transmembrane helices (TMs), six of which (TM4-TM9) form a cavity that accommodates nevanimibe and an endogenous acyl-coenzyme A. This cavity also contains a histidine that has previously been identified as essential for catalytic activity 8 . Our structural data and biochemical analyses provide a physical model to explain the process of cholesterol esterification, as well as details of the interaction between nevanimibe and ACAT1, which may help to accelerate the development of ACAT1 inhibitors to treat related diseases.

Both OGT1 ( O -linked β-N-acetylglucosamine ( O -GlcNAc) transferase isoform 1) and NSL3 (nonspecific lethal protein 3) are crucial components of the MOF (males absent on the first)/NSL histone acetyltransferase complex. We previously described how global histone H4 acetylation levels were modulated by OGT1/ O -GlcNAcylation-mediated NSL3 stability. However, the specific modification site of NSL3 and its molecular mechanism of protein stability remain unknown. Here, we present evidence from biochemical experiments arguing that O -GlcNAcylation of NSL3 at Thr755 is tightly associated with holoenzyme activity of the MOF/NSL complex. Using in vitro O-GlcNAc-transferase assays combined with mass spectrometry, we suppose that the residue Thr755 on NSL3 C-terminus is the major site O-GlcNAc-modified by OGT1. Importantly, O-GlcNAcylation of this site is involved in the regulation of the ubiquitin-degradation of NSL3, because this site mutation (T755A) promotes the ubiquitin-mediated degradation of NSL3. Further in-depth research found that ubiquitin conjugating enzyme E2 S (UBE2S) accelerated the degradation of NSL3 via direct binding to it. Interestingly, OGT1 and UBE2S competitively bind to NSL3, suggesting the coordination of OGT1-UBE2S in regulating NSL3 stability. Furthermore, O-GlcNAcylation of NSL3 Thr755 site regulates the histone H4 acetylation levels at lysine 5, 8, and 16, suggesting that the O -GlcNAcylation of NSL3 at Thr755 is required for maintaining the integrity and holoenzyme activity of the MOF/NSL complex. In colony formation assays, we found that the integrity of the complex impacts the proliferation of the lung carcinoma type II epithelium-like A549 cells. Taken together, our results provide new insight into the elucidation of the molecular mechanism of the MOF/NSL complex., Competing Interests: The authors have declared that no competing interests exist.

Photoreceptor phosphodiesterase 6 (PDE6) is the central effector of the visual excitation pathway in both rod and cone photoreceptors, and PDE6 mutations that alter PDE6 structure or regulation can result in several human retinal diseases. The rod PDE6 holoenzyme consists of two catalytic subunits (Pαβ) whose activity is suppressed in the dark by binding of two inhibitory γ-subunits (Pγ). Upon photoactivation of rhodopsin, the heterotrimeric G protein (transducin) is activated, resulting in binding of the activated transducin α-subunit (Gt α ) to PDE6, displacement of Pγ from the PDE6 active site, and enzyme activation. Although the biochemistry of this pathway is understood, a lack of detailed structural information about the PDE6 activation mechanism hampers efforts to develop therapeutic interventions for managing PDE6-associated retinal diseases. To address this gap, here we used a cross-linking MS-based approach to create a model of the entire interaction surface of Pγ with the regulatory and catalytic domains of Pαβ in its nonactivated state. Following reconstitution of PDE6 and activated Gt α with liposomes and identification of cross-links between Gt α and PDE6 subunits, we determined that the PDE6-Gt α protein complex consists of two Gt α -binding sites per holoenzyme. Each Gt α interacts with the catalytic domains of both catalytic subunits and induces major changes in the interaction sites of the Pγ subunit with the catalytic subunits. These results provide the first structural model for the activated state of the transducin-PDE6 complex during visual excitation, enhancing our understanding of the molecular etiology of inherited retinal diseases., (© 2019 Irwin et al.)

CK2 is a constitutively active protein kinase overexpressed in numerous malignancies. Interaction between CK2α and CK2β subunits is essential for substrate selectivity. The CK2α/CK2β interface has been previously targeted by peptides to achieve functional effects; however, no small molecules modulators were identified due to pocket flexibility and open shape. Here we generated numerous plausible conformations of the interface using the fumigation modeling protocol, and virtually screened a compound library to discover compound 1 that suppressed CK2α/CK2β interaction in vitro and inhibited CK2 in a substrate-selective manner. Orthogonal SPR, crystallography, and NMR experiments demonstrated that 4 and 6, improved analogs of 1, bind to CK2α as predicted. Both inhibitors alter CK2 activity in cells through inhibition of CK2 holoenzyme formation. Treatment with 6 suppressed MDA-MB231 triple negative breast cancer cell growth and induced apoptosis. Altogether, our findings exemplify an innovative computational-experimental approach and identify novel non-peptidic inhibitors of CK2 subunit interface disclosing substrate-selective functional effects.

Prolyl-tRNA synthetase (PRS) is a member of the aminoacyl-tRNA synthetase family that drives protein translation in cells. The apicomplexan PRSs are validated targets of febrifugine (FF) and its halogenated derivative halofuginone (HF). PRSs are of great interest for drug development against Plasmodium falciparum and Toxoplasma gondii. In this study, structures of apo and FF-bound T. gondii (TgPRS) are revealed and the dynamic nature of the conformational changes that occur upon FF binding is unraveled. In addition, this study highlights significant conformational plasticity within two different crystal structures of apo PRSs but not within drug-bound PRSs. The apo PRSs exist in multi-conformational states and manifest pseudo-dimeric structures. In contrast, when FF is bound the PRS dimer adopts a highly symmetrical architecture. It is shown that TgPRS does not display extant fold switching, in contrast to P. falciparum PRS, despite having over 65% sequence identity. Finally, structure-comparison analyses suggest the utility of r.m.s.d. per residue (r.m.s.d. /res ) as a robust tool to detect structural alterations even when the r.m.s.d. is low. Apo TgPRS reveals FF/HF-induced rigidity and this work has implications for drug-design studies that rely on the apo structures of target proteins.

Protein kinase A (PKA) holoenzyme, comprised of a cAMP-binding regulatory (R)-subunit dimer and 2 catalytic (C)-subunits, is the master switch for cAMP-mediated signaling. Of the 4 R-subunits (RIα, RIβ, RIIα, RIIβ), RIα is most essential for regulating PKA activity in cells. Our 2 RIα 2 C 2 holoenzyme states, which show different conformations with and without ATP, reveal how ATP/Mg 2+ functions as a negative orthosteric modulator. Biochemical studies demonstrate how the removal of ATP primes the holoenzyme for cAMP-mediated activation. The opposing competition between ATP/cAMP is unique to RIα. In RIIβ, ATP serves as a substrate and facilitates cAMP-activation. The isoform-specific RI-holoenzyme dimer interface mediated by N3A-N3A' motifs defines multidomain cross-talk and an allosteric network that creates competing roles for ATP and cAMP. Comparisons to the RIIβ holoenzyme demonstrate isoform-specific holoenzyme interfaces and highlights distinct allosteric mechanisms for activation in addition to the structural diversity of the isoforms., Competing Interests: The authors declare no conflict of interest.

The active site (H-cluster) of [FeFe]-hydrogenases is a blueprint for the design of a biologically inspired H 2 -producing catalyst. The maturation process describes the preassembly and uptake of the unique [2Fe H ] cluster into apo-hydrogenase, which is to date not fully understood. In this study, we targeted individual amino acids by site-directed mutagenesis in the [FeFe]-hydrogenase CpI of Clostridium pasteurianum to reveal the final steps of H-cluster maturation occurring within apo-hydrogenase. We identified putative key positions for cofactor uptake and the subsequent structural reorganization that stabilizes the [2Fe H ] cofactor in its functional coordination sphere. Our results suggest that functional integration of the negatively charged [2Fe H ] precursor requires the positive charges and individual structural features of the 2 basic residues of arginine 449 and lysine 358, which mark the entrance and terminus of the maturation channel, respectively. The results obtained for 5 glycine-to-histidine exchange variants within a flexible loop region provide compelling evidence that the glycine residues function as hinge positions in the refolding process, which closes the secondary ligand sphere of the [2Fe H ] cofactor and the maturation channel. The conserved structural motifs investigated here shed light on the interplay between the secondary ligand sphere and catalytic cofactor., Competing Interests: The authors declare no conflict of interest.

Fibrolamellar hepatocellular carcinoma (FLHCC) is driven by J-PKAcα, a kinase fusion chimera of the J domain of DnaJB1 with PKAcα, the catalytic subunit of protein kinase A (PKA). Here we report the crystal structures of the chimeric fusion RIα 2 :J-PKAcα 2 holoenzyme formed by J-PKAcα and the PKA regulatory (R) subunit RIα, and the wild-type (WT) RIα 2 :PKAcα 2 holoenzyme. The chimeric and WT RIα holoenzymes have quaternary structures different from the previously solved WT RIβ and RIIβ holoenzymes. The WT RIα holoenzyme showed the same configuration as the chimeric RIα 2 :J-PKAcα 2 holoenzyme and a distinct second conformation. The J domains are positioned away from the symmetrical interface between the two RIα:J-PKAcα heterodimers in the chimeric fusion holoenzyme and are highly dynamic. The structural and dynamic features of these holoenzymes enhance our understanding of the fusion chimera protein J-PKAcα that drives FLHCC as well as the isoform specificity of PKA., (Published by Elsevier Ltd.)

Bacterial RNA polymerase employs extra-cytoplasmic function (ECF) σ factors to regulate context-specific gene expression programs. Despite being the most abundant and divergent σ factor class, the structural basis of ECF σ factor-mediated transcription initiation remains unknown. Here, we determine a crystal structure of Mycobacterium tuberculosis (Mtb) RNAP holoenzyme comprising an RNAP core enzyme and the ECF σ factor σ H (σ H -RNAP) at 2.7 Å, and solve another crystal structure of a transcription initiation complex of Mtb σ H -RNAP (σ H -RPo) comprising promoter DNA and an RNA primer at 2.8 Å. The two structures together reveal the interactions between σ H and RNAP that are essential for σ H -RNAP holoenzyme assembly as well as the interactions between σ H -RNAP and promoter DNA responsible for stringent promoter recognition and for promoter unwinding. Our study establishes that ECF σ factors and primary σ factors employ distinct mechanisms for promoter recognition and for promoter unwinding.

Bacterial RNA polymerase (RNAP) is essential for gene expression and as such is a valid drug target. Hence, it is imperative to know its structure and dynamics. Here, we present two as-yet-unreported forms of Mycobacterium smegmatis RNAP: core and holoenzyme containing σ A but no other factors. Each form was detected by cryo-electron microscopy in two major conformations. Comparisons of these structures with known structures of other RNAPs reveal a high degree of conformational flexibility of the mycobacterial enzyme and confirm that region 1.1 of σ A is directed into the primary channel of RNAP. Taken together, we describe the conformational changes of unrestrained mycobacterial RNAP. IMPORTANCE We describe here three-dimensional structures of core and holoenzyme forms of mycobacterial RNA polymerase (RNAP) solved by cryo-electron microscopy. These structures fill the thus-far-empty spots in the gallery of the pivotal forms of mycobacterial RNAP and illuminate the extent of conformational dynamics of this enzyme. The presented findings may facilitate future designs of antimycobacterial drugs targeting RNAP., (Copyright © 2019 American Society for Microbiology.)

BbvCI, a Type IIT restriction endonuclease, recognizes and cleaves the seven base pair sequence 5'-CCTCAGC-3', generating 3-base, 5'-overhangs. BbvCI is composed of two protein subunits, each containing one catalytic site. Either site can be inactivated by mutation resulting in enzyme variants that nick DNA in a strand-specific manner. Here we demonstrate that the holoenzyme is labile, with the R1 subunit dissociating at low pH. Crystallization of the R2 subunit under such conditions revealed an elongated dimer with the two catalytic sites located on opposite sides. Subsequent crystallization at physiological pH revealed a tetramer comprising two copies of each subunit, with a pair of deep clefts each containing two catalytic sites appropriately positioned and oriented for DNA cleavage. This domain organization was further validated with single-chain protein constructs in which the two enzyme subunits were tethered via peptide linkers of variable length. We were unable to crystallize a DNA-bound complex; however, structural similarity to previously crystallized restriction endonucleases facilitated creation of an energy-minimized model bound to DNA, and identification of candidate residues responsible for target recognition. Mutation of residues predicted to recognize the central C:G base pair resulted in an altered enzyme that recognizes and cleaves CCTNAGC (N = any base).

The proteasome is an ATP-dependent, 2.5-megadalton molecular machine that is responsible for selective protein degradation in eukaryotic cells. Here we present cryo-electron microscopy structures of the substrate-engaged human proteasome in seven conformational states at 2.8-3.6 Å resolution, captured during breakdown of a polyubiquitylated protein. These structures illuminate a spatiotemporal continuum of dynamic substrate-proteasome interactions from ubiquitin recognition to substrate translocation, during which ATP hydrolysis sequentially navigates through all six ATPases. There are three principal modes of coordinated hydrolysis, featuring hydrolytic events in two oppositely positioned ATPases, in two adjacent ATPases and in one ATPase at a time. These hydrolytic modes regulate deubiquitylation, initiation of translocation and processive unfolding of substrates, respectively. Hydrolysis of ATP powers a hinge-like motion in each ATPase that regulates its substrate interaction. Synchronization of ATP binding, ADP release and ATP hydrolysis in three adjacent ATPases drives rigid-body rotations of substrate-bound ATPases that are propagated unidirectionally in the ATPase ring and unfold the substrate.

Ribonuclease (RNase) P is a ubiquitous ribozyme that cleaves the 5' leader from precursor tRNAs. Here, we report cryo-electron microscopy structures of the human nuclear RNase P alone and in complex with tRNA Val . Human RNase P is a large ribonucleoprotein complex that contains 10 protein components and one catalytic RNA. The protein components form an interlocked clamp that stabilizes the RNA in a conformation optimal for substrate binding. Human RNase P recognizes the tRNA using a double-anchor mechanism through both protein-RNA and RNA-RNA interactions. Structural comparison of the apo and tRNA-bound human RNase P reveals that binding of tRNA induces a local conformational change in the catalytic center, transforming the ribozyme into an active state. Our results also provide an evolutionary model depicting how auxiliary RNA elements in bacterial RNase P, essential for substrate binding, and catalysis, were replaced by the much more complex and multifunctional protein components in higher organisms., (Copyright © 2018 Elsevier Inc. All rights reserved.)

p15PAF is an oncogenic intrinsically disordered protein that regulates DNA replication and lesion bypass by interacting with the human sliding clamp PCNA. In the absence of DNA, p15PAF traverses the PCNA ring via an extended PIP-box that contacts the sliding surface. Here, we probed the atomic-scale structure of p15PAF-PCNA-DNA ternary complexes. Crystallography and MD simulations show that, when p15PAF occupies two subunits of the PCNA homotrimer, DNA within the ring channel binds the unoccupied subunit. The structure of PCNA-bound p15PAF in the absence and presence of DNA is invariant, and solution NMR confirms that DNA does not displace p15PAF from the ring wall. Thus, p15PAF reduces the available sliding surfaces of PCNA, and may function as a belt that fastens the DNA to the clamp during synthesis by the replicative polymerase (pol δ). This constraint, however, may need to be released for efficient DNA lesion bypass by the translesion synthesis polymerase (pol η). Accordingly, our biochemical data show that p15PAF impairs primer synthesis by pol η-PCNA holoenzyme against both damaged and normal DNA templates. In light of our findings, we discuss the possible mechanistic roles of p15PAF in DNA replication and suppression of DNA lesion bypass.

The protein Ser/Thr phosphatase PP1 catalyzes an important fraction of protein dephosphorylation events and forms highly specific holoenzymes through an association with regulatory interactors of protein phosphatase one (RIPPOs). The functional characterization of individual PP1 holoenzymes is hampered by the lack of straightforward strategies for substrate mapping. Because efficient substrate recruitment often involves binding to both PP1 and its associated RIPPO, here we examined whether PP1-RIPPO fusions can be used to trap substrates for further analysis. Fusions of an hypoactive point mutant of PP1 and either of four tested RIPPOs accumulated in HEK293T cells with their associated substrates and were co-immunoprecipitated for subsequent identification of the substrates by immunoblotting or MS analysis. Hypoactive fusions were also used to study RIPPOs themselves as substrates for associated PP1. In contrast, substrate trapping was barely detected with active PP1-RIPPO fusions or with nonfused PP1 or RIPPO subunits. Our results suggest that hypoactive fusions of PP1 subunits represent an easy-to-use tool for substrate identification of individual holoenzymes., (© 2018 Wu et al.)

The enzyme telomerase adds telomeric repeats to chromosome ends to balance the loss of telomeres during genome replication. Telomerase regulation has been implicated in cancer, other human diseases, and ageing, but progress towards clinical manipulation of telomerase has been hampered by the lack of structural data. Here we present the cryo-electron microscopy structure of the substrate-bound human telomerase holoenzyme at subnanometre resolution, showing two flexibly RNA-tethered lobes: the catalytic core with telomerase reverse transcriptase (TERT) and conserved motifs of telomerase RNA (hTR), and an H/ACA ribonucleoprotein (RNP). In the catalytic core, RNA encircles TERT, adopting a well-ordered tertiary structure with surprisingly limited protein-RNA interactions. The H/ACA RNP lobe comprises two sets of heterotetrameric H/ACA proteins and one Cajal body protein, TCAB1, representing a pioneering structure of a large eukaryotic family of ribosome and spliceosome biogenesis factors. Our findings provide a structural framework for understanding human telomerase disease mutations and represent an important step towards telomerase-related clinical therapeutics.

The proteasome is a sophisticated ATP-dependent molecular machine responsible for protein degradation in all known eukaryotic cells. It remains elusive how conformational changes of the AAA-ATPase unfoldase in the regulatory particle (RP) control the gating of the substrate-translocation channel leading to the proteolytic chamber of the core particle (CP). Here we report three alternative states of the ATP-γ-S-bound human proteasome, in which the CP gates are asymmetrically open, visualized by cryo-EM at near-atomic resolutions. At least four nucleotides are bound to the AAA-ATPase ring in these open-gate states. Variation in nucleotide binding gives rise to an axial movement of the pore loops narrowing the substrate-translation channel, which exhibit remarkable structural transitions between the spiral-staircase and saddle-shaped-circle topologies. Gate opening in the CP is thus regulated by nucleotide-driven conformational changes of the AAA-ATPase unfoldase. These findings demonstrate an elegant mechanism of allosteric coordination among sub-machines within the human proteasome holoenzyme.

DNA-dependent protein kinase (DNA-PK) is a serine/threonine protein kinase complex composed of a catalytic subunit (DNA-PKcs) and KU70/80 heterodimer bound to DNA. DNA-PK holoenzyme plays a critical role in non-homologous end joining (NHEJ), the major DNA repair pathway. Here, we determined cryo-electron microscopy structure of human DNA-PK holoenzyme at 6.6 Å resolution. In the complex structure, DNA-PKcs, KU70, KU80 and DNA duplex form a 650-kDa heterotetramer with 1:1:1:1 stoichiometry. The N-terminal α-solenoid (∼2 800 residues) of DNA-PKcs adopts a double-ring fold and connects the catalytic core domain of DNA-PKcs and KU70/80-DNA. DNA-PKcs and KU70/80 together form a DNA-binding tunnel, which cradles ∼30-bp DNA and prevents sliding inward of DNA-PKcs along with DNA duplex, suggesting a mechanism by which the broken DNA end is protected from unnecessary processing. Structural and biochemical analyses indicate that KU70/80 and DNA coordinately induce conformational changes of DNA-PKcs and allosterically stimulate its kinase activity. We propose a model for activation of DNA-PKcs in which allosteric signals are generated upon DNA-PK holoenzyme formation and transmitted to the kinase domain through N-terminal HEAT repeats and FAT domain of DNA-PKcs. Our studies suggest a mechanism for recognition and protection of broken DNA ends and provide a structural basis for understanding the activation of DNA-PKcs and DNA-PK-mediated NHEJ pathway.

Using the energy of ATP hydrolysis, ABC transporters catalyze the trans-membrane transport of molecules. In bacteria, these transporters partner with a high-affinity substrate-binding protein (SBP) to import essential micronutrients. ATP binding by Type I ABC transporters (importers of amino acids, sugars, peptides, and small ions) stabilizes the interaction between the transporter and the SBP, thus allowing transfer of the substrate from the latter to the former. In Type II ABC transporters (importers of trace elements, e.g. vitamin B 12 , heme, and iron-siderophores) the role of ATP remains debatable. Here we studied the interaction between the Yersinia pestis ABC heme importer (HmuUV) and its partner substrate-binding protein (HmuT). Using real-time surface plasmon resonance experiments and interaction studies in membrane vesicles, we find that in the absence of ATP the transporter and the SBP tightly bind. Substrate in excess inhibits this interaction, and ATP binding by the transporter completely abolishes it. To release the stable docked SBP from the transporter hydrolysis of ATP is required. Based on these results we propose a mechanism for heme acquisition by HmuUV-T where the substrate-loaded SBP docks to the nucleotide-free outward-facing conformation of the transporter. ATP binding leads to formation of an occluded state with the substrate trapped in the trans-membrane translocation cavity. Subsequent ATP hydrolysis leads to substrate delivery to the cytoplasm, release of the SBP, and resetting of the system. We propose that other Type II ABC transporters likely share the fundamentals of this mechanism., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Flavin-based electron transfer bifurcation is emerging as a fundamental and powerful mechanism for conservation and deployment of electrochemical energy in enzymatic systems. In this process, a pair of electrons is acquired at intermediate reduction potential ( i.e. intermediate reducing power), and each electron is passed to a different acceptor, one with lower and the other with higher reducing power, leading to "bifurcation." It is believed that a strongly reducing semiquinone species is essential for this process, and it is expected that this species should be kinetically short-lived. We now demonstrate that the presence of a short-lived anionic flavin semiquinone (ASQ) is not sufficient to infer the existence of bifurcating activity, although such a species may be necessary for the process. We have used transient absorption spectroscopy to compare the rates and mechanisms of decay of ASQ generated photochemically in bifurcating NADH-dependent ferredoxin-NADP + oxidoreductase and the non-bifurcating flavoproteins nitroreductase, NADH oxidase, and flavodoxin. We found that different mechanisms dominate ASQ decay in the different protein environments, producing lifetimes ranging over 2 orders of magnitude. Capacity for electron transfer among redox cofactors versus charge recombination with nearby donors can explain the range of ASQ lifetimes that we observe. Our results support a model wherein efficient electron propagation can explain the short lifetime of the ASQ of bifurcating NADH-dependent ferredoxin-NADP + oxidoreductase I and can be an indication of capacity for electron bifurcation.

The ability of the replisome to seamlessly coordinate both high fidelity and translesion DNA synthesis requires a means to regulate recruitment and binding of enzymes from solution. Co-occupancy of multiple DNA polymerases within the replisome has been observed primarily in bacteria and is regulated by posttranslational modifications in eukaryotes, and both cases are coordinated by the processivity clamp. Because of the heterotrimeric nature of the PCNA clamp in some archaea, there is potential to occupy and regulate specific polymerases at defined subunits. In addition to specific PCNA and polymerase interactions (PIP site), we have now identified and characterized a novel protein contact between the Y-family DNA polymerase and the B-family replication polymerase (YB site) bound to PCNA and DNA from Sulfolobus solfataricus. These YB contacts are essential in forming and stabilizing a supraholoenzyme (SHE) complex on DNA, effectively increasing processivity of DNA synthesis. The SHE complex can not only coordinate polymerase exchange within the complex but also provides a mechanism for recruitment of polymerases from solution based on multiequilibrium processes. Our results provide evidence for an archaeal PCNA 'tool-belt' recruitment model of multienzyme function that can facilitate both high fidelity and translesion synthesis within the replisome during DNA replication., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Thymidylate kinase (TMK) is a key enzyme which plays an important role in DNA synthesis. It belongs to the family of nucleoside monophosphate kinases, several of which undergo structure-encoded conformational changes to perform their function. However, the absence of three-dimensional structures for all the different reaction intermediates of a single TMK homolog hinders a clear understanding of its functional mechanism. We herein report the different conformational states along the reaction coordinate of a hyperthermophilic TMK from Aquifex aeolicus, determined via X-ray diffraction and further validated through normal-mode studies. The analyses implicate an arginine residue in the Lid region in catalysis, which was confirmed through site-directed mutagenesis and subsequent enzyme assays on the wild-type protein and mutants. Furthermore, the enzyme was found to exhibit broad specificity toward phosphate group acceptor nucleotides. Our comprehensive analyses of the conformational landscape of TMK, together with associated biochemical experiments, provide insights into the mechanistic details of TMK-driven catalysis, for example, the order of substrate binding and the reaction mechanism for phosphate transfer. Such a study has utility in the design of potent inhibitors for these enzymes., Database: Structural data are available in the PDB under the accession numbers 2PBR, 4S2E, 5H5B, 5XAI, 4S35, 5XB2, 5H56, 5XB3, 5H5K, 5XB5, and 5XBH., (© 2017 Federation of European Biochemical Societies.)

Nonribosomal peptide synthesis involves the interplay between covalent protein modifications, conformational fluctuations, catalysis, and transient protein-protein interactions. Delineating the mechanisms involved in orchestrating these various processes will deepen our understanding of domain-domain communication in nonribosomal peptide synthetases (NRPSs) and lay the groundwork for the rational reengineering of NRPSs by swapping domains handling different substrates to generate novel natural products. Although many structural and biochemical studies of NRPSs exist, few studies have focused on the energetics and dynamics governing the interactions in these systems. Here, we present detailed binding studies of an adenylation domain and its partner carrier protein in apo-, holo-, and substrate-loaded forms. Results from fluorescence anisotropy, isothermal titration calorimetry, and NMR titrations indicated that covalent modifications to a carrier protein modulate domain communication, suggesting that chemical modifications to carrier proteins during NRPS synthesis may impart directionality to sequential NRPS domain interactions. Comparison of the structure and dynamics of an apo-aryl carrier protein with those of its modified forms revealed structural fluctuations induced by post-translational modifications and mediated by modulations of protein dynamics. The results provide a comprehensive molecular description of a carrier protein throughout its life cycle and demonstrate how a network of dynamic residues can propagate the molecular impact of chemical modifications throughout a protein and influence its affinity toward partner domains., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

To investigate the function of the pa4079 gene from the opportunistic pathogen Pseudomonas aeruginosa PAO1, we determined its crystal structure and confirmed it to be a NAD(P)-dependent short-chain dehydrogenase/reductase. Structural similarity and activity for a broad range of substrates indicate that PA4079 functions as a carbonyl reductase. Comparison of apo- and holo-PA4079 shows that NADP stabilizes the active site specificity loop, and small molecule binding induces rotation of the Tyr183 side chain by approximately 90° out of the active site. Quantitative real-time PCR results show that pa4079 maintains high expression levels during antibiotic exposure. This work provides a starting point for understanding substrate recognition and selectivity by PA4079, as well as its possible reduction of antimicrobial drugs., Database: Structural data are available in the Protein Data Bank (PDB) under the following accession numbers: apo PA4079 (condition I), 5WQM; apo PA4079 (condition II), 5WQN; PA4079 + NADP (condition I), 5WQO; PA4079 + NADP (condition II), 5WQP., (© 2017 Federation of European Biochemical Societies.)

Since their initial characterization over 30 years ago, it has been believed that the archaeal B-family DNA polymerases are single-subunit enzymes. This contrasts with the multi-subunit B-family replicative polymerases of eukaryotes. Here we reveal that the highly studied PolB1 from Sulfolobus solfataricus exists as a heterotrimeric complex in cell extracts. Two small subunits, PBP1 and PBP2, associate with distinct surfaces of the larger catalytic subunit and influence the enzymatic properties of the DNA polymerase. Thus, multi-subunit replicative DNA polymerase holoenzymes are present in all three domains of life. We reveal the architecture of the assembly by a combination of cross-linking coupled with mass spectrometry, X-ray crystallography and single-particle electron microscopy. The small subunits stabilize the holoenzyme assembly and the acidic tail of one small subunit mitigates the ability of the enzyme to perform strand-displacement synthesis, with important implications for lagging strand DNA synthesis.