SDS22 coordinates the assembly of holoenzymes from nascent protein phosphatase-1.

SDS22 forms an inactive complex with nascent protein phosphatase PP1 and Inhibitor-3. SDS22:PP1:Inhibitor-3 is a substrate for the ATPase p97/VCP, which liberates PP1 for binding to canonical regulatory subunits. The exact role of SDS22 in PP1-holoenzyme assembly remains elusive. Here, we show that SDS22 stabilizes nascent PP1. In the absence of SDS22, PP1 is gradually lost, resulting in substrate hyperphosphorylation and a proliferation arrest. Similarly, we identify a female individual with a severe neurodevelopmental disorder bearing an unstable SDS22 mutant, associated with decreased PP1 levels. We furthermore find that SDS22 directly binds to Inhibitor-3 and that this is essential for the stable assembly of SDS22:PP1: Inhibitor-3, the recruitment of p97/VCP, and the extraction of SDS22 during holoenzyme assembly. SDS22 with a disabled Inhibitor-3 binding site co-transfers with PP1 to canonical regulatory subunits, thereby forming non-functional holoenzymes. Our data show that SDS22, through simultaneous interaction with PP1 and Inhibitor-3, integrates the major steps of PP1 holoenzyme assembly.

Hub stability in the calcium calmodulin-dependent protein kinase II.

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 ß isoforms, either standalone or within an ß 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 ß hub polymorphs, encompassing the curvature of both polymorphs. The MD explains how an open hub structure also obtained from the ß 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 αß heterooligomers for their dynamic localization within synapses in neurons.

Structure and flexibility of the DNA polymerase holoenzyme of vaccinia virus.

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.

Structural basis for the inhibition mechanism of the DNA polymerase holoenzyme from mpox virus.

There are three key components at the core of the mpox virus (MPXV) DNA polymerase holoenzyme: DNA polymerase F8, processivity factors A22, and the Uracil-DNA glycosylase E4. The holoenzyme is recognized as a vital antiviral target because MPXV replicates in the cytoplasm of host cells. Nucleotide analogs such as cidofovir and cytarabine (Ara-C) have shown potential in curbing MPXV replication and they also display promise against other poxviruses. However, the mechanism behind their inhibitory effects remains unclear. Here, we present the cryo-EM structure of the DNA polymerase holoenzyme F8/A22/E4 bound with its competitive inhibitor Ara-C-derived cytarabine triphosphate (Ara-CTP) at an overall resolution of 3.0 Å and reveal its inhibition mechanism. Ara-CTP functions as a direct chain terminator in proximity to the deoxycytidine triphosphate (dCTP)-binding site. The extra hydrogen bond formed with Asn665 makes it more potent in binding than dCTP. Asn665 is conserved among eukaryotic B-family polymerases.

Replication of [AT/TA]25 Microsatellite Sequences by Human DNA Polymerase δ Holoenzymes Is Dependent on dNTP and RPA Levels.

Fragile sites are unstable genomic regions that are prone to breakage during stressed DNA replication. Several common fragile sites (CFS) contain A+T-rich regions including perfect [AT/TA] microsatellite repeats that may collapse into hairpins when in single-stranded DNA (ssDNA) form and coincide with chromosomal hotspots for breakage and rearrangements. While many factors contribute to CFS instability, evidence exists for replication stalling within [AT/TA] microsatellite repeats. Currently, it is unknown how stress causes replication stalling within [AT/TA] microsatellite repeats. To investigate this, we utilized FRET to characterize the structures of [AT/TA]25 sequences and also reconstituted lagging strand replication to characterize the progression of pol δ holoenzymes through A+T-rich sequences. The results indicate that [AT/TA]25 sequences adopt hairpins that are unwound by the major ssDNA-binding complex, RPA, and the progression of pol δ holoenzymes through A+T-rich sequences saturated with RPA is dependent on the template sequence and dNTP concentration. Importantly, the effects of RPA on the replication of [AT/TA]25 sequences are dependent on dNTP concentration, whereas the effects of RPA on the replication of A+T-rich, nonstructure-forming sequences are independent of dNTP concentration. Collectively, these results reveal complexities in lagging strand replication and provide novel insights into how [AT/TA] microsatellite repeats contribute to genome instability.

Oligomerization states of the Mycobacterium tuberculosis RNA polymerase core and holoenzymes.

During the past few decades, a wealth of knowledge has been made available for the transcription machinery in bacteria from the structural, functional and mechanistic point of view. However, comparatively little is known about the homooligomerization of the multisubunit M. tuberculosis RNA polymerase (RNAP) enzyme and its functional relevance. While E. coli RNAP has been extensively studied, many aspects of RNAP of the deadly pathogenic M. tuberculosis are still unclear. We used biophysical and biochemical methods to study the oligomerization states of the core and holoenzymes of M. tuberculosis RNAP. By size exclusion chromatography and negative staining Transmission Electron Microscopy (TEM) studies and quantitative analysis of the TEM images, we demonstrate that the in vivo reconstituted RNAP core enzyme (α2ßß'ω) can also exist as dimers in vitro. Using similar methods, we also show that the holoenzyme (core + σA) does not dimerize in vitro and exist mostly as monomers. It is tempting to suggest that the oligomeric changes that we see in presence of σA factor might have functional relevance in the cellular process. Although reported previously in E. coli, to our knowledge we report here for the first time the study of oligomeric nature of M. tuberculosis RNAP in presence and absence of σA factor.

Emerging Roles of B56 Phosphorylation and Binding Motif in PP2A-B56 Holoenzyme Biological Function.

Protein serine/threonine phosphatase 2A (PP2A) regulates diverse cellular processes via the formation of ~100 heterotrimeric holoenzymes. However, a scarcity of knowledge on substrate recognition by various PP2A holoenzymes has greatly prevented the deciphering of PP2A function in phosphorylation-mediated signaling in eukaryotes. The review summarized the contribution of B56 phosphorylation to PP2A-B56 function and proposed strategies for intervening B56 phosphorylation to treat diseases associated with PP2A-B56 dysfunction; it especially analyzed recent advancements in LxxIxEx B56-binding motifs that provide the molecular details of PP2A-B56 binding specificity and, on this basis, explored the emerging role of PP2A-B56 in the mitosis process, virus attack, and cancer development through LxxIxE motif-mediated PP2A-B56 targeting. This review provides theoretical support for discriminatingly targeting specific PP2A holoenzymes to guide PP2A activity against specific pathogenic drivers.

Re-investigation of in vitro activity of acetohydroxyacid synthase I holoenzyme from Escherichia coli.

Acetohydroxyacid synthase (AHAS) is one of the key enzymes of the biosynthesis of branched-chain amino acids, it is also an effective target for the screening of herbicides and antibiotics. In this study we present a method for preparing Escherichia coli AHAS I holoenzyme (EcAHAS I) with exceptional stability, which provides a solid ground for us to re-investigate the in vitro catalytic properties of the protein. The results show EcAHAS I synthesized in this way exhibits similar function to Bacillus subtilis acetolactate synthase in its catalysis with pyruvate and 2-ketobutyrate (2-KB) as dual-substrate, producing four 2-hydroxy-3-ketoacids including (S)-2-acetolactate, (S)-2-aceto-2-hydroxybutyrate, (S)-2-propionyllactate, and (S)-2-propionyl-2-hydroxybutyrate. Quantification of the reaction indicates that the two substrates almost totally consume, and compound (S)-2-aceto-2- hydroxybutyrate forms in the highest yield among the four major products. Moreover, the protein also condenses two molecules of 2-KB to furnish (S)-2-propionyl-2-hydroxybutyrate. Further exploration manifests that EcAHAS I ligates pyruvate/2-KB and nitrosobenzene to generate two arylhydroxamic acids N-hydroxy-N-phenylacetamide and N-hydroxy-N-phenyl- propionamide. These findings enhance our comprehension of the catalytic characteristics of EcAHAS I. Furthermore, the application of this enzyme as a catalyst in construction of C-N bonds displays promising potential.

Real-time single-molecule imaging of CaMKII-calmodulin interactions.

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 CaMKIIß isoform using single-molecule total internal reflection fluorescence microscopy. Rhodamine-CAM associations/dissociations to surface-immobilized Venus-CaMKIIß 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 ß isoforms. The study of mutant ß 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.

Cell Cycle-Specific Protein Phosphatase 1 (PP1) Substrates Identification Using Genetically Modified Cell Lines.

The identification of protein phosphatase 1 (PP1) holoenzyme substrates has proven to be a challenging task. PP1 can form different holoenzyme complexes with a variety of regulatory subunits, and many of those are cell cycle regulated. Although several methods have been used to identify PP1 substrates, their cell cycle specificity is still an unmet need. Here, we present a new strategy to investigate PP1 substrates throughout the cell cycle using clustered regularly interspersed short palindromic repeats (CRISPR)-Cas9 genome editing and generate cell lines with endogenously tagged PP1 regulatory subunit (regulatory interactor of protein phosphatase one, RIPPO). RIPPOs are tagged with the auxin-inducible degron (AID) or ascorbate peroxidase 2 (APEX2) modules, and PP1 substrate identification is conducted by SILAC proteomic-based approaches. Proteins in close proximity to RIPPOs are first identified through mass spectrometry (MS) analyses using the APEX2 system; then a list of differentially phosphorylated proteins upon RIPPOs rapid degradation (achieved via the AID system) is compiled via SILAC phospho-mass spectrometry. The "in silico" overlap between the two proteomes will be enriched for PP1 putative substrates. Several methods including fluorescence resonance energy transfer (FRET), proximity ligation assays (PLA), and in vitro assays can be used as substrate validations approaches.

Emerging roles of the Protein Phosphatase 1 (PP1) in the context of viral infections.

Protein Phosphatase 1 (PP1) is a major serine/threonine phosphatase in eukaryotes, participating in several cellular processes and metabolic pathways. Due to their low substrate specificity, PP1's catalytic subunits do not exist as free entities but instead bind to Regulatory Interactors of Protein Phosphatase One (RIPPO), which regulate PP1's substrate specificity and subcellular localization. Most RIPPOs bind to PP1 through combinations of short linear motifs (4-12 residues), forming highly specific PP1 holoenzymes. These PP1-binding motifs may, hence, represent attractive targets for the development of specific drugs that interfere with a subset of PP1 holoenzymes. Several viruses exploit the host cell protein (de)phosphorylation machinery to ensure efficient virus particle formation and propagation. While the role of many host cell kinases in viral life cycles has been extensively studied, the targeting of phosphatases by viral proteins has been studied in less detail. Here, we compile and review what is known concerning the role of PP1 in the context of viral infections and discuss how it may constitute a putative host-based target for the development of novel antiviral strategies.

The four subunits of rabbit skeletal muscle lactate dehydrogenase do not exert their catalytic action additively.

Oligomeric enzymes containing multiple active sites are usually considered to perform their catalytic action at higher rates when compared with their monomeric counterparts. This implies, in turn, that the activity performed by different holoenzyme subunits features additivity. Nevertheless, the extent of this additivity occurring in holoenzymes is far from being adequately understood. To tackle this point, we used tetrameric rabbit lactate dehydrogenase (rbLDH) as a model system to assay the reduction of pyruvate catalysed by this enzyme at the expense of ß-NADH under pre-steady-state conditions. In particular, we observed the kinetics of reactions triggered by concentrations of ß-NADH equimolar to 1, 2, 3, or all 4 subunits of the rbLDH holoenzyme, in the presence of an excess of pyruvate. Surprisingly, when the concentration of the limiting reactant exceeded that of a single holoenzyme subunit, we observed a sharp slowdown of the enzyme conformational rearrangements associated to the generation and the release of l-lactate. Furthermore, using a model to interpret the complex kinetics observed under the highest concentration of the limiting reactant, we estimated the diversity of the rates describing the action of the different rbLDH subunits.

B56δ long-disordered arms form a dynamic PP2A regulation interface coupled with global allostery and Jordan's syndrome mutations.

Intrinsically disordered regions (IDR) and short linear motifs (SLiMs) play pivotal roles in the intricate signaling networks governed by phosphatases and kinases. B56δ (encoded by PPP2R5D) is a regulatory subunit of protein phosphatase 2A (PP2A) with long IDRs that harbor a substrate-mimicking SLiM and multiple phosphorylation sites. De novo missense mutations in PPP2R5D cause intellectual disabilities (ID), macrocephaly, Parkinsonism, and a broad range of neurological symptoms. Our single-particle cryo-EM structures of the PP2A-B56δ holoenzyme reveal that the long, disordered arms at the B56δ termini fold against each other and the holoenzyme core. This architecture suppresses both the phosphatase active site and the substrate-binding protein groove, thereby stabilizing the enzyme in a closed latent form with dual autoinhibition. The resulting interface spans over 190 Šand harbors unfavorable contacts, activation phosphorylation sites, and nearly all residues with ID-associated mutations. Our studies suggest that this dynamic interface is coupled to an allosteric network responsive to phosphorylation and altered globally by mutations. Furthermore, we found that ID mutations increase the holoenzyme activity and perturb the phosphorylation rates, and the severe variants significantly increase the mitotic duration and error rates compared to the normal variant.

Protein Kinase CK2α', More than a Backup of CK2α.

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 CK2ß subunits. These three proteins exist in a free form, bound to other cellular proteins, as tetrameric holoenzymes composed of CK2α2/ß2, CK2αα'/ß2, or CK2α'2/ß2 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.

piRNA processing by a trimeric Schlafen-domain nuclease.

Transposable elements are genomic parasites that expand within and spread between genomes1. PIWI proteins control transposon activity, notably in the germline2,3. 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 (m7G-cap) and a uracil at position three. We also demonstrate how PUCH interacts with PETISCO, a complex that binds to piRNA precursors4, 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 immunity5, exposing a molecular link between immune responses in mammals and deeply conserved RNA-based mechanisms that control transposable elements.

PP2A-B55alpha controls keratinocyte adhesion through dephosphorylation of the Desmoplakin C-terminus.

Critical for the maintenance of epidermal integrity and function are attachments between intermediate filaments (IF) and intercellular junctions called desmosomes. The desmosomal cytoplasmic plaque protein desmoplakin (DP) is essential for anchoring IF to the junction. DP-IF interactions are regulated by a phospho-regulatory motif within the DP C-terminus controlling keratinocyte intercellular adhesion. Here we identify the protein phosphatase 2A (PP2A)-B55α holoenzyme as the major serine/threonine phosphatase regulating DP's C-terminus and consequent intercellular adhesion. Using a combination of chemical and genetic approaches, we show that the PP2A-B55α holoenzyme interacts with DP at intercellular membranes in 2D- and 3D- epidermal models and human skin samples. Our experiments demonstrate that PP2A-B55α regulates the phosphorylation status of junctional DP and is required for maintaining strong desmosome-mediated intercellular adhesion. These data identify PP2A-B55α as part of a regulatory module capable of tuning intercellular adhesion strength and a candidate disease target in desmosome-related disorders of the skin and heart.

Structures of the holoenzyme TglHI required for 3-thiaglutamate biosynthesis.

Structural diverse natural products like ribosomally synthesized and posttranslationally modified peptides (RiPPs) display a wide range of biological activities. Currently, the mechanism of an uncommon reaction step during the biosynthesis of 3-thiaglutamate (3-thiaGlu) is poorly understood. The removal of the ß-carbon from the Cys in the TglA-Cys peptide catalyzed by the TglHI holoenzyme remains elusive. Here, we present three crystal structures of TglHI complexes with and without bound iron, which reveal that the catalytic pocket is formed by the interaction of TglH-TglI and that its activation is conformation dependent. Biochemical assays suggest a minimum of two iron ions in the active cluster, and we identify the position of a third iron site. Collectively, our study offers insights into the activation and catalysis mechanisms of the non-heme dioxygen-dependent holoenzyme TglHI. Additionally, it highlights the evolutionary and structural conservation in the DUF692 family of biosynthetic enzymes that produce diverse RiPPs.

CaMKII autophosphorylation can occur between holoenzymes without subunit exchange.

The dodecameric protein kinase CaMKII is expressed throughout the body. The alpha isoform is responsible for synaptic plasticity and participates in memory through its phosphorylation of synaptic proteins. Its elaborate subunit organization and propensity for autophosphorylation allow it to preserve neuronal plasticity across space and time. The prevailing hypothesis for the spread of CaMKII activity, involving shuffling of subunits between activated and naive holoenzymes, is broadly termed subunit exchange. In contrast to the expectations of previous work, we found little evidence for subunit exchange upon activation, and no effect of restraining subunits to their parent holoenzymes. Rather, mass photometry, crosslinking mass spectrometry, single molecule TIRF microscopy and biochemical assays identify inter-holoenzyme phosphorylation (IHP) as the mechanism for spreading phosphorylation. The transient, activity-dependent formation of groups of holoenzymes is well suited to the speed of neuronal activity. Our results place fundamental limits on the activation mechanism of this kinase.

Regulation and role of the PP2A-B56 holoenzyme family in cancer.

Protein phosphatase 2A (PP2A) inactivation is common in cancer, leading to sustained activation of pro-survival and growth-promoting pathways. PP2A consists of a scaffolding A-subunit, a catalytic C-subunit, and a regulatory B-subunit. The functional complexity of PP2A holoenzymes arises mainly through the vast repertoire of regulatory B-subunits, which determine both their substrate specificity and their subcellular localization. Therefore, a major challenge for developing more effective therapeutic strategies for cancer is to identify the specific PP2A complexes to be targeted. Of note, the development of small molecules specifically directed at PP2A-B56α has opened new therapeutic avenues in both solid and hematological tumors. Here, we focus on the B56/PR61 family of PP2A regulatory subunits, which have a central role in directing PP2A tumor suppressor activity. We provide an overview of the mechanisms controlling the formation and regulation of these complexes, the pathways they control, and the mechanisms underlying their deregulation in cancer.

Imaging single CaMKII holoenzymes at work by high-speed atomic force microscopy.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) plays a pivotal role in synaptic plasticity. It is a dodecameric serine/threonine kinase that has been highly conserved across metazoans for over a million years. Despite the extensive knowledge of the mechanisms underlying CaMKII activation, its behavior at the molecular level has remained unobserved. In this study, we used high-speed atomic force microscopy to visualize the activity-dependent structural dynamics of rat/hydra/C. elegans CaMKII with nanometer resolution. Our imaging results revealed that the dynamic behavior is dependent on CaM binding and subsequent pT286 phosphorylation. Among the species studies, only rat CaMKIIα with pT286/pT305/pT306 exhibited kinase domain oligomerization. Furthermore, we revealed that the sensitivity of CaMKII to PP2A in the three species differs, with rat, C. elegans, and hydra being less dephosphorylated in that order. The evolutionarily acquired features of mammalian CaMKIIα-specific structural arrangement and phosphatase tolerance may differentiate neuronal function between mammals and other species.