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Phosphorylation of substrates by cyclin-dependent kinases (CDKs) is the driving force of cell cycle progression. Several CDK-activating cyclins are involved, yet how they contribute to substrate specificity is still poorly understood. Here, we discover that a positively charged pocket in cyclin B1, which is exclusively conserved within B-type cyclins and binds phosphorylated serine- or threonine-residues, is essential for correct execution of mitosis. HeLa cells expressing pocket mutant cyclin B1 are strongly delayed in anaphase onset due to multiple defects in mitotic spindle function and timely activation of the E3 ligase APC/C. Pocket integrity is essential for APC/C phosphorylation particularly at non-consensus CDK1 sites and full in vitro ubiquitylation activity. Our results support a model in which cyclin B1's pocket facilitates sequential substrate phosphorylations involving initial priming events that assist subsequent pocket-dependent phosphorylations even at non-consensus CDK1 motifs. How cyclins contribute to CDK1 substrate specificity during cell division is poorly understood. Here, the authors show that a phosphate-binding pocket in cyclin B1 is critical for accurate CDK1 substrate phosphorylation ensuring mitotic fidelity. [ABSTRACT FROM AUTHOR]

Autophagy is relevant for diverse processes in eukaryotic cells, making its regulation of fundamental importance. The formation and maturation of autophagosomes require a complex choreography of numerous factors. The endosomal sorting complex required for transport (ESCRT) is implicated in the final step of autophagosomal maturation by sealing of the phagophore membrane. ESCRT-III components were shown to mediate membrane scission by forming filaments that interact with cellular membranes. However, the molecular mechanisms underlying the recruitment of ESCRTs to non-endosomal membranes remain largely unknown. Here we focus on the ESCRT-associated protein ALG2-interacting protein X (ALIX) and identify Ca2+-dependent lipid binding protein 1 (CaLB1) as its interactor. Our findings demonstrate that CaLB1 interacts with AUTOPHAGY8 (ATG8) and PI(3)P, a phospholipid found in autophagosomal membranes. Moreover, CaLB1 and ALIX localize with ATG8 on autophagosomes upon salt treatment and assemble together into condensates. The depletion of CaLB1 impacts the maturation of salt-induced autophagosomes and leads to reduced delivery of autophagosomes to the vacuole. Here, we propose a crucial role of CaLB1 in augmenting phase separation of ALIX, facilitating the recruitment of ESCRT-III to the site of phagophore closure thereby ensuring efficient maturation of autophagosomes. The study demonstrates that the C2 domain-containing protein CaLB1 interacts with and induces phase separation of the ESCRT protein ALIX on autophagosomes. This modulates autophagosome maturation, particularly in response to salt stress, in Arabidopsis. [ABSTRACT FROM AUTHOR]

Intrinsically disordered regions (IDRs) are essential for membrane receptor regulation but often remain unresolved in structural studies. TRPV4, a member of the TRP vanilloid channel family involved in thermo- and osmosensation, has a large N-terminal IDR of approximately 150 amino acids. With an integrated structural biology approach, we analyze the structural ensemble of the TRPV4 IDR and the network of antagonistic regulatory elements it encodes. These modulate channel activity in a hierarchical lipid-dependent manner through transient long-range interactions. A highly conserved autoinhibitory patch acts as a master regulator by competing with PIP2 binding to attenuate channel activity. Molecular dynamics simulations show that loss of the interaction between the PIP2-binding site and the membrane reduces the force exerted by the IDR on the structured core of TRPV4. This work demonstrates that IDR structural dynamics are coupled to TRPV4 activity and highlights the importance of IDRs for TRP channel function and regulation. An integrated structural biology approach uncovers the structural complexity of the intrinsically disordered region (IDR) within the TRPV4 ion channel. Multiple stimulatory and inhibitory elements were identified within the IDR that modulate channel activity in a lipid-dependent manner. [ABSTRACT FROM AUTHOR]

RNA ligases are present across all forms of life. While enzymatic RNA ligation between 5′-PO4 and 3′-OH termini is prevalent in viruses, fungi, and plants, such RNA ligases are yet to be identified in vertebrates. Here, using a nucleotide-based chemical probe targeting human AMPylated proteome, we have enriched and identified the hitherto uncharacterised human protein chromosome 12 open reading frame 29 (C12orf29) as a human enzyme promoting RNA ligation between 5′-PO4 and 3′-OH termini. C12orf29 catalyses ATP-dependent RNA ligation via a three-step mechanism, involving tandem auto- and RNA AMPylation. Knock-out of C12ORF29 gene impedes the cellular resilience to oxidative stress featuring concurrent RNA degradation, which suggests a role of C12orf29 in maintaining RNA integrity. These data provide the groundwork for establishing a human RNA repair pathway. RNA ligases are present across all forms of life. Here, the hitherto uncharacterised human protein C12orf29 was identified as a human enzyme promoting RNA ligation between 5′-PO4 and 3′-OH termini. This data provides the groundwork for establishing a human RNA repair pathway. [ABSTRACT FROM AUTHOR]

Covalent attachment of ubiquitin (Ub) to proteins is a highly versatile posttranslational modification. Moreover, Ub is not only a modifier but itself is modified by phosphorylation and lysine acetylation. However, the functional consequences of Ub acetylation are poorly understood. By generation and comprehensive characterization of all seven possible mono-acetylated Ub variants, we show that each acetylation site has a particular impact on Ub structure. This is reflected in selective usage of the acetylated variants by different E3 ligases and overlapping but distinct interactomes, linking different acetylated variants to different cellular pathways. Notably, not only electrostatic but also steric effects contribute to acetylation-induced changes in Ub structure and, thus, function. Finally, we provide evidence that p300 acts as a position-specific Ub acetyltransferase and HDAC6 as a general Ub deacetylase. Our findings provide intimate insights into the structural and functional consequences of Ub acetylation and highlight the general importance of Ub acetylation. Ubiquitin is not only a posttranslational modifier but itself is subject to modifications, such as acetylation. Characterization of distinct acetylated ubiquitin variants reveals that each acetylation site has a particular impact on ubiquitin structure and its protein-protein interaction properties. [ABSTRACT FROM AUTHOR]

The nucleotides diadenosine triphosphate (Ap3A) and diadenosine tetraphosphate (Ap4A) are formed in prokaryotic and eukaryotic cells. Since their concentrations increase significantly upon cellular stress, they are considered to be alarmones triggering stress adaptive processes. However, their cellular roles remain elusive. To elucidate the proteome-wide interactome of Ap3A and Ap4A and thereby gain insights into their cellular roles, we herein report the development of photoaffinity-labeling probes and their employment in chemical proteomics. We demonstrate that the identified ApnA interactors are involved in many fundamental cellular processes including carboxylic acid and nucleotide metabolism, gene expression, various regulatory processes and cellular response mechanisms and only around half of them are known nucleotide interactors. Our results highlight common functions of these ApnAs across the domains of life, but also identify those that are different for Ap3A or Ap4A. This study provides a rich source for further functional studies of these nucleotides and depicts useful tools for characterization of their regulatory mechanisms in cells. Diadenosine polyphosphates (ApAs) are involved in cellular stress signaling but only a few molecular targets have been characterized so far. Here, the authors develop ApnA-based photoaffinity-labeling probes and use them to identify Ap3A and Ap4A binding proteins in human cell lysates. [ABSTRACT FROM AUTHOR]

Decoding the role of histone posttranslational modifications (PTMs) is key to understand the fundamental process of epigenetic regulation. This is well studied for PTMs of core histones but not for linker histone H1 in general and its ubiquitylation in particular due to a lack of proper tools. Here, we report on the chemical synthesis of site-specifically mono-ubiquitylated H1.2 and identify its ubiquitin-dependent interactome on a proteome-wide scale. We show that site-specific ubiquitylation of H1 at position K64 modulates interactions with deubiquitylating enzymes and the deacetylase SIRT1. Moreover, it affects H1-dependent chromatosome assembly and phase separation resulting in a more open chromatosome conformation generally associated with a transcriptionally active chromatin state. In summary, we propose that site-specific ubiquitylation plays a general regulatory role for linker histone H1. While the role of specific posttranslational modifications (PTMs) is increasingly well understood for core histones, this is not the case for linker histone H1. Here the authors show that site-specific ubiquitylation of H1 results in distinct interactomes, regulates phase separation, and modulates assembly of chromatosomes. [ABSTRACT FROM AUTHOR]

The attachment of differently linked ubiquitin (Ub) chains of varying length to proteins is a prevalent posttranslational modification in eukaryotic cells. We established an affinity enrichment approach for the identification of linkage- and length-selective ubiquitin binding proteins and could show that in addition to the linkage, the length of a Ub chain is an important determinant for Ub chain recognition. [ABSTRACT FROM AUTHOR]

AKAP79/150 is essential for coordinating second messenger-responsive enzymes in processes including synaptic long-term depression. Ca2+ directly regulates AKAP79 through its effector calmodulin (CaM), but the molecular basis of this regulation was previously unknown. Here, we report that CaM recognizes a '1-4-7-8' pattern of hydrophobic amino acids starting at Trp79 in AKAP79. Cross-linking coupled to mass spectrometry assisted mapping of the interaction site. Removal of the CaM-binding sequence in AKAP79 prevents formation of a Ca2+-sensitive interface between AKAP79 and calcineurin, and increases resting cellular PKA phosphorylation. We determined a crystal structure of CaM bound to a peptide encompassing its binding site in AKAP79. CaM adopts a highly compact conformation in which its open Ca2+-activated C-lobe and closed N-lobe cooperate to recognize a mixed α/310 helix in AKAP79. The structure guided a bioinformatic screen to identify potential sites in other proteins that may employ similar motifs for interaction with CaM. [ABSTRACT FROM AUTHOR]

Protein aggregates are the hallmark of stressed and ageing cells, and characterize several pathophysiological states. Healthy metazoan cells effectively eliminate intracellular protein aggregates, indicating that efficient disaggregation and/or degradation mechanisms exist. However, metazoans lack the key heat-shock protein disaggregase HSP100 of non-metazoan HSP70-dependent protein disaggregation systems, and the human HSP70 system alone, even with the crucial HSP110 nucleotide exchange factor, has poor disaggregation activity in vitro. This unresolved conundrum is central to protein quality control biology. Here we show that synergic cooperation between complexed J-protein co-chaperones of classes A and B unleashes highly efficient protein disaggregation activity in human and nematode HSP70 systems. Metazoan mixed-class J-protein complexes are transient, involve complementary charged regions conserved in the J-domains and carboxy-terminal domains of each J-protein class, and are flexible with respect to subunit composition. Complex formation allows J-proteins to initiate transient higher order chaperone structures involving HSP70 and interacting nucleotide exchange factors. A network of cooperative class A and B J-protein interactions therefore provides the metazoan HSP70 machinery with powerful, flexible, and finely regulatable disaggregase activity and a further level of regulation crucial for cellular protein quality control. [ABSTRACT FROM AUTHOR]

We describe a method that integrates data derived from different mass spectrometry (MS)-based techniques with a modeling strategy for structural characterization of protein assemblies. We encoded structural data derived from native MS, bottom-up proteomics, ion mobility-MS and chemical cross-linking MS into modeling restraints to compute the most likely structure of a protein assembly. We used the method to generate near-native models for three known structures and characterized an assembly intermediate of the proteasomal base. [ABSTRACT FROM AUTHOR]

The anaphase-promoting complex or cyclosome (APC/C) is an unusually large E3 ubiquitin ligase responsible for regulating defined cell cycle transitions. Information on how its 13 constituent proteins are assembled, and how they interact with co-activators, substrates and regulatory proteins is limited. Here, we describe a recombinant expression system that allows the reconstitution of holo APC/C and its sub-complexes that, when combined with electron microscopy, mass spectrometry and docking of crystallographic and homology-derived coordinates, provides a precise definition of the organization and structure of all essential APC/C subunits, resulting in a pseudo-atomic model for 70% of the APC/C. A lattice-like appearance of the APC/C is generated by multiple repeat motifs of most APC/C subunits. Three conserved tetratricopeptide repeat (TPR) subunits (Cdc16, Cdc23 and Cdc27) share related superhelical homo-dimeric architectures that assemble to generate a quasi-symmetrical structure. Our structure explains how this TPR sub-complex, together with additional scaffolding subunits (Apc1, Apc4 and Apc5), coordinate the juxtaposition of the catalytic and substrate recognition module (Apc2, Apc11 and Apc10 (also known as Doc1)), and TPR-phosphorylation sites, relative to co-activator, regulatory proteins and substrates. [ABSTRACT FROM AUTHOR]

The covalent attachment of the cytokine-inducible ubiquitin-like modifier HLA-F adjacent transcript 10 (FAT10) to hundreds of substrate proteins leads to their rapid degradation by the 26 S proteasome independently of ubiquitylation. Here, we identify another function of FAT10, showing that it interferes with the activation of SUMO1/2/3 in vitro and down-regulates SUMO conjugation and the SUMO-dependent formation of promyelocytic leukemia protein (PML) bodies in cells. Mechanistically, we show that FAT10 directly binds to and impedes the activity of the heterodimeric SUMO E1 activating enzyme AOS1/UBA2 by competing very efficiently with SUMO for activation and thioester formation. Nevertheless, activation of FAT10 by AOS1/UBA2 does not lead to covalent conjugation of FAT10 with substrate proteins which relies on its cognate E1 enzyme UBA6. Hence, we report that one ubiquitin-like modifier (FAT10) inhibits the conjugation and function of another ubiquitin-like modifier (SUMO) by impairing its activation. FAT10 is an ubiquitin-like modifier that targets proteins to proteasomal degradation. Here, the authors show that FAT10 also regulates SUMO activation in vitro and in cells, providing evidence for functional crosstalk between two ubiquitin-like modifiers. [ABSTRACT FROM AUTHOR]

Deregulation of the ubiquitin ligase E6AP is causally linked to the development of human disease, including cervical cancer. In complex with the E6 oncoprotein of human papillomaviruses, E6AP targets the tumor suppressor p53 for degradation, thereby contributing to carcinogenesis. Moreover, E6 acts as a potent activator of E6AP by a yet unknown mechanism. However, structural information explaining how the E6AP-E6-p53 enzyme-substrate complex is assembled, and how E6 stimulates E6AP, is largely missing. Here, we develop and apply different crosslinking mass spectrometry-based approaches to study the E6AP-E6-p53 interplay. We show that binding of E6 induces conformational rearrangements in E6AP, thereby positioning E6 and p53 in the immediate vicinity of the catalytic center of E6AP. Our data provide structural and functional insights into the dynamics of the full-length E6AP-E6-p53 enzyme-substrate complex, demonstrating how E6 can stimulate the ubiquitin ligase activity of E6AP while facilitating ubiquitin transfer from E6AP onto p53. Oncoprotein E6 facilitates the E6AP-catalyzed ubiquitination of p53. Here, the authors study the structural basis of this process by qualitative and quantitative cross-linking mass spectrometry, providing insights into E6AP-E6-p53 complex assembly and the conformational dynamics that enable p53 ubiquitination. [ABSTRACT FROM AUTHOR]