ABSTRACT
Polyubiquitination by E2 and E3 enzymes is a predominant mechanism regulating protein function. Some RING E3s, including anaphase-promoting complex/cyclosome (APC), catalyze polyubiquitination by sequential reactions with two different E2s. An initiating E2 ligates ubiquitin to an E3-bound substrate. Another E2 grows a polyubiquitin chain on the ubiquitin-primed substrate through poorly defined mechanisms. Here we show that human APC's RING domain is repurposed for dual functions in polyubiquitination. The canonical RING surface activates an initiating E2-ubiquitin intermediate for substrate modification. However, APC engages and activates its specialized ubiquitin chain-elongating E2 UBE2S in ways that differ from current paradigms. During chain assembly, a distinct APC11 RING surface helps deliver a substrate-linked ubiquitin to accept another ubiquitin from UBE2S. Our data define mechanisms of APC/UBE2S-mediated polyubiquitination, reveal diverse functions of RING E3s and E2s, and provide a framework for understanding distinctive RING E3 features specifying ubiquitin chain elongation.
Subject(s)
Apc11 Subunit, Anaphase-Promoting Complex-Cyclosome/metabolism , Apc2 Subunit, Anaphase-Promoting Complex-Cyclosome/metabolism , Peptide Biosynthesis, Nucleic Acid-Independent , Polyubiquitin/biosynthesis , Ubiquitin-Conjugating Enzymes/metabolism , Ubiquitination/physiology , Amino Acid Sequence , Apc4 Subunit, Anaphase-Promoting Complex-Cyclosome/metabolism , Cell Cycle Checkpoints , HeLa Cells , Humans , Molecular Sequence Data , Polyubiquitin/genetics , Protein Structure, TertiaryABSTRACT
Molecular machines or macromolecular complexes are supramolecular assemblies of biomolecules with a variety of functions. Structure determination of these complexes in a purified state is often tedious owing to their compositional complexity and the associated relative structural instability. To improve the stability of macromolecular complexes in vitro, we present a generic method that optimizes the stability, homogeneity and solubility of macromolecular complexes by sparse-matrix screening of their thermal unfolding behavior in the presence of various buffers and small molecules. The method includes the automated analysis of thermal unfolding curves based on a biophysical unfolding model for complexes. We found that under stabilizing conditions, even large multicomponent complexes reveal an almost ideal two-state unfolding behavior. We envisage an improved biochemical understanding of purified macromolecules as well as a substantial boost in successful macromolecular complex structure determination by both X-ray crystallography and cryo-electron microscopy.
Subject(s)
Algorithms , Models, Chemical , Models, Molecular , Multiprotein Complexes/chemistry , Multiprotein Complexes/ultrastructure , Software , Binding Sites , Computer Simulation , Crystallization , Protein Binding , Protein Conformation , Protein FoldingABSTRACT
For many E3 ligases, a mobile RING (Really Interesting New Gene) domain stimulates ubiquitin (Ub) transfer from a thioester-linked E2â¼Ub intermediate to a lysine on a remotely bound disordered substrate. One such E3 is the gigantic, multisubunit 1.2-MDa anaphase-promoting complex/cyclosome (APC), which controls cell division by ubiquitinating cell cycle regulators to drive their timely degradation. Intrinsically disordered substrates are typically recruited via their KEN-box, D-box, and/or other motifs binding to APC and a coactivator such as CDH1. On the opposite side of the APC, the dynamic catalytic core contains the cullin-like subunit APC2 and its RING partner APC11, which collaborates with the E2 UBCH10 (UBE2C) to ubiquitinate substrates. However, how dynamic RING-E2â¼Ub catalytic modules such as APC11-UBCH10â¼Ub collide with distally tethered disordered substrates remains poorly understood. We report structural mechanisms of UBCH10 recruitment to APC(CDH1) and substrate ubiquitination. Unexpectedly, in addition to binding APC11's RING, UBCH10 is corecruited via interactions with APC2, which we visualized in a trapped complex representing an APC(CDH1)-UBCH10â¼Ub-substrate intermediate by cryo-electron microscopy, and in isolation by X-ray crystallography. To our knowledge, this is the first structural view of APC, or any cullin-RING E3, with E2 and substrate juxtaposed, and it reveals how tripartite cullin-RING-E2 interactions establish APC's specificity for UBCH10 and harness a flexible catalytic module to drive ubiquitination of lysines within an accessible zone. We propose that multisite interactions reduce the degrees of freedom available to dynamic RING E3-E2â¼Ub catalytic modules, condense the search radius for target lysines, increase the chance of active-site collision with conformationally fluctuating substrates, and enable regulation.
Subject(s)
Anaphase-Promoting Complex-Cyclosome/chemistry , Apc1 Subunit, Anaphase-Promoting Complex-Cyclosome/chemistry , Apc11 Subunit, Anaphase-Promoting Complex-Cyclosome/chemistry , DNA Helicases/chemistry , DNA-Binding Proteins/chemistry , Ubiquitin-Conjugating Enzymes/chemistry , Ubiquitin/chemistry , Anaphase-Promoting Complex-Cyclosome/metabolism , Apc1 Subunit, Anaphase-Promoting Complex-Cyclosome/metabolism , Apc11 Subunit, Anaphase-Promoting Complex-Cyclosome/metabolism , Crystallography, X-Ray , DNA Helicases/metabolism , DNA-Binding Proteins/metabolism , Humans , Ubiquitin/metabolism , Ubiquitin-Conjugating Enzymes/metabolismABSTRACT
The spindle pole body of the budding yeast Saccharomyces cerevisiae has served as a model system for understanding microtubule organizing centers, yet very little is known about the molecular structure of its components. We report here the structure of the C-terminal domain of the core component Cnm67 at 2.3 Å resolution. The structure determination was aided by a novel approach to crystallization of proteins containing coiled-coils that utilizes globular domains to stabilize the coiled-coils. This enhances their solubility in Escherichia coli and improves their crystallization. The Cnm67 C-terminal domain (residues Asn-429-Lys-581) exhibits a previously unseen dimeric, interdigitated, all α-helical fold. In vivo studies demonstrate that this domain alone is able to localize to the spindle pole body. In addition, the structure reveals a large functionally indispensable positively charged surface patch that is implicated in spindle pole body localization. Finally, the C-terminal eight residues are disordered but are critical for protein folding and structural stability.
Subject(s)
Cytoskeletal Proteins/chemistry , Protein Folding , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae/chemistry , Spindle Apparatus/chemistry , Crystallography, X-Ray , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolism , Protein Stability , Protein Structure, Tertiary , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Spindle Apparatus/genetics , Spindle Apparatus/metabolism , Structure-Activity RelationshipABSTRACT
The active site of myosin contains a group of highly conserved amino acid residues whose roles in nucleotide hydrolysis and energy transduction might appear to be obvious from the initial structural and kinetic analyses but become less clear on deeper investigation. One such residue is Ser236 (Dictyostelium discoideum myosin II numbering) which was proposed to be involved in a hydrogen transfer network during gamma-phosphate hydrolysis of ATP, which would imply a critical function in ATP hydrolysis and motility. The S236A mutant protein shows a comparatively small decrease in hydrolytic activity and motility, and thus this residue does not appear to be essential. To understand better the contribution of Ser236 to the function of myosin, structural and kinetic studies have been performed on the S236A mutant protein. The structures of the D. discoideum motor domain (S1dC) S236A mutant protein in complex with magnesium pyrophosphate, MgAMPPNP, and MgADP.vanadate have been determined. In contrast to the previous structure of wild-type S1dC, the S236A.MgAMPPNP complex crystallized in the closed state. Furthermore, transient-state kinetics showed a 4-fold reduction of the nucleotide release step, suggesting that the mutation stabilizes a closed active site. The structures show that a water molecule approximately adopts the location of the missing hydroxyl of Ser236 in the magnesium pyrophosphate and MgAMPPNP structures. This study suggests that the S236A mutant myosin proceeds via a different structural mechanism than wild-type myosin, where the alternate mechanism is able to maintain near normal transient-state kinetic values.
Subject(s)
Adenylyl Imidodiphosphate/chemistry , Adenylyl Imidodiphosphate/physiology , Myosin Type II/chemistry , Myosin Type II/physiology , Myosins/chemistry , Myosins/physiology , Serine/chemistry , Serine/physiology , Actins/chemistry , Actins/physiology , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/chemistry , Adenosine Triphosphate/physiology , Animals , Binding Sites/genetics , Catalytic Domain/genetics , Crystallography, X-Ray , Dictyostelium , Hydrogen Bonding , Myosin Type II/genetics , Myosins/genetics , Serine/genetics , Structure-Activity RelationshipABSTRACT
The anaphase-promoting complex/cyclosome (APC/C) is a massive E3 ligase that controls mitosis by catalyzing ubiquitination of key cell cycle regulatory proteins. The APC/C assembly contains two subcomplexes: the "Platform" centers around a cullin-RING-like E3 ligase catalytic core; the "Arc Lamp" is a hub that mediates transient association with regulators and ubiquitination substrates. The Arc Lamp contains the small subunits APC16, CDC26, and APC13, and tetratricopeptide repeat (TPR) proteins (APC7, APC3, APC6, and APC8) that homodimerize and stack with quasi-2-fold symmetry. Within the APC/C complex, APC3 serves as center for regulation. APC3's TPR motifs recruit substrate-binding coactivators, CDC20 and CDH1, via their C-terminal conserved Ile-Arg (IR) tail sequences. Human APC3 also binds APC16 and APC7 and contains a >200-residue loop that is heavily phosphorylated during mitosis, although the basis for APC3 interactions and whether loop phosphorylation is required for ubiquitination are unclear. Here, we map the basis for human APC3 assembly with APC16 and APC7, report crystal structures of APC3Δloop alone and in complex with the C-terminal domain of APC16, and test roles of APC3's loop and IR tail binding surfaces in APC/C-catalyzed ubiquitination. The structures show how one APC16 binds asymmetrically to the symmetric APC3 dimer and, together with biochemistry and prior data, explain how APC16 recruits APC7 to APC3, show how APC3's C-terminal domain is rearranged in the full APC/C assembly, and visualize residues in the IR tail binding cleft important for coactivator-dependent ubiquitination. Overall, the results provide insights into assembly, regulation, and interactions of TPR proteins and the APC/C.
Subject(s)
Apc3 Subunit, Anaphase-Promoting Complex-Cyclosome/chemistry , Apc3 Subunit, Anaphase-Promoting Complex-Cyclosome/metabolism , Proteins/chemistry , Proteins/metabolism , Amino Acid Sequence , Apc7 Subunit, Anaphase-Promoting Complex-Cyclosome/chemistry , Apc7 Subunit, Anaphase-Promoting Complex-Cyclosome/metabolism , Cell Cycle , Cell Cycle Proteins , Crystallography, X-Ray , Humans , Models, Molecular , Protein Conformation , Protein Interaction Maps , Protein Multimerization , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/metabolismABSTRACT
The anaphase-promoting complex/cyclosome (APC/C) is a ~1.5-MDa multiprotein E3 ligase enzyme that regulates cell division by promoting timely ubiquitin-mediated proteolysis of key cell-cycle regulatory proteins. Inhibition of human APC/C(CDH1) during interphase by early mitotic inhibitor 1 (EMI1) is essential for accurate coordination of DNA synthesis and mitosis. Here, we report a hybrid structural approach involving NMR, electron microscopy and enzymology, which reveal that EMI1's 143-residue C-terminal domain inhibits multiple APC/C(CDH1) functions. The intrinsically disordered D-box, linker and tail elements, together with a structured zinc-binding domain, bind distinct regions of APC/C(CDH1) to synergistically both block the substrate-binding site and inhibit ubiquitin-chain elongation. The functional importance of intrinsic structural disorder is explained by enabling a small inhibitory domain to bind multiple sites to shut down various functions of a 'molecular machine' nearly 100 times its size.