ABSTRACT
The linear ubiquitin chain assembly complex (LUBAC) is a key nuclear factor-κB (NF-κB) pathway component that produces linear polyubiquitin chains. The HOIL-1L subunit of LUBAC has been shown to bind linear chains; however, detailed structural and functional analyses on the binding between LUBAC and linear chains have not been performed. In this study, we found that the Npl4 zinc finger (NZF) domain of HOIL-1L specifically binds linear polyubiquitin chains and determined the crystal structure of the HOIL-1L NZF domain in complex with linear diubiquitin at 1.7-Å resolution. The HOIL-1L NZF domain consists of a zinc-coordinating "NZF core" region and an additional α-helical "NZF tail" region. The HOIL-1L NZF core binds both the canonical Ile44-centered hydrophobic surface on the distal ubiquitin and a Phe4-centered hydrophobic patch on the proximal ubiquitin, representing a mechanism for the specific recognition of linear chains. The NZF tail binds the proximal ubiquitin to enhance the binding affinity. These recognition mechanisms were supported by the accompanying in vitro and in vivo structure-based mutagenesis experiments.
Subject(s)
Carrier Proteins/chemistry , Nuclear Proteins/chemistry , Ubiquitin-Protein Ligases/chemistry , Amino Acid Sequence , Animals , Crystallography, X-Ray/methods , Humans , Mice , Molecular Conformation , Molecular Sequence Data , NF-kappa B/metabolism , Protein Conformation , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Surface Plasmon Resonance/methods , Transcription FactorsABSTRACT
The exocyst complex is a hetero-octameric protein complex that functions during cell polarization by tethering the secretory vesicle to the target membrane. The yeast exocyst subunit Sec3 binds to phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) and the small GTPases Rho1 and Cdc42 via its N-terminal domain (Sec3-N), and these interactions target Sec3 to the plasma membrane. Here we report the crystal structure of the Sec3-N in complex with Rho1 at 2.6-A resolution. Sec3-N adopts a pleckstrin homology (PH) fold, despite having no detectable sequence homology with other PH domains of known structure. Clusters of conserved basic residues constitute a positively charged cleft, which was identified as a binding site for PtdIns(4,5)P(2). Residues Phe77, Ile115 and Leu131 of Sec3 bind to an extended hydrophobic surface formed around switch regions I and II of Rho1. To our knowledge, these are the first structural insights into how an exocyst subunit might interact with both protein and phospholipid factors on the target membrane.
Subject(s)
Phosphatidylinositols/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Yeasts/chemistry , Yeasts/metabolism , rho GTP-Binding Proteins/metabolism , Crystallography, X-Ray , Models, Molecular , Protein Structure, Quaternary , Protein Subunits/chemistry , Protein Subunits/metabolismABSTRACT
Tail anchored (TA) proteins, which are important for numerous cellular processes, are defined by a single transmembrane domain (TMD) near the C-terminus. The membrane insertion of TA proteins is mediated by the highly conserved ATPase Get3. Here we report the crystal structures of Get3 in ADP-bound and nucleotide-free forms at 3.0 A and 2.8 A resolutions, respectively. Get3 consists of a nucleotide binding domain and a helical domain. Both structures exhibit a Zn(2+)-mediated homodimer in a head-to-head orientation, representing an open dimer conformation. Our cross-link experiments indicated the closed dimer-stimulating ATP hydrolysis, which might be coupled with TA-protein release. Further, our coexpression-based binding assays using a model TA protein Sec22p revealed the direct interaction between the helical domain of Get3 and the Sec22p TMD. This interaction is independent of ATP and dimer formation. Finally, we propose a structural mechanism that links ATP hydrolysis with the TA-protein insertion mediated by the conserved DTAPTGH motif.
Subject(s)
Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/metabolism , Cell Membrane/metabolism , Guanine Nucleotide Exchange Factors/chemistry , Guanine Nucleotide Exchange Factors/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/metabolism , Adenosine Triphosphate/metabolism , Binding Sites , Cell Membrane/drug effects , Cross-Linking Reagents/pharmacology , Crystallography, X-Ray , Hydrolysis/drug effects , Models, Biological , Mutation/genetics , Protein Binding/drug effects , Protein Multimerization/drug effects , Protein Structure, Secondary , Protein Structure, Tertiary , Saccharomyces cerevisiae/drug effects , Zinc/metabolismABSTRACT
TAB2 and TAB3 activate the Jun N-terminal kinase and nuclear factor-kappaB pathways through the specific recognition of Lys 63-linked polyubiquitin chains by its Npl4 zinc-finger (NZF) domain. Here we report crystal structures of the TAB2 and TAB3 NZF domains in complex with Lys 63-linked diubiquitin at 1.18 and 1.40 A resolutions, respectively. Both NZF domains bind to the distal ubiquitin through a conserved Thr-Phe dipeptide that has been shown to be important for the interaction of the NZF domain of Npl4 with monoubiquitin. In contrast, a surface specific to TAB2 and TAB3 binds the proximal ubiquitin. Both the distal and proximal binding sites of the TAB2 and TAB3 NZF domains recognize the Ile 44-centred hydrophobic patch on ubiquitin but do not interact with the Lys 63-linked isopeptide bond. Mutagenesis experiments show that both binding sites are required to enable binding of Lys 63-linked diubiquitin. We therefore propose a mechanism for the recognition of Lys 63-linked polyubiquitin chains by TAB2 and TAB3 NZF domains in which diubiquitin units are specifically recognized by a single NZF domain.
Subject(s)
Adaptor Proteins, Signal Transducing/chemistry , Polyubiquitin/chemistry , Animals , Binding Sites , Crystallography, X-Ray/methods , JNK Mitogen-Activated Protein Kinases/metabolism , Lysine/chemistry , Mice , Models, Molecular , NF-kappa B/metabolism , Protein Binding , Protein Structure, Tertiary , Ubiquitin/chemistry , Zinc FingersABSTRACT
NEMO is essential for activation of the NF-kappaB signaling pathway, which is regulated by ubiquitination of proteins. The C-terminal leucine zipper of NEMO and its adjacent coiled-coil region (CC2-LZ) reportedly bind to linear ubiquitin chains with 1 microM affinity and to Lys 63-linked chains with 100 microM affinity. Here we report the crystal structure of the CC2-LZ region of mouse NEMO in complex with Lys 63-linked di-ubiquitin (K63-Ub(2)) at 2.7A resolution. The ubiquitin-binding region consists of a 130A-long helix and forms a parallel coiled-coil dimer. The Ile 44-centered hydrophobic patch of ubiquitin is recognized in the middle of the NEMO ubiquitin-binding region. NEMO interacts with each K63-Ub(2)via a single ubiquitin-binding site, consistent with low affinity binding with K63-Ub(2).
Subject(s)
Intracellular Signaling Peptides and Proteins/chemistry , Lysine/chemistry , Protein Structure, Secondary , Protein Structure, Tertiary , Ubiquitin/chemistry , Amino Acid Sequence , Animals , Binding Sites , Crystallography, X-Ray , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Lysine/metabolism , Mice , Models, Molecular , Molecular Sequence Data , Protein Binding , Sequence Alignment , Ubiquitin/genetics , Ubiquitin/metabolismABSTRACT
RAP80 has a key role in the recruitment of the Abraxas-BRCC36-BRCA1-BARD1 complex to DNA-damage foci for DNA repair through specific recognition of Lys 63-linked polyubiquitinated proteins by its tandem ubiquitin-interacting motifs (UIMs). Here, we report the crystal structure of the RAP80 tandem UIMs (RAP80-UIM1-UIM2) in complex with Lys 63-linked di-ubiquitin at 2.2 A resolution. The two UIMs, UIM1 and UIM2, and the alpha-helical inter-UIM region together form a continuous 60 A-long alpha-helix. UIM1 and UIM2 bind to the proximal and distal ubiquitin moieties, respectively. Both UIM1 and UIM2 of RAP80 recognize an Ile 44-centered hydrophobic patch on ubiquitin but neither UIM interacts with the Lys 63-linked isopeptide bond. Our structure suggests that the inter-UIM region forms a 12 A-long alpha-helix that ensures that the UIMs are arranged to enable specific binding of Lys 63-linked di-ubiquitin. This was confirmed by pull-down analyses using RAP80-UIM1-UIM2 mutants of various length inter-UIM regions. Further, we show that the Epsin1 tandem UIM, which has an inter-UIM region similar to that of RAP80-UIM1-UIM2, also selectively binds Lys 63-linked di-ubiquitin.
Subject(s)
Cell Cycle Proteins/chemistry , Cell Cycle Proteins/metabolism , Lysine/metabolism , Polyubiquitin/metabolism , Transcription Factors/chemistry , Transcription Factors/metabolism , Amino Acid Motifs , Animals , Carrier Proteins/chemistry , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Cycle Proteins/genetics , Crystallography, X-Ray , DNA-Binding Proteins , Histone Chaperones , Lysine/chemistry , Mice , Models, Molecular , Molecular Sequence Data , Mutant Proteins/chemistry , Mutant Proteins/genetics , Mutant Proteins/metabolism , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Polyubiquitin/chemistry , Protein Binding , Protein Conformation , Sequence Alignment , Transcription Factors/geneticsABSTRACT
Deubiquitinating enzymes (DUBs) remove ubiquitin from conjugated substrates to regulate various cellular processes. The Zn(2+)-dependent DUBs AMSH and AMSH-LP regulate receptor trafficking by specifically cleaving Lys 63-linked polyubiquitin chains from internalized receptors. Here we report the crystal structures of the human AMSH-LP DUB domain alone and in complex with a Lys 63-linked di-ubiquitin at 1.2 A and 1.6 A resolutions, respectively. The AMSH-LP DUB domain consists of a Zn(2+)-coordinating catalytic core and two characteristic insertions, Ins-1 and Ins-2. The distal ubiquitin interacts with Ins-1 and the core, whereas the proximal ubiquitin interacts with Ins-2 and the core. The core and Ins-1 form a catalytic groove that accommodates the Lys 63 side chain of the proximal ubiquitin and the isopeptide-linked carboxy-terminal tail of the distal ubiquitin. This is the first reported structure of a DUB in complex with an isopeptide-linked ubiquitin chain, which reveals the mechanism for Lys 63-linkage-specific deubiquitination by AMSH family members.
