pFind Studio: a computational solution for mass spectrometry-based proteomics
2018
Nature2018. Myers, JB et al.
Portland State Univ, Dept Chem, Portland, OR 97207 USA.
ABSTRACT:Gap junctions establish direct pathways for cell-to-cell communication through the assembly of twelve connexin subunits that form intercellular channels connecting neighbouring cells. Co-assembly of different connexin isoforms produces channels with unique properties and enables communication across cell types. Here we used single-particle cryo-electron microscopy to investigate the structural basis of connexin co-assembly in native lens gap junction channels composed of connexin 46 and connexin 50 (Cx46/50). We provide the first comparative analysis to connexin 26 (Cx26), which-together with computational studies-elucidates key energetic features governing gap junction permselectivity. Cx46/50 adopts an open-state conformation that is distinct from the Cx26 crystal structure, yet it appears to be stabilized by a conserved set of hydrophobic anchoring residues. 'Hot spots' of genetic mutations linked to hereditary cataract formation map to the core structural-functional elements identified in Cx46/50, suggesting explanations for many of the disease-causing effects.
Use: pLink
NATURE2018. Sanghai, ZA et al.
Rockefeller Univ, Lab Prot & Nucle Acid Chem, New York, NY 10065 USA.
ABSTRACT:Early co-transcriptional events during eukaryotic ribosome assembly result in the formation of precursors of the small (40S) and large (60S) ribosomal subunits1. A multitude of transient assembly factors regulate and chaperone the systematic folding of pre-ribosomal RNA subdomains. However, owing to a lack of structural information, the role of these factors during early nucleolar 60S assembly is not fully understood. Here we report cryo-electron microscopy (cryo-EM) reconstructions of the nucleolar pre-60S ribosomal subunit in different conformational states at resolutions of up to 3.4 angstrom. These reconstructions reveal how steric hindrance and molecular mimicry are used to prevent both premature folding states and binding of later factors. This is accomplished by the concerted activity of 21 ribosome assembly factors that stabilize and remodel pre-ribosomal RNA and ribosomal proteins. Among these factors, three Brix-domain proteins and their binding partners form a ring-like structure at ribosomal RNA (rRNA) domain boundaries to support the architecture of the maturing particle. The existence of mutually exclusive conformations of these pre-60S particles suggests that the formation of the polypeptide exit tunnel is achieved through different folding pathways during subsequent stages of ribosome assembly. These structures rationalize previous genetic and biochemical data and highlight the mechanisms that drive eukaryotic ribosome assembly in a unidirectional manner.
Use: pLink
Nature Microbiology2018. Ozcan, A et al.
Max Planck Inst Terr Microbiol, Marburg, Germany.
ABSTRACT:Type IV CRISPR-Cas modules belong to class 1 prokaryotic adaptive immune systems, which are defined by the presence of multisubunit effector complexes. They usually lack the known Cas proteins involved in adaptation and target cleavage, and their function has not been experimentally addressed. To investigate RNA and protein components of this CRISPR-Cas type, we located a complete type IV cas gene locus and an adjacent CRISPR array on a megaplasmid of Aromatoleum aromaticum EbN1, which contains an additional type I-C system on its chromosome. RNA sequencing analyses verified CRISPR RNA (crRNA) production and maturation for both systems. Type IV crRNAs were shown to harbour unusually short 7 nucleotide 5'-repeat tags and stable 3' hairpin structures. A unique Cas6 variant (Csf5) was identified that generates crRNAs that are specifically incorporated into type IV CRISPR-ribonucleoprotein (crRNP) complexes. Structures of RNA-bound Csf5 were obtained. Recombinant production and purification of the type IV Cas proteins, together with electron microscopy, revealed that Csf2 acts as a helical backbone for type IV crRNPs that include Csf5, Csf3 and a large subunit (Csf1). Mass spectrometry analyses identified protein-protein and protein-RNA contact sites. These results highlight evolutionary connections between type IV and type I CRISPR-Cas systems and demonstrate that type IV CRISPR-Cas systems employ crRNA-guided effector complexes.
Use: pLink
Nature Structural & Molecular Biology2018. Mei, KR et al.
Univ Penn, Dept Biol, Philadelphia, PA 19104 USA.
ABSTRACT:The exocyst is an evolutionarily conserved octameric protein complex that mediates the tethering of post-Golgi secretory vesicles to the plasma membrane during exocytosis and is implicated in many cellular processes such as cell polarization, cytokinesis, ciliogenesis and tumor invasion. Using cryo-EM and chemical cross-linking MS (CXMS), we solved the structure of the Saccharomyces cerevisiae exocyst complex at an average resolution of 4.4 angstrom. Our model revealed the architecture of the exocyst and led to the identification of the helical bundles that mediate the assembly of the complex at its core. Sequence analysis suggests that these regions are evolutionarily conserved across eukaryotic systems. Additional cell biological data suggest a mechanism for exocyst assembly that leads to vesicle tethering at the plasma membrane.
Use: pLink
Cell Research2018. Xizi Chen, Mengjie Liu et al.
Fudan University Shanghai Cancer Center, Institutes of Biomedical Sciences, Shanghai Medical College of Fudan University, Shanghai 200032, China;
ABSTRACT:Mechanistic targetofrapamycin (mTOR)complex2(mTORC2) plays an essential role in regulating cell proliferation through phosphorylating AGC protein kinase family members, including AKT, PKC and SGK1. The functional corecomplexconsistsofmTOR, mLST8, and two mTORC2-specific components, Rictor and mSin1. Here we investigated the intermolecular interactions within mTORC2complexand determined its cryo-electron microscopystructureat 4.9 A resolution. Thestructurereveals a hollow rhombohedral fold with a2-fold symmetry. The dimerizedmTORserves as a scaffold for thecomplexassembly. The N-terminal halfofRictor is composedofhelical repeat clusters and binds tomTORthrough multiple contacts. mSin1 is located close to the FRB domain and catalytic cavityofmTOR. Rictor and mSin1 together generate steric hindrance to inhibit bindingofFKBP12-rapamycin tomTOR, revealing the mechanism for rapamycin insensitivityofmTORC2. ThemTORdimer in mTORC2 shows more compact conformation than thatofmTORC1 (rapamycin sensitive), which might result from the interaction betweenmTORand Rictor-mSin1. Structural comparison shows that bindingofRictor and Raptor (mTORC1-specific component) tomTORis mutually exclusive. Our study provides a basis for understanding the assemblyofmTORC2 and a framework to further characterize the regulatory mechanismofmTORC2 pathway.
Use: pLink
Cell Reports2018. Tuttle, LM et al.
Fred Hutchinson Canc Res Ctr, Div Basic Sci, Seattle, WA 98109 USA.
ABSTRACT:Transcription activation domains (ADs) are inherently disordered proteins that often target multiple coactivator complexes, but the specificity of these interactions is not understood. Efficient transcription activation by yeast Gcn4 requires its tandem ADs and four activator-binding domains (ABDs) on its target, the Mediator subunit Med15. Multiple ABDs are a common feature of coactivator complexes. We find that the large Gcn4-Med15 complex is heterogeneous and contains nearly all possible AD-ABD interactions. Gcn4-Med15 forms via a dynamic fuzzy protein- protein interface, where ADs bind the ABDs in multiple orientations via hydrophobic regions that gain helicity. This combinatorial mechanism allows individual low-affinity and specificity interactions to generate a biologically functional, specific, and higher affinity complex despite lacking a defined protein- protein interface. This binding strategy is likely representative of many activators that target multiple coactivators, as it allows great flexibility in combinations of activators that can cooperate to regulate genes with variable coactivator requirements.
Use: pLink
nature structural & molecular biology2018. Bodnar, NO et al.
Harvard Med Sch, Howard Hughes Med Inst, Boston, MA 02115 USA.
ABSTRACT:Many polyubiquitinated proteins are extracted from membranes or complexes by the conserved ATPase Cdc48 (in yeast; p97 or VCP in mammals) before proteasomal degradation. Each Cdc48 hexamer contains two stacked ATPase rings (D1 and D2) and six N-terminal (N) domains. Cdc48 binds various cofactors, including the Ufd1-Npl4 heterodimer. Here, we report structures of the Cdc48-Ufd1-Npl4 complex from Chaetomium thermophilum. Npl4 interacts through its UBX-like domain with a Cdc48 N domain, and it uses two Zn2+-finger domains to anchor the enzymatically inactive Mpr1-Pad1 N-terminal (MPN) domain, homologous to domains found in several isopeptidases, to the top of the D1 ATPase ring. The MPN domain of Npl4 is located above Cdc48's central pore, a position similar to the MPN domain from deubiquitinase Rpn11 in the proteasome. Our results indicate that Npl4 is unique among Cdc48 cofactors and suggest a mechanism for binding and translocation of polyubiquitinated substrates into the ATPase.
Use: pLink
NATURE COMMUNICATIONS2018. Oroz, J et al.
German Ctr Neurodegenerat Dis DZNE, Von Siebold Str 3a, D-37075 Gottingen, Germany.
ABSTRACT:The molecular chaperone Hsp90 is critical for the maintenance of cellular homeostasis and represents a promising drug target. Despite increasing knowledge on the structure of Hsp90, the molecular basis of substrate recognition and pro-folding by Hsp90/co-chaperone complexes remains unknown. Here, we report the solution structures of human full-length Hsp90 in complex with the PPIase FKBP51, as well as the 280 kDa Hsp90/FKBP51 complex bound to the Alzheimer's disease-related protein Tau. We reveal that the FKBP51/Hsp90 complex, which synergizes to promote toxic Tau oligomers in vivo, is highly dynamic and stabilizes the extended conformation of the Hsp90 dimer resulting in decreased Hsp90 ATPase activity. Within the ternary Hsp90/FKBP51/Tau complex, Hsp90 serves as a scaffold that traps the PPIase and nucleates multiple conformations of Tau's proline-rich region next to the PPIase catalytic pocket in a phosphorylation-dependent manner. Our study defines a conceptual model for dynamic Hsp90/co-chaperone/client recognition.
Use: pLink
Cell reports2018. Kecman, T et al.
Univ Oxford, Dept Biochem, Oxford OX1 3QU, England.
ABSTRACT:Termination of RNA polymerase II (Pol II) transcription is a key step that is important for 3' end formation of functional mRNA, mRNA release, and Pol II recycling. Even so, the underlying termination mechanism is not yet understood. Here, we demonstrate that the conserved and essential termination factor Seb1 is found on Pol II near the end of the RNA exit channel and the Rpb4/7 stalk. Furthermore, the Seb1 interaction surface with Pol II largely overlaps with that of the elongation factor Spt5. Notably, Seb1 co-transcriptional recruitment is dependent on Spt5 dephosphorylation by the conserved PP1 phosphatase Dis2, which also dephosphorylates threonine 4 within the Pol II heptad repeated C-terminal domain. We propose that Dis2 orchestrates the transition from elongation to termination phase during the transcription cycle by mediating elongation to termination factor exchange and dephosphorylation of Pol II C-terminal domain.
Use: pLink
Scientific reports2018. Petriman, NA et al.
Albert Ludwigs Univ Freiburg, Fac Med, ZBMZ, Inst Biochem & Mol Biol, D-79104 Freiburg, Germany.
ABSTRACT:YidC/Oxa1/Alb3 are essential proteins that operate independently or cooperatively with the Sec machinery during membrane protein insertion in bacteria, archaea and eukaryotic organelles. Although the interaction between the bacterial SecYEG translocon and YidC has been observed in multiple studies, it is still unknown which domains of YidC are in contact with the SecYEG translocon. By in vivo and in vitro site-directed and para-formaldehyde cross-linking we identified the auxiliary transmembrane domain 1 of E. coli YidC as a major contact site for SecY and SecG. Additional SecY contacts were observed for the tightly packed globular domain and the C1 loop of YidC, which reveals that the hydrophilic cavity of YidC faces the lateral gate of SecY. Surprisingly, YidC-SecYEG contacts were only observed when YidC and SecYEG were present at about stoichiometric concentrations, suggesting that the YidC-SecYEG contact in vivo is either very transient or only observed for a very small SecYEG sub-population. This is different for the YidC-SRP and YidC-FtsY interaction, which involves the C1 loop of YidC and is efficiently observed even at sub-stoichiometric concentrations of SRP/FtsY. In summary, our data provide a first detailed view on how YidC interacts with the SecYEG translocon and the SRP-targeting machinery.
Use: pLink