scholarly journals Molecular dynamics of C99-bound γ-secretase reveal two binding modes with distinct compactness, stability, and active-site retention: implications for Aβ production

2019 ◽  
Vol 476 (7) ◽  
pp. 1173-1189 ◽  
Author(s):  
Budheswar Dehury ◽  
Ning Tang ◽  
Kasper P. Kepp
Keyword(s):  
Structural Basis ◽  
Binding Modes ◽  
Wild Type ◽  
Dynamic Simulations ◽  
Aβ Peptides ◽  
Cryo Electron Microscopy ◽  
Compact State ◽  
Β Sheet ◽  
Aβ Production

Abstract The membrane protease γ-secretase cleaves the C99 fragment of the amyloid precursor protein, thus producing the Aβ peptides central to Alzheimer's disease. Cryo-electron microscopy has provided the topology but misses the membrane and loop parts that contribute to substrate binding. We report here an essentially complete atomic model of C99 within wild-type γ-secretase that respects all the experimental constraints and additionally describes loop, helix, and C99 substrate dynamics in a realistic all-atom membrane. Our model represents the matured auto-cleaved state required for catalysis. From two independent 500-ns molecular dynamic simulations, we identify two conformation states of C99 in equilibrium, a compact and a loose state. Our simulations provide a basis for C99 processing and Aβ formation and explain the production of longer and shorter Aβ, as the compact state retains C99 for longer and thus probably trims to shorter Aβ peptides. We expect pathogenic presenilin mutations to stabilize the loose over the compact state. The simulations detail the role of the Lys53–Lys54–Lys55 anchor for C99 binding, a loss of helicity of bound C99, and positioning of Thr48 and Leu49 leading to alternative trimming pathways on opposite sides of the C99 helix in three amino acid steps. The C99 binding topology resembles that of C83-bound γ-secretase without membrane but lacks a presenilin 1-C99 β-sheet, which could be induced by C83's stronger binding. The loose state should be selectively disfavored by γ-secretase modulators to increase C99 trimming and reduce the formation of longer Aβ, a strategy that is currently much explored but has lacked a structural basis.

Role of the N-terminal region and of β-sheet residue Thr29 on the activity of the ω2 global regulator from the broad-host range Streptococcus pyogenes plasmid pSM19035

2005 ◽  
Vol 386 (9) ◽  
Author(s):  
Karin Welfle ◽  
Florencia Pratto ◽  
Rolf Misselwitz ◽  
Joachim Behlke ◽  
Juan C. Alonso ◽  
...  
Keyword(s):  
Host Range ◽  
Streptococcus Pyogenes ◽  
Regulatory Protein ◽  
Terminal Region ◽  
Broad Host Range ◽  
Wild Type ◽  
Global Regulator ◽  
Β Sheet

AbstractThe dimeric regulatory protein wild-type ω (wt ω

scholarly journals Structural basis for VIPP1 oligomerization and maintenance of thylakoid membrane integrity

2020 ◽  
Author(s):  
Tilak Kumar Gupta ◽  
Sven Klumpe ◽  
Karin Gries ◽  
Steffen Heinz ◽  
Wojciech Wietrzynski ◽  
...  
Keyword(s):  
Membrane Integrity ◽  
Point Mutations ◽  
Binding Pocket ◽  
Thylakoid Membranes ◽  
Lipid Binding ◽  
Structural Basis ◽  
Amphipathic Helices ◽  
Cryo Electron Microscopy

AbstractVesicle-inducing protein in plastids (VIPP1) is essential for the biogenesis and maintenance of thylakoid membranes, which transform light into life. However, it is unknown how VIPP1 performs its vital membrane-shaping function. Here, we use cryo-electron microscopy to determine structures of cyanobacterial VIPP1 rings, revealing how VIPP1 monomers flex and interweave to form basket-like assemblies of different symmetries. Three VIPP1 monomers together coordinate a non-canonical nucleotide binding pocket that is required for VIPP1 oligomerization. Inside the ring’s lumen, amphipathic helices from each monomer align to form large hydrophobic columns, enabling VIPP1 to bind and curve membranes. In vivo point mutations in these hydrophobic surfaces cause extreme thylakoid swelling under high light, indicating an essential role of VIPP1 lipid binding in resisting stress-induced damage. Our study provides a structural basis for understanding how the oligomerization of VIPP1 drives the biogenesis of thylakoid membranes and protects these life-giving membranes from environmental stress.

scholarly journals Analysis of the Secondary Structure of β-Amyloid (Aβ42) Fibrils by Systematic Proline Replacement

2004 ◽  
Vol 279 (50) ◽  
pp. 52781-52788 ◽  
Author(s):  
Akira Morimoto ◽  
Kazuhiro Irie ◽  
Kazuma Murakami ◽  
Yuichi Masuda ◽  
Hajime Ohigashi ◽  
...  
Keyword(s):  
Amino Acid ◽  
Secondary Structure ◽  
Amyloid Fibrils ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Aβ Peptides ◽  
Amyloid Peptides ◽  
Aggregation Inhibitors ◽  
Β Amyloid ◽  
Β Sheet

Amyloid fibrils in Alzheimer's disease mainly consist of 40- and 42-mer β-amyloid peptides (Aβ40 and Aβ42) that exhibit aggregative ability and neurotoxicity. Although the aggregates of Aβ peptides are rich in intermolecular β-sheet, the precise secondary structure of Aβ in the aggregates remains unclear. To identify the amino acid residues involved in the β-sheet formation, 34 proline-substituted mutants of Aβ42 were synthesized and their aggregative ability and neurotoxicity on PC12 cells were examined. Prolines are rarely present in β-sheet, whereas they are easily accommodated in β-turn as a Pro-Xcorner. Among the mutants at positions 15-32, only E22P-Aβ42 extensively aggregated with stronger neurotoxicity than wild-type Aβ42, suggesting that the residues at positions 15-21 and 24-32 are involved in the β-sheet and that the turn at positions 22 and 23 plays a crucial role in the aggregation and neurotoxicity of Aβ42. The C-terminal proline mutants (A42P-, I41P-, and V40P-Aβ42) hardly aggregated with extremely weak cytotoxicity, whereas the C-terminal threonine mutants (A42T- and I41T-Aβ42) aggregated potently with significant cytotoxicity. These results indicate that the hydrophobicity of the C-terminal two residues of Aβ42 is not related to its aggregative ability and neurotoxicity, rather the C-terminal three residues adopt the β-sheet. These results demonstrate well the large difference in aggregative ability and neurotoxicity between Aβ42 and Aβ40. In contrast, the proline mutants at the N-terminal 13 residues showed potent aggregative ability and neurotoxicity similar to those of wild-type Aβ42. The identification of the β-sheet region of Aβ42 is a basis for designing new aggregation inhibitors of Aβ peptides.

scholarly journals Calcium Binding Is Required for Calmodulin Function in Aspergillus nidulans

2002 ◽  
Vol 1 (1) ◽  
pp. 119-125 ◽  
Author(s):  
James D. Joseph ◽  
Anthony R. Means
Keyword(s):  
Amino Acid ◽  
Aspergillus Nidulans ◽  
Calcium Binding ◽  
Structural Basis ◽  
Wild Type ◽  
Endogenous Protein ◽  
Chimeric Molecules ◽  
Overlay Assay

ABSTRACT To explore the structural basis for the essential role of calmodulin (CaM) in Aspergillus nidulans, we have compared the biochemical and in vivo properties of A. nidulans CaM (AnCaM) with those of heterologous CaMs. Neither Saccharomyces cerevisiae CaM (ScCaM) nor a Ca2+ binding mutant of A. nidulans CaM (1234) interacts appreciably with A. nidulans CaM binding proteins by an overlay assay or activates two essential CaMKs, CMKA and CMKB. In contrast, although vertebrate CaM (VCaM) binds a spectrum of proteins similar to that for AnCaM, it is unable to fully activate CMKA and CMKB, displaying a higher K CaM and reduced V max for both enzymes. In correlation with the biochemical analysis, neither ScCaM nor 1234 can support A. nidulans growth in the absence of the endogenous protein, whereas VCaM only partially complements the absence of wild-type CaM. Analysis of VCaM and AnCaM chimeras demonstrates that amino acid variations in both N- and C-terminal domains contribute to the inability of VCaM to activate CMKB, but differences in the N terminus are largely responsible for the reduced activity towards CMKA. In vivo, the chimeric molecules support growth equivalently, but only to levels intermediate between those of VCaM and AnCaM, suggesting that the reduced ability to activate the CaMKs is not solely responsible for the inability of VCaM to complement the absence of the wild-type protein. Thus, not only is Ca2+ binding required for CaM function in A. nidulans, but the essential in vivo functions of A. nidulans CaM are uniquely sensitive to the subtle amino acid variations present in vertebrate CaM.

scholarly journals Structural Insights into Sphingosine-1-phosphate Receptor Activation

2022 ◽  
Author(s):  
Leiye Yu ◽  
Licong He ◽  
Bing Gan ◽  
Rujuan Ti ◽  
Qingjie Xiao ◽  
...  
Keyword(s):  
Transition Process ◽  
Receptor Activation ◽  
Activation Mechanism ◽  
Structural Basis ◽  
Lymphoid Tissues ◽  
Binding Modes ◽  
Dynamic Simulations ◽  
S1p Receptors ◽  
Sphingosine 1 Phosphate ◽  
Key Steps

As a critical sphingolipid metabolite, sphingosine-1-phosphate (S1P) plays an essential role in immune and vascular systems. There are five S1P receptors, designated as S1PR1-5, encoded in the human genome, and their activities are governed by endogenous S1P, lipid-like S1P mimics, or non-lipid-like therapeutic molecules. Among S1PRs, S1PR1 stands out due to its non-redundant functions, such as the egress of T and B cells from the thymus and secondary lymphoid tissues, making it a potential therapeutic target. However, the structural basis of S1PR1 activation and regulation by various agonists remains unclear. Here we reported four atomic resolution cryo-EM structures of Gi-coupled human S1PR1 complexes: bound to endogenous agonist d18:1 S1P, benchmark lipid-like S1P mimic phosphorylated Fingolimod ((S)-FTY720-P), or non-lipid-like therapeutic molecule CBP-307 in two binding modes. Our results revealed the similarities and differences of activation of S1PR1 through distinct ligands binding to the amphiphilic orthosteric pocket. We also proposed a two-step "shallow to deep" transition process of CBP-307 for S1PR1 activation. Both binding modes of CBP-307 could activate S1PR1, but from shallow to deep transition may trigger the rotation of the N-terminal helix of Gαi and further stabilize the complex by increasing the Gαi interaction with the cell membrane. We combine with extensive biochemical analysis and molecular dynamic simulations to suggest key steps of S1P binding and receptor activation. The above results decipher the common feature of the S1PR1 agonist recognition and activation mechanism and will firmly promote the development of therapeutics targeting S1P receptors.

scholarly journals Structural basis of the regulation of the normal and oncogenic methylation of nucleosomal histone H3 Lys36 by NSD2

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ko Sato ◽  
Amarjeet Kumar ◽  
Keisuke Hamada ◽  
Chikako Okada ◽  
Asako Oguni ◽  
...  
Keyword(s):  
Histone H3 ◽  
Structural Basis ◽  
Wild Type ◽  
Cryo Electron Microscopy ◽  
Nucleosomal Dna ◽  
Oncogenic Mutations ◽  
Binding Cleft ◽  
Dynamics Simulations ◽  
Nucleosome Binding ◽  
Nucleosomal Histone

AbstractDimethylated histone H3 Lys36 (H3K36me2) regulates gene expression, and aberrant H3K36me2 upregulation, resulting from either the overexpression or point mutation of the dimethyltransferase NSD2, is found in various cancers. Here we report the cryo-electron microscopy structure of NSD2 bound to the nucleosome. Nucleosomal DNA is partially unwrapped, facilitating NSD2 access to H3K36. NSD2 interacts with DNA and H2A along with H3. The NSD2 autoinhibitory loop changes its conformation upon nucleosome binding to accommodate H3 in its substrate-binding cleft. Kinetic analysis revealed that two oncogenic mutations, E1099K and T1150A, increase NSD2 catalytic turnover. Molecular dynamics simulations suggested that in both mutants, the autoinhibitory loop adopts an open state that can accommodate H3 more often than the wild-type. We propose that E1099K and T1150A destabilize the interactions that keep the autoinhibitory loop closed, thereby enhancing catalytic turnover. Our analyses guide the development of specific inhibitors of NSD2.

scholarly journals Cryo-EM structures of undocked innexin-6 hemichannels in phospholipids

2020 ◽  
Vol 6 (7) ◽  
pp. eaax3157 ◽  
Author(s):  
Batuujin Burendei ◽  
Ruriko Shinozaki ◽  
Masakatsu Watanabe ◽  
Tohru Terada ◽  
Kazutoshi Tani ◽  
...  
Keyword(s):  
Gap Junctions ◽  
Structural Basis ◽  
Wild Type ◽  
Amino Terminal ◽  
Functional Assays ◽  
Cryo Electron Microscopy ◽  
Dynamics Simulations ◽  
Open States ◽  
Insight Into ◽  
Junction Proteins

Gap junctions form intercellular conduits with a large pore size whose closed and open states regulate communication between adjacent cells. The structural basis of the mechanism by which gap junctions close, however, remains uncertain. Here, we show the cryo–electron microscopy structures of Caenorhabditis elegans innexin-6 (INX-6) gap junction proteins in an undocked hemichannel form. In the nanodisc-reconstituted structure of the wild-type INX-6 hemichannel, flat double-layer densities obstruct the channel pore. Comparison of the hemichannel structures of a wild-type INX-6 in detergent and nanodisc-reconstituted amino-terminal deletion mutant reveals that lipid-mediated amino-terminal rearrangement and pore obstruction occur upon nanodisc reconstitution. Together with molecular dynamics simulations and electrophysiology functional assays, our results provide insight into the closure of the INX-6 hemichannel in a lipid bilayer before docking of two hemichannels.

scholarly journals Experimental evidence for the essential role of the C-terminal residue in the strict aminopeptidase activity of the thiol aminopeptidase PepC, a bacterial bleomycin hydrolase

1997 ◽  
Vol 328 (2) ◽  
pp. 343-347 ◽  
Author(s):  
Luis MATA ◽  
Marta ERRA-PUJADA ◽  
Jean-Claude GRIPON ◽  
Michel-Yves MISTOU
Keyword(s):  
Essential Role ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
Aminopeptidase Activity ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Terminal Residue ◽  
Bleomycin Hydrolase ◽  
Eukaryotic Organisms

PepCs isolated from lactic acid bacteria and bleomycin hydrolases of eukaryotic organisms are strict aminopeptidases which belong to the papain family of thiol peptidases. The structural basis of the enzymic specificity of the lactococcal PepC has been investigated by site-directed mutagenesis. The deletion of the C-terminal residue (Ala-435) abolished the aminopeptidase activity, whereas this deletion led to a new peptidase specificity. The enzymic properties of wild-type and mutant PepCs demonstrate that the terminal α-carboxy group plays a key role in the strict aminopeptidase activity.

Pathway and stability of protein folding

1991 ◽  
Vol 332 (1263) ◽  
pp. 171-176 ◽  
Keyword(s):  
Protein Folding ◽  
Protein Engineering ◽  
Hydrogen Exchange ◽  
Hydrophobic Core ◽  
Wild Type ◽  
Kinetic Measurements ◽  
Mutant Proteins ◽  
Folding Process ◽  
Β Sheet

We describe an experimental approach to the problem of protein folding and stability which measures interaction energies and maps structures of intermediates and transition states during the folding pathway. The strategy is based on two steps. First, protein engineering is used to remove interactions that stabilize defined positions in barnase, the RNAse from Bacillus amyloliquefaciens . The consequent changes in stability are measured from the changes in free energy of unfolding of the protien. Second, each mutation is used as a probe of the structure around the wild-type side chain during the folding process. Kinetic measurements are made on the folding and unfolding of wild-type and mutant proteins. The kinetic and thermodynamic data are combined and analysed to show the role of individual side chains in the stabilization of the folded, transition and intermediate states of the protein. The protein engineering experiments are corroborated by nuclear magnetic resonance studies of hydrogen exchange during the folding process. Folding is a multiphasic process in which α-helices and β-sheet are formed relatively early. Formation of the hydrophobic core by docking helix and sheet is (partly) rate determining. The final steps involve the forming of loops and the capping of the N-termini of helices.

scholarly journals Structural basis for differential recognition of phosphohistidine-containing peptides by 1-pHis and 3-pHis monoclonal antibodies

2021 ◽  
Vol 118 (6) ◽  
pp. e2010644118
Author(s):  
Rajasree Kalagiri ◽  
Robyn L. Stanfield ◽  
Jill Meisenhelder ◽  
James J. La Clair ◽  
Stephen R. Fuhs ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Structural Information ◽  
Phosphate Group ◽  
Structural Basis ◽  
Binding Modes ◽  
Peptide Backbone ◽  
Wide Range ◽  
Complementarity Determining Regions ◽  
Histidine Phosphorylation

In 2015, monoclonal antibodies (mAbs) that selectively recognize the 1-pHis or 3-pHis isoforms of phosphohistidine were developed by immunizing rabbits with degenerate Ala/Gly peptides containing the nonhydrolyzable phosphohistidine (pHis) analog- phosphotriazolylalanine (pTza). Here, we report structures of five rabbit mAbs bound to cognate pTza peptides: SC1-1 and SC50-3 that recognize 1-pHis, and their 3-pHis–specific counterparts, SC39-4, SC44-8, and SC56-2. These cocrystal structures provide insights into the binding modes of the pTza phosphate group that are distinct for the 1- and 3-pHis mAbs with the selectivity arising from specific contacts with the phosphate group and triazolyl ring. The mode of phosphate recognition in the 3-pHis mAbs recapitulates the Walker A motif, as present in kinases. The complementarity-determining regions (CDRs) of four of the Fabs interact with the peptide backbone rather than peptide side chains, thus conferring sequence independence, whereas SC44-8 shows a proclivity for binding a GpHAGA motif mediated by a sterically complementary CDRL3 loop. Specific hydrogen bonding with the triazolyl ring precludes recognition of pTyr and other phosphoamino acids by these mAbs. Kinetic binding experiments reveal that the affinity of pHis mAbs for pHis and pTza peptides is submicromolar. Bound pHis mAbs also shield the pHis peptides from rapid dephosphorylation. The epitope–paratope interactions illustrate how these anti-pHis antibodies are useful for a wide range of research techniques and this structural information can be utilized to improve the specificity and affinity of these antibodies toward a variety of pHis substrates to understand the role of histidine phosphorylation in healthy and diseased states.

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