scholarly journals Structural Mechanism for Regulation of Bcl-2 protein Noxa by phosphorylation

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Christine B. Karim ◽  
L. Michel Espinoza-Fonseca ◽  
Zachary M. James ◽  
Eric A. Hanse ◽  
Jeffrey S. Gaynes ◽  
...  
Keyword(s):  
Md Simulations ◽  
Salt Bridges ◽  
Binding Assays ◽  
Peptide Backbone ◽  
Structural Mechanism ◽  
Binding Partner ◽  
Flexible Loop ◽  
Bh3 Domain ◽  
N Terminus ◽  
Β Sheets

Abstract We showed previously that phosphorylation of Noxa, a 54-residue Bcl-2 protein, at serine 13 (Ser13) inhibited its ability to promote apoptosis through interactions with canonical binding partner, Mcl-1. Using EPR spectroscopy, molecular dynamics (MD) simulations and binding assays, we offer evidence that a structural alteration caused by phosphorylation partially masks Noxa’s BH3 domain, inhibiting the Noxa-Mcl-1 interaction. EPR of unphosphorylated Noxa, with spin-labeled amino acid TOAC incorporated within the BH3 domain, revealed equilibrium between ordered and dynamically disordered states. Mcl-1 further restricted the ordered component for non-phosphorylated Noxa, but left the pSer13 Noxa profile unchanged. Microsecond MD simulations indicated that the BH3 domain of unphosphorylated Noxa is housed within a flexible loop connecting two antiparallel β-sheets, flanked by disordered N- and C-termini and Ser13 phosphorylation creates a network of salt-bridges that facilitate the interaction between the N-terminus and the BH3 domain. EPR showed that a spin label inserted near the N-terminus was weakly immobilized in unphosphorylated Noxa, consistent with a solvent-exposed helix/loop, but strongly constrained in pSer13 Noxa, indicating a more ordered peptide backbone, as predicted by MD simulations. Together these studies reveal a novel mechanism by which phosphorylation of a distal serine inhibits a pro-apoptotic BH3 domain and promotes cell survival.

scholarly journals Identification of TAZ as a Binding Partner of the Polyomavirus T Antigens

2004 ◽  
Vol 78 (22) ◽  
pp. 12657-12664 ◽  
Author(s):  
Yu Tian ◽  
Dawei Li ◽  
Jean Dahl ◽  
John You ◽  
Thomas Benjamin
Keyword(s):  
Dna Replication ◽  
Host Range ◽  
T Antigen ◽  
Binding Motif ◽  
Wild Type ◽  
T Antigens ◽  
Ww Domain ◽  
Binding Partner ◽  
N Terminus ◽  
The Common

ABSTRACT A polyomavirus mutant isolated by the tumor host range selection procedure (19) has a three-amino-acid deletion (Δ2-4) in the common N terminus of the T antigens. To search for a cellular protein bound by wild-type but not the mutant T antigen(s), a yeast two-hybrid screen of a mouse embryo cDNA library was carried out with a bait of wild-type small T antigen (sT) fused N terminally to the DNA-binding domain of Gal4. TAZ, a transcriptional coactivator with a WW domain and PDZ-binding motif (17), was identified as a binding partner. TAZ bound in vivo to all three T antigens with different apparent affinities estimated as 1:7:100 (large T antigen [lT]:middle T antigen [mT]:sT). The Δ2-4 mutant T antigens showed no detectable binding. The sT and mT of the host range transformation-defective (hr-t) mutant NG59 with an alteration in the common sT/mT region (179 D→NI) and a normal N terminus also failed to bind TAZ, while the unaltered lT bound but with reduced affinity compared to that seen in a wild-type virus infection. The WW domain but not the PDZ-binding motif of TAZ was essential for T antigen binding. The Δ2-4 mutant was defective in viral DNA replication. Forced overexpression of TAZ blocked wild-type DNA replication in a manner dependent on the binding site for the polyomavirus enhancer-binding protein 2α. Wild-type polyomavirus T antigens effectively block transactivation by TAZ. The functional significance of TAZ interactions with polyomavirus T antigens is discussed.

scholarly journals Surface Signaling in Ferric Citrate Transport Gene Induction: Interaction of the FecA, FecR, and FecI Regulatory Proteins

2000 ◽  
Vol 182 (3) ◽  
pp. 637-646 ◽  
Author(s):  
Sabine Enz ◽  
Susanne Mahren ◽  
Uwe H. Stroeher ◽  
Volkmar Braun
Keyword(s):  
Nitrilotriacetic Acid ◽  
Ferric Citrate ◽  
Binding Assays ◽  
Surface Binding ◽  
Citrate Transport ◽  
C Terminus ◽  
N Terminus ◽  
Surface Signaling

ABSTRACT In Escherichia coli, transcription of the ferric citrate transport genes fecABCDE is controlled by a novel signal transduction mechanism that starts at the cell surface. Binding of ferric citrate to the outer membrane protein FecA initiates a signal that is transmitted by FecR across the cytoplasmic membrane into the cytoplasm where FecI, the sigma factor, is activated. Interaction between the signaling proteins was demonstrated by utilizing two methods. In in vitro binding assays, FecR that was His tagged at the N terminus [(His)10-FecR] and bound to a Ni-nitrilotriacetic acid agarose column was able to retain FecA, and FecR that was His tagged at the C terminus [FecR-(His)6] retained FecI on the column. An N-terminally truncated, induction-negative but transport-active FecA protein did not bind to (His)10-FecR. The in vivo assay involved the determination of the FecA, FecR, and FecI interacting domains with the bacterial two-hybrid Lex-based system. FecA1–79 interacts with FecR101–317 and FecR1–85 interacts with FecI1–173. These data clearly support a model that proposes interaction of the periplasmic N terminus of FecA with the periplasmic C-terminal portion of FecR and interaction of the cytoplasmic N terminus of FecR with FecI, which results in FecI activation.

scholarly journals Critical Sequence Hot-spots for Binding of nCOV-2019 to ACE2 as Evaluated by Molecular Simulations

2020 ◽  
Author(s):  
Mahdi Ghorbani ◽  
Bernard R. Brooks ◽  
Jeffery B. Klauda
Keyword(s):  
Md Simulations ◽  
Host Cells ◽  
Cell Surface Receptor ◽  
Receptor Protein ◽  
Structural Mechanism ◽  
The World ◽  
Surface Receptor ◽  
Escape Mutants ◽  
The Stability ◽  
Novel Coronavirus

AbstractThe novel coronavirus (nCOV-2019) outbreak has put the world on edge, causing millions of cases and hundreds of thousands of deaths all around the world, as of June 2020, let alone the societal and economic impacts of the crisis. The spike protein of nCOV-2019 resides on the virion’s surface mediating coronavirus entry into host cells by binding its receptor binding domain (RBD) to the host cell surface receptor protein, angiotensin converter enzyme (ACE2). Our goal is to provide a detailed structural mechanism of how nCOV-2019 recognizes and establishes contacts with ACE2 and its difference with an earlier coronavirus SARS-COV in 2002 via extensive molecular dynamics (MD) simulations. Numerous mutations have been identified in the RBD of nCOV-2019 strains isolated from humans in different parts of the world. In this study, we investigated the effect of these mutations as well as other Ala-scanning mutations on the stability of RBD/ACE2 complex. It is found that most of the naturally-occurring mutations to the RBD either strengthen or have the same binding affinity to ACE2 as the wild-type nCOV-2019. This may have implications for high human-to-human transmission of coronavirus in regions where these mutations have been found as well as any vaccine design endeavors since these mutations could act as antibody escape mutants. Furthermore, in-silico Ala-scanning and long-timescale MD simulations, highlight the crucial role of the residues at the interface of RBD and ACE2 that may be used as potential pharmacophores for any drug development endeavors. From an evolutional perspective, this study also identifies how the virus has evolved from its predecessor SARS-COV and how it could further evolve to become more infectious.

scholarly journals THE INTERACTION OF PROTONATED OCTOPAMINE AND NOREPINEPHRINE WITH Β1-ADRENERGIC RECEPTOR: MOLECULAR DOCKING AND DYNAMICAL SIMULATION

2020 ◽  
pp. 13-25
Author(s):  
Žiko Milanović ◽  
Dušan Dimić ◽  
Jasmina Dimitrić Marković ◽  
Marijana Stanojević-Pirković ◽  
Edina Avdović ◽  
...  
Keyword(s):  
Molecular Docking ◽  
Adrenergic Receptor ◽  
Md Simulations ◽  
Protonated Form ◽  
Free Energy Calculations ◽  
Amino Acid Residues ◽  
Salt Bridges ◽  
Docking Simulations ◽  
Dynamical Simulation ◽  
G Protein Coupled

In the current study, the interaction mechanisms between protonated neurotransmitters: octopamine (4-(2-amino-1-hydroxyethyl)phenol) and norepinephrine (4-[(1R)-2-amino-1-hydroxyethyl]benzene-1,2-diol) with the β-1 adrenergic receptor (β1AR) were examined by molecular docking, molecular dynamics (MD) simulations and MM/PBSA free energy calculations. The investigated receptor belongs to the G-protein coupled receptor group. The investigation was carried out at physiological pH=7.4. It was estimated that both compounds exist in the protonated form in the water at physiological pH. It was found that both protonated neurotransmitters established similar interactions with amino acid residues of the receptor, such as salt bridges, conventional hydrogen bonds, π-σ, and T-shaped π-π interactions, as shown by molecular docking simulations. As the initial structures for MD simulation with a total time of 10ns the most stable docking structures were used. The presented results are expected to provide some useful information for the design of specific β1AR agonists.

scholarly journals Insights into channel modulation mechanism of RYR1 mutants using Ca2+ imaging and molecular dynamics

2019 ◽  
Vol 152 (1) ◽  
Author(s):  
Toshiko Yamazawa ◽  
Haruo Ogawa ◽  
Takashi Murayama ◽  
Maki Yamaguchi ◽  
Hideto Oyamada ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Md Simulations ◽  
Hek293 Cells ◽  
Salt Bridges ◽  
Open Probability ◽  
Release Channel ◽  
Functional Studies ◽  
Function Relationship ◽  
Interdomain Interactions ◽  
Relationship Of

Type 1 ryanodine receptor (RYR1) is a Ca2+ release channel in the sarcoplasmic reticulum in skeletal muscle and plays an important role in excitation–contraction coupling. Mutations in the RYR1 gene cause severe muscle diseases such as malignant hyperthermia (MH), which is a disorder of CICR via RYR1. Thus far, >300 mutations in RYR1 have been reported in patients with MH. However, owing to a lack of comprehensive analysis of the structure–function relationship of mutant RYR1, the mechanism remains largely unknown. Here, we combined functional studies and molecular dynamics (MD) simulations of RYR1 bearing disease-associated mutations at the N-terminal region. When expressed in HEK293 cells, the mutant RYR1 caused abnormalities in Ca2+ homeostasis. MD simulations of WT and mutant RYR1s were performed using crystal structure of the N-terminal domain (NTD) monomer, consisting of A, B, and C domains. We found that the mutations located around the interdomain region differentially affected hydrogen bonds/salt bridges. Particularly, mutations at R402, which increase the open probability of the channel, cause clockwise rotation of BC domains with respect to the A domain by alteration of the interdomain interactions. Similar results were also obtained with artificial mutations that mimic alteration of the interactions. Our results reveal the importance of interdomain interactions within the NTD in the regulation of the RYR1 channel and provide insights into the mechanism of MH caused by the mutations at the NTD.

scholarly journals Conversion between parallel and antiparallel β-sheets in wild-type and Iowa mutant fibrils

2017 ◽  
Author(s):  
Wenhui Xi ◽  
Ulrich H.E. Hansmann
Keyword(s):  
Replica Exchange ◽  
Mutant Form ◽  
Salt Bridges ◽  
Wild Type ◽  
Β Sheets

AbstractUsing a variant of Hamilton-Replica-Exchange we study for wild type and Iowa mutant Aβ40 the conversion between fibrils with antiparallel β-sheets, and such with parallel β-sheets. We show that wild type and mutant form distinct salt bridges that in turn stabilize different fibril organizations. The conversion between the two fibril forms leads to the release of small aggregates that in the Iowa mutant may shift the equilibrium from fibrils to more toxic oligomers.

scholarly journals Dynamical Behavior of the Human Ferroportin Homologue from Bdellovibrio bacteriovorus: Insight into the Ligand Recognition Mechanism

2020 ◽  
Vol 21 (18) ◽  
pp. 6785
Author(s):  
Valentina Tortosa ◽  
Maria Carmela Bonaccorsi di Patti ◽  
Federico Iacovelli ◽  
Andrea Pasquadibisceglie ◽  
Mattia Falconi ◽  
...  
Keyword(s):  
Dynamical Behavior ◽  
Md Simulations ◽  
Major Facilitator Superfamily ◽  
Ligand Recognition ◽  
Salt Bridges ◽  
Recognition Mechanism ◽  
Bdellovibrio Bacteriovorus ◽  
Physiological Relevance ◽  
Bacteria And Fungi ◽  
Major Facilitator

Members of the major facilitator superfamily of transporters (MFS) play an essential role in many physiological processes such as development, neurotransmission, and signaling. Aberrant functions of MFS proteins are associated with several diseases, including cancer, schizophrenia, epilepsy, amyotrophic lateral sclerosis and Alzheimer’s disease. MFS transporters are also involved in multidrug resistance in bacteria and fungi. The structures of most MFS members, especially those of members with significant physiological relevance, are yet to be solved. The lack of structural and functional information impedes our detailed understanding, and thus the pharmacological targeting, of these transporters. To improve our knowledge on the mechanistic principles governing the function of MSF members, molecular dynamics (MD) simulations were performed on the inward-facing and outward-facing crystal structures of the human ferroportin homologue from the Gram-negative bacterium Bdellovibrio bacteriovorus (BdFpn). Several simulations with an excess of iron ions were also performed to explore the relationship between the protein’s dynamics and the ligand recognition mechanism. The results reinforce the existence of the alternating-access mechanism already described for other MFS members. In addition, the reorganization of salt bridges, some of which are conserved in several MFS members, appears to be a key molecular event facilitating the conformational change of the transporter.

scholarly journals Binding of protofibrillar Aβ trimers to lipid bilayer surface enhances Aβ structural stability and causes membrane thinning

2017 ◽  
Vol 19 (40) ◽  
pp. 27556-27569 ◽  
Author(s):  
Xuewei Dong ◽  
Yunxiang Sun ◽  
Guanghong Wei ◽  
Ruth Nussinov ◽  
Buyong Ma
Keyword(s):  
Lipid Bilayer ◽  
Structural Stability ◽  
Salt Bridges ◽  
Membrane Interactions ◽  
Aβ Oligomers ◽  
Membrane Perturbation ◽  
Β Sheets ◽  
Membrane Thinning

Aβ–membrane interactions enhance structural stability of protofibrillar Aβ oligomers by stabilizing β-sheets and D23–K28 salt-bridges, and cause membrane perturbation by decreasing membrane's local thickness.

scholarly journals Mutagenesis of the BH3 Domain of BAX Identifies Residues Critical for Dimerization and Killing

1998 ◽  
Vol 18 (10) ◽  
pp. 6083-6089 ◽  
Author(s):  
Kun Wang ◽  
Atan Gross ◽  
Gabriel Waksman ◽  
Stanley J. Korsmeyer
Keyword(s):  
Site Directed Mutagenesis ◽  
Initial Assessment ◽  
Agonist Activity ◽  
Binding Assays ◽  
Yeast Two Hybrid ◽  
Bh3 Domain ◽  
Protein Partners ◽  
Killing Function ◽  
Α Helix ◽  
Two Hybrid

ABSTRACT The BCL-2 family of proteins is comprised of proapoptotic as well as antiapoptotic members (S. N. Farrow and R. Brown, Curr. Opin. Genet. Dev. 6:45–49, 1996). A prominent death agonist, BAX, forms homodimers and heterodimerizes with multiple antiapoptotic members. Death agonists have an amphipathic α helix, called BH3; however, the initial assessment of BH3 in BAX has yielded conflicting results. Our BAX deletion constructs and minimal domain constructs indicated that the BH3 domain was required for BAX homodimerization and heterodimerization with BCL-2, BCL-XL, and MCL-1. An extensive site-directed mutagenesis of BH3 revealed that substitutions along the hydrophobic face of BH3, especially charged substitutions, had the greatest affects on dimerization patterns and death agonist activity. Particularly instructive was the BAX mutant mIII-1 (L63A, G67A, L70A, and M74A), which replaced the hydrophobic face of BH3 with alanines, preserving its amphipathic nature. BAXmIII-1 failed to form heterodimers or homodimers by yeast two-hybrid or immunoprecipitation analysis yet retained proapoptotic activity. This suggests that BAX’s killing function reflects mechanisms beyond its binding to BCL-2 or BCL-XL to inhibit them or simply displace other protein partners. Notably, BAXmIII-1 was found predominantly in mitochondrial membranes, where it was homodimerized as assessed by homobifunctional cross-linkers. This characteristic of BAXmIII-1 correlates with its capacity to induce mitochondrial dysfunction, caspase activation, and apoptosis. These data are consistent with a model in which BAX death agonist activity may require an intramembranous conformation of this molecule that is not assessed accurately by classic binding assays.

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