scholarly journals Cryptic adaptor protein interactions regulate DNA replication initiation

2018 ◽  
Author(s):  
Lindsay A. Matthews ◽  
Lyle A. Simmons
Keyword(s):  
Dna Replication ◽  
Complex Formation ◽  
Signaling Pathways ◽  
Protein Interactions ◽  
Protein Complex ◽  
Adaptor Protein ◽  
Single Amino Acid ◽  
Full Description ◽  
Human Pathogen ◽  
Helicase Loading

AbstractDNA replication is a fundamental biological process that is tightly regulated in all living cells. In bacteria, the master regulator DnaA controls when and where replication begins by building a step-wise complex that loads the replicative helicase onto chromosomal DNA. In many bacteria, DnaA requires the adaptor proteins DnaD and DnaB to aid DnaA during helicase loading. How DnaA, its adaptors, and the helicase form a complex at the origin is largely unknown. In this study, we addressed this long-standing question by disassembling the initiation proteins into their individual domains and testing all possible pair-wise combinations in a bacterial two-hybrid assay. Here we report a full description of the cryptic interaction sites used by the helicase loading machinery from Bacillus subtilis. In addition, we investigated how complex formation of the helicase loading machinery is regulated by the checkpoint protein SirA, which is a potent replication inhibitor in sporulating cells. We found that SirA and the DnaD adaptor bind overlapping sites on DnaA, and therefore SirA acts as a competitive inhibitor to block initiation. The interaction between DnaA and DnaD was also mapped to the same DnaA surface in the human pathogen Staphylococcus aureus, demonstrating the broad conservation of this interface. Therefore, our approach has unveiled key protein interactions essential for initiation and is widely applicable for mapping interactions in other signaling pathways that are governed by cryptic binding surfaces.Author SummaryIn order to proliferate, bacteria must first build a step-wise protein complex on their chromosomes that determines when and where DNA replication begins. This protein complex is assembled through dynamic interactions that have been difficult to study and remain largely uncharacterized. Here we show that by deconstructing the proteins into their constituent domains, the interactions used to build the initiation complex can be readily detected and mapped to single amino acid resolution. Using this approach, we demonstrate that DNA replication is controlled through conformational changes that dictate the availability of interaction surfaces. In addition, negative regulators can also block DNA replication by influencing complex formation so that cells survive inhospitable conditions. Initiation proteins from the model organism B. subtilis and the human pathogen S. aureus were both used to underscore the general applicability of the results to different bacterial systems. Furthermore, our general strategy for mapping dynamic protein interactions is suitable for many different signaling pathways that are controlled through cryptic interaction surfaces.

scholarly journals The adaptor protein Ste50 directly modulates yeast MAPK signaling specificity through differential connections of its RA domain

2019 ◽  
Vol 30 (6) ◽  
pp. 794-807
Author(s):  
Nusrat Sharmeen ◽  
Traian Sulea ◽  
Malcolm Whiteway ◽  
Cunle Wu
Keyword(s):  
Signaling Pathways ◽  
Protein Interactions ◽  
Mapk Pathway ◽  
Adaptor Protein ◽  
Filamentous Growth ◽  
Mapk Signaling ◽  
Yeast Cells ◽  
Signaling Specificity ◽  
Signaling Complex ◽  
And Function

Discriminating among diverse environmental stimuli is critical for organisms to ensure their proper development, homeostasis, and survival. Saccharomyces cerevisiae regulates mating, osmoregulation, and filamentous growth using three different MAPK signaling pathways that share common components and therefore must ensure specificity. The adaptor protein Ste50 activates Ste11p, the MAP3K of all three modules. Its Ras association (RA) domain acts in both hyperosmolar and filamentous growth pathways, but its connection to the mating pathway is unknown. Genetically probing the domain, we found mutants that specifically disrupted mating or HOG-signaling pathways or both. Structurally these residues clustered on the RA domain, forming distinct surfaces with a propensity for protein–protein interactions. GFP fusions of wild-type (WT) and mutant Ste50p show that WT is localized to the shmoo structure and accumulates at the growing shmoo tip. The specifically pheromone response–defective mutants are severely impaired in shmoo formation and fail to localize ste50p, suggesting a failure of association and function of Ste50 mutants in the pheromone-signaling complex. Our results suggest that yeast cells can use differential protein interactions with the Ste50p RA domain to provide specificity of signaling during MAPK pathway activation.

scholarly journals APPL1 is a multifunctional endosomal signaling adaptor protein

2017 ◽  
Vol 45 (3) ◽  
pp. 771-779 ◽  
Author(s):  
Nicole L. Diggins ◽  
Donna J. Webb
Keyword(s):  
Signaling Pathways ◽  
Protein Interactions ◽  
Leucine Zipper ◽  
Adaptor Protein ◽  
Adaptor Proteins ◽  
Ph Domain ◽  
Protein Protein Interactions ◽  
Multiple Protein ◽  
Signaling Receptors ◽  
Ptb Domain

Endosomal adaptor proteins are important regulators of signaling pathways underlying many biological processes. These adaptors can integrate signals from multiple pathways via localization to specific endosomal compartments, as well as through multiple protein–protein interactions. One such adaptor protein that has been implicated in regulating signaling pathways is the adaptor protein containing a pleckstrin homology (PH) domain, phosphotyrosine-binding (PTB) domain, and leucine zipper motif 1 (APPL1). APPL1 localizes to a subset of Rab5-positive endosomes through its Bin–Amphiphysin–Rvs and PH domains, and it coordinates signaling pathways through its interaction with many signaling receptors and proteins through its PTB domain. This review discusses our current understanding of the role of APPL1 in signaling and trafficking, as well as highlights recent work into the function of APPL1 in cell migration and adhesion.

scholarly journals Regulation of Complex Formation of POB1/Epsin/Adaptor Protein Complex 2 by Mitotic Phosphorylation

2000 ◽  
Vol 275 (24) ◽  
pp. 18399-18406 ◽  
Author(s):  
Kenji Kariya ◽  
Shinya Koyama ◽  
Shintaro Nakashima ◽  
Takafumi Oshiro ◽  
Kenji Morinaka ◽  
...  
Keyword(s):  
Complex Formation ◽  
Protein Complex ◽  
Adaptor Protein ◽  
Mitotic Phosphorylation

scholarly journals Cryo-EM structure of an activated GPCR-G protein complex in lipid nanodiscs

2020 ◽  
Author(s):  
Gerhard Wagner ◽  
Meng Zhang ◽  
Miao Gui ◽  
Zi-Fu Wang ◽  
Christoph Gorgulla ◽  
...  
Keyword(s):  
Complex Formation ◽  
G Protein ◽  
Lipid Bilayer ◽  
Lipid Bilayers ◽  
Protein Interactions ◽  
Protein Complex ◽  
Lipid Membrane ◽  
Protein Complexes ◽  
Structural Basis ◽  
Downstream Signaling

Abstract G protein coupled receptors (GPCRs) are the largest superfamily of transmembrane proteins and the targets of over 30% of currently marketed pharmaceuticals. Although several structures have been solved for GPCR-G protein complexes, structural studies of the complex in a physiological lipid membrane environment are lacking. Here, we report cryo-EM structures of lipid bilayer-bound complexes of neurotensin, neurotensin receptor 1, and Gai1b1g1 protein in two conformational states, resolved to 4.1 and 4.2 Å resolution. The structures were determined in lipid bilayer without any stabilizing antibodies/nanobodies, and thus provide a native-like platform for understanding the structural basis of GPCR-G protein complex formation. Our structures reveal an extended network of protein-protein interactions at the GPCR-G protein interface compared to in detergent micelles, defining roles for the lipid membrane in modulating the structure and dynamics of complex formation, and providing a molecular explanation for the stronger interaction between GPCR and G protein in lipid bilayers. We propose a detailed allosteric mechanism for GDP release, providing new insights into the activation of G proteins for downstream signaling.

scholarly journals Modeling and simulating networks of interdependent protein interactions

2017 ◽  
Author(s):  
Bianca K. Stöecker ◽  
Johannes Köester ◽  
Eli Zamir ◽  
Sven Rahmann
Keyword(s):  
Complex Formation ◽  
Protein Interaction ◽  
Protein Interactions ◽  
Protein Complex ◽  
Simulation Software ◽  
Protein Interaction Networks ◽  
Interaction Networks ◽  
Mathematical Framework ◽  
Link Type ◽  
Protein Complex Formation

AbstractProtein interactions are fundamental building blocks of biochemical reaction systems underlying cellular functions. The complexity and functionality of these systems emerge not only from the protein interactions themselves but also from the dependencies between these interactions, e.g., allosteric effects, mutual exclusion or steric hindrance. Therefore, formal models for integrating and using information about such dependencies are of high interest. We present an approach for endowing protein networks with interaction dependencies using propositional logic, thereby obtaining constrained protein interaction networks (“constrained networks”). The construction of these networks is based on public interaction databases and known as well as text-mined interaction dependencies. We present an efficient data structure and algorithm to simulate protein complex formation in constrained networks. The efficiency of the model allows a fast simulation and enables the analysis of many proteins in large networks. Therefore, we are able to simulate perturbation effects (knockout and overexpression of single or multiple proteins, changes of protein concentrations). We illustrate how our model can be used to analyze a partially constrained human adhesome network. Comparing complex formation under known dependencies against without dependencies, we find that interaction dependencies limit the resulting complex sizes. Further we demonstrate that our model enables us to investigate how the interplay of network topology and interaction dependencies influences the propagation of perturbation effects. Our simulation software CPINSim (for Constrained Protein Interaction Network Simulator) is available under the MIT license at http://github.com/BiancaStoecker/cpinsimandviaBioconda (https://bioconda.github.io).Author summaryProteins are the main molecular tools of cells. They do not act individually, but rather collectively in order to peform complex cellular actions. Recent years have led to a relatively good understanding about which proteins may interact, both in general and in specific conditions, leading to the definition of protein interaction networks. However, the reality is more complex, and protein interactions are not independent of each other. Instead, several potential interaction partners of a specific protein may compete for the same binding domain, making all of these interactions mutually exclusive. Additionally, a binding of a protein to another one can enable or prevent their interactions with other proteins, even if those interactions are mediated by different domains. Hence, understanding how the dependencies (or constraints) of protein interactions affect the behaviour of the system is an important and timely goal, as data is now becoming available. Here we present a mathematical framework to formalize such interaction constraints and incorporate them into the simulation of protein complex formation. With our framework, we are able to better understand how perturbations of single proteins (knockout or overexpression) impact other proteins in the network.

scholarly journals ERK and β-Arrestin Interaction

2014 ◽  
Vol 20 (3) ◽  
pp. 341-349 ◽  
Author(s):  
Haifeng Eishingdrelo ◽  
Wei Sun ◽  
Hua Li ◽  
Li Wang ◽  
Alex Eishingdrelo ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Protein Interactions ◽  
Tyrosine Kinases ◽  
Intracellular Signaling ◽  
Kinase Inhibitors ◽  
Adaptor Protein ◽  
Direct Interaction ◽  
Signal Pathways ◽  
Surface Receptors ◽  
Extracellular Stimuli

β-Arrestin, a signal adaptor protein, mediates intracellular signal transductions through protein-protein interactions by bringing two or more proteins in proximity. Extracellular signal-regulated kinase (ERK), a protein kinase in the family of mitogen-activated protein kinases (MAPKs), is involved in various receptor signal pathways. Interaction of ERK with β-arrestin or formation of ERK/β-arrestin signal complex occurs in response to activation of a variety of cell surface receptors. The ERK/β-arrestin signal complex may be a common transducer to converge a variety of extracellular stimuli to similar downstream intracellular signaling pathways. By using a cell-based protein-protein interaction LinkLight assay technology, we demonstrate a direct interaction between ERK and β-arrestin in response to extracellular stimuli, which can be sensitively and quantitatively monitored. Activations of G protein–coupled receptors (GPCRs), receptor tyrosine kinases (RTKs), and cytokine receptors promote formation of the ERK/β-arrestin signal complex. Our data indicate that the ERK/β-arrestin signal complex is a common transducer that participates in a variety of receptor signaling pathways. Furthermore, we demonstrate that receptor antagonists or kinase inhibitors can block the agonist-induced ERK and β-arrestin interaction. Thus, the ERK/β-arrestin interaction assay is useful for screening of new receptor modulators.

scholarly journals Cryo-EM structure of an activated GPCR-G protein complex in lipid nanodiscs

2020 ◽  
Author(s):  
Meng Zhang ◽  
Miao Gui ◽  
Zi-Fu Wang ◽  
Christoph Gorgulla ◽  
James J Yu ◽  
...  
Keyword(s):  
Complex Formation ◽  
G Protein ◽  
G Proteins ◽  
Lipid Bilayers ◽  
Protein Interactions ◽  
Protein Complex ◽  
Lipid Membrane ◽  
Complex Structure ◽  
Underlying Mechanism ◽  
Structural Basis

AbstractG protein coupled receptors (GPCRs) are the largest superfamily of transmembrane proteins and the targets of over 30% of currently marketed pharmaceuticals1,2. Although several structures have been solved for GPCR-G protein complexes3–17, structural studies of the complex in a physiological lipid membrane environment are lacking. Additionally, most previous studies required additional antibodies/nanobodies and/or engineered G proteins for complex stabilization. In the absence of a native complex structure, the underlying mechanism of G protein activation leading to GDP/GTP exchange remains unclear. Here, we report cryo-EM structures of lipid bilayer-bound complexes of neurotensin, neurotensin receptor 1, and Gαi1β1γ1 protein in two conformational states, resolved to 4.1 and 4.2 Å resolution. The structures were determined without any stabilizing antibodies/nanobodies, and thus provide a native-like platform for understanding the structural basis of GPCR-G protein complex formation. Our structures reveal an extended network of protein-protein interactions at the GPCR-G protein interface compared to in detergent micelles, defining roles for the lipid membrane in modulating the structure and dynamics of complex formation, and providing a molecular explanation for the stronger interaction between GPCR and G protein in lipid bilayers. We propose a detailed allosteric mechanism for GDP release, providing new insights into the activation of G proteins for downstream signaling under near native conditions.

Phospholipid-Protein Interactions in the Formation of Prothrombin Activator

1965 ◽  
Vol 14 (03/04) ◽  
pp. 431-444 ◽  
Author(s):  
E. R Cole ◽  
J. L Koppel ◽  
J. H Olwin
Keyword(s):  
Complex Formation ◽  
Protein Interactions ◽  
Calcium Ions ◽  
Fixed Number ◽  
Factor V ◽  
Clotting Factors ◽  
Number Of Binding Sites ◽  
C Complex ◽  
Autoprothrombin C

SummarySince Ac-globulin (factor V) is involved in the formation of prothrombin activator, its ability to complex with phospholipids was studied. Purified bovine Ac-globulin was complexed to asolectin, there being presumably a fixed number of binding sites on the phospholipid micelle for Ac-globulin. In contrast to the requirement for calcium ions in the formation of complexes between asolectin and autoprothrombin C, calcium ions were not required for complex formation between asolectin and Ac-globulin to occur ; in fact, the presence of calcium prevented complex formation occurring, the degree of inhibition being dependent on the calcium concentration. By treating isolated, pre-formed aso- lectin-Ac-globulin complexes with calcium chloride solutions, Ac-globulin could be recovered in a much higher state of purity and essentially free of asolectin.Complete activators were formed by first preparing the asolectin-calcium- autoprothrombin C complex and then reacting the complex with Ac-globulin. A small amount of this product was very effective as an activator of purified prothrombin without further addition of calcium or any other cofactor. If the autoprothrombin C preparation used to prepare the complex was free of traces of prothrombin, the complete activator was stable for several hours at room temperature. Stable preparations of the complete activator were centrifuged, resulting in the sedimentation of most of the activity. Experimental evidence also indicated that activator activity was highest when autoprothrombin C and Ac-globulin were complexed to the same phospholipid micelle, rather than when the two clotting factors were complexed to separate micelles. These data suggested that the in vivo prothrombin activator may be a sedimentable complex composed of a thromboplastic enzyme, calcium, Ac-globulin and phospholipid.

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