Protein−Protein Interactions:  Interface Structure, Binding Thermodynamics, and Mutational Analysis

1997 ◽  
Vol 97 (5) ◽  
pp. 1233-1250 ◽  
Author(s):  
Wesley E. Stites
Keyword(s):  
Protein Interactions ◽  
Mutational Analysis ◽  
Interface Structure ◽  
Protein Protein Interactions ◽  
Binding Thermodynamics

Leucine periodicity of U2 small nuclear ribonucleoprotein particle (snRNP) A' protein is implicated in snRNP assembly via protein-protein interactions

1991 ◽  
Vol 11 (3) ◽  
pp. 1578-1589
Author(s):  
L D Fresco ◽  
D S Harper ◽  
J D Keene
Keyword(s):  
Protein Interactions ◽  
Leucine Zipper ◽  
Tandem Repeats ◽  
Mutational Analysis ◽  
Particle Density ◽  
Protein Protein Interactions ◽  
Small Nuclear Ribonucleoprotein ◽  
Cell Extracts ◽  
Associated Proteins ◽  
Nuclear Ribonucleoprotein

Recombinant A' protein could be reconstituted into U2 small nuclear ribonucleoprotein particles (snRNPs) upon addition to HeLa cell extracts as determined by coimmunoprecipitation and particle density; however, direct binding to U2 RNA could not be demonstrated except in the presence of the U2 snRNP B" protein. Mutational analysis indicated that a central core region of A' was required for particle reconstitution. This region consists of five tandem repeats of approximately 24 amino acids each that exhibit a periodicity of leucine and asparagine residues that is distinct from the leucine zipper. Similar leucine-rich (Leu-Leu motif) repeats are characteristic of a diverse array of soluble and membrane-associated proteins from yeasts to humans but have not been reported previously to reside in nuclear proteins. Several of these proteins, including Toll, chaoptin, RNase/angiogenin inhibitors, lutropin-choriogonadotropin receptor, carboxypeptidase N, adenylyl cyclase, CD14, and human immunodeficiency virus type 1 Rev, may be involved in protein-protein interactions. Our findings suggest that in cell extracts the Leu-Leu motif of A' is required for reconstitution with U2 snRNPs and perhaps with other components involved in splicing through protein-protein interactions.

scholarly journals Leucine periodicity of U2 small nuclear ribonucleoprotein particle (snRNP) A' protein is implicated in snRNP assembly via protein-protein interactions.

1991 ◽  
Vol 11 (3) ◽  
pp. 1578-1589 ◽  
Author(s):  
L D Fresco ◽  
D S Harper ◽  
J D Keene
Keyword(s):  
Protein Interactions ◽  
Leucine Zipper ◽  
Tandem Repeats ◽  
Mutational Analysis ◽  
Particle Density ◽  
Protein Protein Interactions ◽  
Small Nuclear Ribonucleoprotein ◽  
Cell Extracts ◽  
Associated Proteins ◽  
Nuclear Ribonucleoprotein

Recombinant A' protein could be reconstituted into U2 small nuclear ribonucleoprotein particles (snRNPs) upon addition to HeLa cell extracts as determined by coimmunoprecipitation and particle density; however, direct binding to U2 RNA could not be demonstrated except in the presence of the U2 snRNP B" protein. Mutational analysis indicated that a central core region of A' was required for particle reconstitution. This region consists of five tandem repeats of approximately 24 amino acids each that exhibit a periodicity of leucine and asparagine residues that is distinct from the leucine zipper. Similar leucine-rich (Leu-Leu motif) repeats are characteristic of a diverse array of soluble and membrane-associated proteins from yeasts to humans but have not been reported previously to reside in nuclear proteins. Several of these proteins, including Toll, chaoptin, RNase/angiogenin inhibitors, lutropin-choriogonadotropin receptor, carboxypeptidase N, adenylyl cyclase, CD14, and human immunodeficiency virus type 1 Rev, may be involved in protein-protein interactions. Our findings suggest that in cell extracts the Leu-Leu motif of A' is required for reconstitution with U2 snRNPs and perhaps with other components involved in splicing through protein-protein interactions.

scholarly journals SKEMPI 2.0: An updated benchmark of changes in protein-protein binding energy, kinetics and thermodynamics upon mutation

2018 ◽  
Author(s):  
Justina Jankauskaitė ◽  
Brian Jiménez-García ◽  
Justas Dapkūnas ◽  
Juan Fernández-Recio ◽  
Iain H. Moal
Keyword(s):  
Binding Energy ◽  
Protein Interactions ◽  
Binding Free Energy ◽  
Molecular Complexes ◽  
Protein Protein Interactions ◽  
Binding Thermodynamics ◽  
Binding Data ◽  
Enthalpy And Entropy Changes ◽  
Enthalpy And Entropy ◽  
The Relationship

AbstractMotivationUnderstanding the relationship between the sequence, structure, binding energy, binding kinetics and binding thermodynamics of protein-protein interactions is crucial to understanding cellular signaling, the assembly and regulation of molecular complexes, the mechanisms through which mutations lead to disease, and protein engineering.ResultsWe present SKEMPI 2.0, a major update to our database of binding free energy changes upon mutation for structurally resolved protein-protein interactions. This version now contains manually curated binding data for 7085 mutations, an increase of 133%, including changes in kinetics for 1844 mutations, enthalpy and entropy changes for 443 mutations, and 440 mutations which abolish detectable binding.AvailabilityThe database is available at https://life.bsc.es/pid/skempi2/

scholarly journals The Glycoprotein Cytoplasmic Tail of Uukuniemi Virus (Bunyaviridae) Interacts with Ribonucleoproteins and Is Critical for Genome Packaging

2007 ◽  
Vol 81 (7) ◽  
pp. 3198-3205 ◽  
Author(s):  
Anna K. Överby ◽  
Ralf F. Pettersson ◽  
Etienne P. A. Neve
Keyword(s):  
Amino Acids ◽  
Protein Interactions ◽  
Mutational Analysis ◽  
Cytoplasmic Tail ◽  
Protein Protein Interactions ◽  
Genome Packaging ◽  
Glycoprotein Precursor ◽  
Glycoprotein G ◽  
Rescue System ◽  
Polymerase I

ABSTRACT We have analyzed the importance of specific amino acids in the cytoplasmic tail of the glycoprotein GN for packaging of ribonucleoproteins (RNPs) into virus-like particles (VLPs) of Uukuniemi virus (UUK virus), a member of the Bunyaviridae family. In order to study packaging, we added the GN/GC glycoprotein precursor (p110) to a polymerase I-driven minigenome rescue system to generate VLPs that are released into the supernatant. These particles can infect new cells, and reporter gene expression can be detected. To determine the role of UUK virus glycoproteins in RNP packaging, we performed an alanine scan of the glycoprotein GN cytoplasmic tail (amino acids 1 to 81). First, we discovered three regions in the tail (amino acids 21 to 25, 46 to 50, and 71 to 81) which are important for minigenome transfer by VLPs. Further mutational analysis identified four amino acids that were important for RNP packaging. These amino acids are essential for the binding of nucleoproteins and RNPs to the glycoprotein without affecting the morphology of the particles. No segment-specific interactions between the RNA and the cytoplasmic tail could be observed. We propose that VLP systems are useful tools for analyzing protein-protein interactions important for packaging of viral genome segments, assembly, and budding of other members of the Bunyaviridae family.

scholarly journals Functional and Physical Interactions between AML1 Proteins and an ETS Protein, MEF: Implications for the Pathogenesis of t(8;21)-Positive Leukemias

1999 ◽  
Vol 19 (5) ◽  
pp. 3635-3644 ◽  
Author(s):  
Shifeng Mao ◽  
Richard C. Frank ◽  
Jin Zhang ◽  
Yasushi Miyazaki ◽  
Stephen D. Nimer
Keyword(s):  
Dna Binding ◽  
Protein Interactions ◽  
Myeloid Leukemia ◽  
Mutational Analysis ◽  
Myeloid Differentiation ◽  
Protein Protein Interactions ◽  
Ets Proteins ◽  
Ets Family ◽  
Physical Interactions ◽  
Definitive Hematopoiesis

ABSTRACT The AML1 and ETS families of transcription factors play critical roles in hematopoiesis; AML1, and its non-DNA-binding heterodimer partner CBFβ, are essential for the development of definitive hematopoiesis in mice, whereas the absence of certain ETS proteins creates specific defects in lymphopoiesis or myelopoiesis. The promoter activities of numerous genes expressed in hematopoietic cells are regulated by AML1 proteins or ETS proteins. MEF (for myeloid ELF-1-like factor) is a recently cloned ETS family member that, like AML1B, can strongly transactivate several of these promoters, which led us to examine whether MEF functionally or physically interacts with AML1 proteins. In this study, we demonstrate direct interactions between MEF and AML1 proteins, including the AML1/ETO fusion protein, in t(8;21)-positive acute myeloid leukemia (AML) cells. Using mutational analysis, we identified a novel ETS-interacting subdomain (EID) in the C-terminal portion of the Runt homology domain (RHD) in AML1 proteins and determined that the N-terminal region of MEF was responsible for its interaction with AML1. MEF and AML1B synergistically transactivated an interleukin 3 promoter reporter gene construct, yet the activating activity of MEF was abolished when MEF was coexpressed with AML1/ETO. The repression by AML1/ETO was independent of DNA binding but depended on its ability to interact with MEF, suggesting that AML1/ETO can repress genes not normally regulated by AML1 via protein-protein interactions. Interference with MEF function by AML1/ETO may lead to dysregulation of genes important for myeloid differentiation, thereby contributing to the pathogenesis of t(8;21) AML.

scholarly journals Physical Interaction between Coat Morphogenetic Proteins SpoVID and CotE Is Necessary for Spore Encasement in Bacillus subtilis

2012 ◽  
Vol 194 (18) ◽  
pp. 4941-4950 ◽  
Author(s):  
Melissa de Francesco ◽  
Jake Z. Jacobs ◽  
Filipa Nunes ◽  
Mónica Serrano ◽  
Peter T. McKenney ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Protein Interactions ◽  
Mutational Analysis ◽  
Physical Interaction ◽  
Spore Coat ◽  
Coat Proteins ◽  
Protein Protein Interactions ◽  
Content Type ◽  
Morphogenetic Protein ◽  
Physical Interactions

ABSTRACTEndospore formation byBacillus subtilisis a complex and dynamic process. One of the major challenges of sporulation is the assembly of a protective, multilayered, proteinaceous spore coat, composed of at least 70 different proteins. Spore coat formation can be divided into two distinct stages. The first is the recruitment of proteins to the spore surface, dependent on the morphogenetic protein SpoIVA. The second step, known as encasement, involves the migration of the coat proteins around the circumference of the spore in successive waves, a process dependent on the morphogenetic protein SpoVID and the transcriptional regulation of individual coat genes. We provide genetic and biochemical evidence supporting the hypothesis that SpoVID promotes encasement of the spore by establishing direct protein-protein interactions with other coat morphogenetic proteins. It was previously demonstrated that SpoVID directly interacts with SpoIVA and the inner coat morphogenetic protein, SafA. Here, we show by yeast two-hybrid and pulldown assays that SpoVID also interacts directly with the outer coat morphogenetic protein, CotE. Furthermore, by mutational analysis, we identified a specific residue in the N-terminal domain of SpoVID that is essential for the interaction with CotE but dispensable for the interaction with SafA. We propose an updated model of coat assembly and spore encasement that incorporates several physical interactions between the principal coat morphogenetic proteins.

scholarly journals Mapping of a Domain Required for Protein-Protein Interactions and Inhibitory Activity of a Helicobacter pylori Dominant-Negative VacA Mutant Protein

2006 ◽  
Vol 74 (4) ◽  
pp. 2093-2101 ◽  
Author(s):  
Victor J. Torres ◽  
Mark S. McClain ◽  
Timothy L. Cover
Keyword(s):  
Amino Acids ◽  
Helicobacter Pylori ◽  
Protein Interactions ◽  
Inhibitory Activity ◽  
Mammalian Cells ◽  
Mutational Analysis ◽  
Dominant Negative ◽  
Mutant Protein ◽  
Wild Type ◽  
Protein Protein Interactions

ABSTRACT The Helicobacter pylori VacA toxin is an 88-kDa secreted protein that causes multiple alterations in mammalian cells and is considered an important virulence factor in the pathogenesis of peptic ulcer disease and gastric cancer. We have shown previously that a VacA mutant protein lacking amino acids 6 to 27 (Δ6-27p88 VacA) is able to inhibit many activities of wild-type VacA in a dominant-negative manner. Analysis of a panel of C-terminally truncated Δ6-27p88 VacA proteins indicated that a fragment containing amino acids 1 to 478 (Δ6-27p48) exhibited a dominant-negative phenotype similar to that of the full-length Δ6-27p88 VacA protein. In contrast, a shorter VacA fragment lacking amino acids 6 to 27 (Δ6-27p33) did not exhibit detectable inhibitory activity. The Δ6-27p48 protein physically interacted with wild-type p88 VacA, whereas the Δ6-27p33 protein did not. Mutational analysis indicated that amino acids 351 to 360 are required for VacA protein-protein interactions and for dominant-negative inhibitory activity. The C-terminal portion (p55 domain) of wild-type p88 VacA could complement either Δ6-27p33 or Δ(6-27/351-360)p48, reconstituting dominant-negative inhibitory activity. Collectively, our data provide strong evidence that the inhibitory properties of dominant-negative VacA mutant proteins are dependent on interactions between the mutant VacA proteins and wild-type VacA, and they allow mapping of a domain involved in the formation of oligomeric VacA complexes.

scholarly journals Mutational Analysis of STE5 in the Yeast Saccharomyces cerevisiae: Application of a Differential Interaction Trap Assay for Examining Protein-Protein Interactions

Genetics ◽  
1997 ◽  
Vol 147 (2) ◽  
pp. 479-492 ◽  
Author(s):  
Carla Inouye ◽  
Namrita Dhillon ◽  
Tim Durfee ◽  
Patricia C Zambryski ◽  
Jeremy Thorner
Keyword(s):  
Protein Interactions ◽  
Mutational Analysis ◽  
Mapk Cascade ◽  
Mitogen Activated Protein Kinase ◽  
Missense Mutations ◽  
Protein Protein Interactions ◽  
Yeast Saccharomyces Cerevisiae ◽  
Mitogen Activated Protein

Ste5 is essential for the yeast mating pheromone response pathway and is thought to function as a scaffold that organizes the components of the mitogen-activated protein kinase (MAPK) cascade. A new method was developed to isolate missense mutations in Ste5 that differentially affect the ability of Ste5 to interact with either of two MAPK cascade constituents, the MEKK (Ste11) and the MEK (Ste7). Mutations that affect association with Ste7 or with Ste11 delineate discrete regions of Ste5 that are critical for each interaction. Co-immunoprecipitation analysis, examining the binding in vitro of Ste5 to Ste11, Ste7, Ste4 (G protein, β subunit), and Fus3 (MAPK), confirmed that each mutation specifically affects the interaction of Ste5 with only one protein. When expressed in a ste5Δ cell, mutant Ste5 proteins that are defective in their ability to interact with either Ste11 or Ste7 result in a markedly reduced mating proficiency. One mutation that clearly weakened (but did not eliminate) interaction of Ste5 with Ste7 permitted mating at wild-type efficiency, indicating that an efficacious signal is generated even when Ste5 associates with only a small fraction of (or only transiently with) Ste7. Ste5 mutants defective in association with Ste11 or Ste7 showed strong interallelic complementation when co-expressed, suggesting that the functional form of Ste5 in vivo is an oligomer.

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