scholarly journals De novocoiled-coil peptides as scaffolds for disrupting protein–protein interactions

2018 ◽  
Vol 9 (39) ◽  
pp. 7656-7665 ◽  
Author(s):  
Jordan M. Fletcher ◽  
Katherine A. Horner ◽  
Gail J. Bartlett ◽  
Guto G. Rhys ◽  
Andrew J. Wilson ◽  
...  
Keyword(s):  
Protein Binding ◽  
Protein Interactions ◽  
Coiled Coils ◽  
Protein Protein Interactions ◽  
Binding Motifs ◽  
Helical Protein

Homo- and hetero-dimeric coiled coils as scaffolds for the presentation of α-helical protein-binding motifs.

scholarly journals Coiled-coil networking shapes cell molecular machinery

2012 ◽  
Vol 23 (19) ◽  
pp. 3911-3922 ◽  
Author(s):  
Yongqiang Wang ◽  
Xinlei Zhang ◽  
Hong Zhang ◽  
Yi Lu ◽  
Haolong Huang ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Critical Role ◽  
Coiled Coil ◽  
Coiled Coils ◽  
Protein Protein Interactions ◽  
Full Spectrum ◽  
Kinetochore Assembly ◽  
Genome Wide ◽  
Molecular Machinery ◽  
Systematic Understanding

The highly abundant α-helical coiled-coil motif not only mediates crucial protein–protein interactions in the cell but is also an attractive scaffold in synthetic biology and material science and a potential target for disease intervention. Therefore a systematic understanding of the coiled-coil interactions (CCIs) at the organismal level would help unravel the full spectrum of the biological function of this interaction motif and facilitate its application in therapeutics. We report the first identified genome-wide CCI network in Saccharomyces cerevisiae, which consists of 3495 pair-wise interactions among 598 predicted coiled-coil regions. Computational analysis revealed that the CCI network is specifically and functionally organized and extensively involved in the organization of cell machinery. We further show that CCIs play a critical role in the assembly of the kinetochore, and disruption of the CCI network leads to defects in kinetochore assembly and cell division. The CCI network identified in this study is a valuable resource for systematic characterization of coiled coils in the shaping and regulation of a host of cellular machineries and provides a basis for the utilization of coiled coils as domain-based probes for network perturbation and pharmacological applications.

scholarly journals ArabidopsisImmunophilin-like TWD1 Functionally Interacts with Vacuolar ABC Transporters

2004 ◽  
Vol 15 (7) ◽  
pp. 3393-3405 ◽  
Author(s):  
Markus Geisler ◽  
Marjolaine Girin ◽  
Sabine Brandt ◽  
Vincent Vincenzetti ◽  
Sonia Plaza ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Abc Transporters ◽  
Loss Of Function ◽  
Protein Protein Interactions ◽  
Binding Motifs ◽  
Calmodulin Binding ◽  
C Terminus ◽  
Domain Mapping ◽  
Tetratricopeptide Repeat Domain

Previously, the immunophilin-like protein TWD1 from Arabidopsis has been demonstrated to interact with the ABC transporters AtPGP1 and its closest homologue, AtPGP19. Physiological and biochemical investigation of pgp1/pgp19 and of twd1 plants suggested a regulatory role of TWD1 on AtPGP1/AtPGP19 transport activities. To further understand the dramatic pleiotropic phenotype that is caused by loss-of-function mutation of the TWD1 gene, we were interested in other TWD1 interacting proteins. AtMRP1, a multidrug resistance-associated (MRP/ABCC)-like ABC transporter, has been isolated in a yeast two-hybrid screen. We demonstrate molecular interaction between TWD1 and ABC transporters AtMRP1 and its closest homologue, AtMRP2. Unlike AtPGP1, AtMRP1 binds to the C-terminal tetratricopeptide repeat domain of TWD1, which is well known to mediate protein-protein interactions. Domain mapping proved that TWD1 binds to a motif of AtMRP1 that resembles calmodulin-binding motifs; and calmodulin binding to the C-terminus of MRP1 was verified. By membrane fractionation and GFP-tagging, we localized AtMRP1 to the central vacuolar membrane and the TWD1-AtMRP1 complex was verified in vivo by coimmunoprecipitation. We were able to demonstrate that TWD1 binds to isolated vacuoles and has a significant impact on the uptake of metolachlor-GS and estradiol-β-glucuronide, well-known substrates of vacuolar transporters AtMRP1 and AtMRP2.

scholarly journals Mechanism of the G-protein mimetic nanobody binding to a muscarinic G-protein-coupled receptor

2018 ◽  
Vol 115 (12) ◽  
pp. 3036-3041 ◽  
Author(s):  
Yinglong Miao ◽  
J. Andrew McCammon
Keyword(s):  
Protein Binding ◽  
G Protein ◽  
Protein Interactions ◽  
Intracellular Signaling ◽  
Md Simulations ◽  
Binding Pocket ◽  
Signaling Proteins ◽  
Protein Protein Interactions ◽  
Intracellular Loops ◽  
G Protein Coupled

Protein–protein binding is key in cellular signaling processes. Molecular dynamics (MD) simulations of protein–protein binding, however, are challenging due to limited timescales. In particular, binding of the medically important G-protein-coupled receptors (GPCRs) with intracellular signaling proteins has not been simulated with MD to date. Here, we report a successful simulation of the binding of a G-protein mimetic nanobody to the M2 muscarinic GPCR using the robust Gaussian accelerated MD (GaMD) method. Through long-timescale GaMD simulations over 4,500 ns, the nanobody was observed to bind the receptor intracellular G-protein-coupling site, with a minimum rmsd of 2.48 Å in the nanobody core domain compared with the X-ray structure. Binding of the nanobody allosterically closed the orthosteric ligand-binding pocket, being consistent with the recent experimental finding. In the absence of nanobody binding, the receptor orthosteric pocket sampled open and fully open conformations. The GaMD simulations revealed two low-energy intermediate states during nanobody binding to the M2 receptor. The flexible receptor intracellular loops contribute remarkable electrostatic, polar, and hydrophobic residue interactions in recognition and binding of the nanobody. These simulations provided important insights into the mechanism of GPCR–nanobody binding and demonstrated the applicability of GaMD in modeling dynamic protein–protein interactions.

scholarly journals HippDB: a database of readily targeted helical protein-protein interactions

2013 ◽  
Vol 29 (21) ◽  
pp. 2806-2807 ◽  
Author(s):  
C. M. Bergey ◽  
A. M. Watkins ◽  
P. S. Arora
Keyword(s):  
Protein Interactions ◽  
Protein Protein Interactions ◽  
Helical Protein

scholarly journals A Multiplexed Bacterial Two-Hybrid for Rapid Characterization of Protein-Protein Interactions and Iterative Protein Design

2020 ◽  
Author(s):  
W. Clifford Boldridge ◽  
Ajasja Ljubetič ◽  
Hwangbeom Kim ◽  
Nathan Lubock ◽  
Dániel Szilágyi ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Coiled Coil ◽  
Building Blocks ◽  
Coiled Coils ◽  
Next Generation ◽  
Protein Protein Interactions ◽  
Library Size ◽  
Large Sets ◽  
Synthetic Cell ◽  
Two Hybrid

AbstractMyriad biological functions require protein-protein interactions (PPIs), and engineered PPIs are crucial for applications ranging from drug design to synthetic cell circuits. Understanding and engineering specificity in PPIs is particularly challenging as subtle sequence changes can drastically alter specificity. Coiled-coils are small protein domains that have long served as a simple model for studying the sequence-determinants of specificity and have been used as modular building blocks to build large protein nanostructures and synthetic circuits. Despite their simple rules and long-time use, building large sets of well-behaved orthogonal pairs that can be used together is still challenging because predictions are often inaccurate, and, as the library size increases, it becomes difficult to test predictions at scale. To address these problems, we first developed a method called the Next-Generation Bacterial Two-Hybrid (NGB2H), which combines gene synthesis, a bacterial two-hybrid assay, and a high-throughput next-generation sequencing readout, allowing rapid exploration of interactions of programmed protein libraries in a quantitative and scalable way. After validating the NGB2H system on previously characterized libraries, we designed, built, and tested large sets of orthogonal synthetic coiled-coils. In an iterative set of experiments, we assayed more than 8,000 PPIs, used the dataset to train a novel linear model-based coiled-coil scoring algorithm, and then characterized nearly 18,000 interactions to identify the largest set of orthogonal PPIs to date with twenty-two on-target interactions.

scholarly journals High-resolution structures of a heterochiral coiled coil

2015 ◽  
Vol 112 (43) ◽  
pp. 13144-13149 ◽  
Author(s):  
David E. Mortenson ◽  
Jay D. Steinkruger ◽  
Dale F. Kreitler ◽  
Dominic V. Perroni ◽  
Gregory P. Sorenson ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Structural Information ◽  
Coiled Coil ◽  
Coiled Coils ◽  
Side Chain ◽  
Amino Acid Residues ◽  
Protein Protein Interactions ◽  
Polypeptide Chains ◽  
Packing Interactions

Interactions between polypeptide chains containing amino acid residues with opposite absolute configurations have long been a source of interest and speculation, but there is very little structural information for such heterochiral associations. The need to address this lacuna has grown in recent years because of increasing interest in the use of peptides generated from d amino acids (d peptides) as specific ligands for natural proteins, e.g., to inhibit deleterious protein–protein interactions. Coiled–coil interactions, between or among α-helices, represent the most common tertiary and quaternary packing motif in proteins. Heterochiral coiled–coil interactions were predicted over 50 years ago by Crick, and limited experimental data obtained in solution suggest that such interactions can indeed occur. To address the dearth of atomic-level structural characterization of heterochiral helix pairings, we report two independent crystal structures that elucidate coiled-coil packing between l- and d-peptide helices. Both structures resulted from racemic crystallization of a peptide corresponding to the transmembrane segment of the influenza M2 protein. Networks of canonical knobs-into-holes side-chain packing interactions are observed at each helical interface. However, the underlying patterns for these heterochiral coiled coils seem to deviate from the heptad sequence repeat that is characteristic of most homochiral analogs, with an apparent preference for a hendecad repeat pattern.

scholarly journals A High-Throughput Solid-Phase Microplate Protein-Binding Assay to Investigate Interactions between Myofilament Proteins

2011 ◽  
Vol 2011 ◽  
pp. 1-8 ◽  
Author(s):  
Brandon J. Biesiadecki ◽  
J.-P. Jin
Keyword(s):  
Protein Binding ◽  
Protein Interactions ◽  
Protein Modification ◽  
Binding Assay ◽  
Solid Phase ◽  
Regulatory Protein ◽  
Protein Isoforms ◽  
Protein Protein Interactions ◽  
Protein Binding Assay ◽  
Phase Protein

To understand the structure-function relationship of muscle-regulatory-protein isoforms, mutations, and posttranslational modifications, it is necessary to probe functional effects at the level of the protein-protein interaction. Traditional methodologies assessing such protein-protein interactions are laborious and require significant amounts of purified protein, while many current methodologies require costly and specialized equipment or modification of the proteins, which may affect their interaction. To address these issues, we developed a novel method of microplate-based solid-phase protein-binding assay over the recent years. This method assesses specific protein-protein interactions at physiological conditions, utilizes relatively small amounts of protein, is free of protein modification, and does not require specialized instrumentation. Here we present detailed methodology for the solid-phase protein-binding assay with examples that we have successfully applied to quantify interactions of myofilament-regulatory proteins. We further provide considerations for optimization of the assay conditions and its broader application in studies of other protein-protein interactions.

scholarly journals Homologue Scanning Mutagenesis Reveals Cd66 Receptor Residues Required for Neisserial Opa Protein Binding

1999 ◽  
Vol 190 (3) ◽  
pp. 331-340 ◽  
Author(s):  
Martine P. Bos ◽  
Daniel Hogan ◽  
Robert J. Belland
Keyword(s):  
Epithelial Cells ◽  
Protein Binding ◽  
Protein Interactions ◽  
Human Neutrophils ◽  
Loss Of Function ◽  
Protein Protein Interactions ◽  
Binding Characteristics ◽  
Protein Receptor ◽  
Strain Ms11 ◽  
Differential Binding

The immunoglobulin-like family of CD66 antigens, present on human neutrophils and epithelial cells, are used as receptors for adhesins expressed by the pathogenic Neisseriae. N. gonorrhoeae strain MS11 can express 11 isoforms of these adhesins, called opacity-related (Opa) proteins. Each MS11 Opa protein recognizes a distinct spectrum of CD66 receptors. CD66–Opa binding is mediated by the NH2-terminal domain of the receptor and occurs through protein–protein interactions. In this report, we have investigated the molecular basis for the binding between the CD66 and Opa protein families by mapping amino acids in CD66 receptors that determine Opa protein binding. We performed homologue scanning mutagenesis between CD66e, which binds multiple Opa variants, and CD66b, which binds none, and tested both loss-of-function by CD66e and gain-of-function by CD66b in solution assays and in assays involving full-length receptors expressed by epithelial cells. We found that three residues in the CD66e N-domain are required for maximal Opa protein receptor activity. Opa proteins that recognize the same spectrum of native CD66 molecules showed differential binding of receptors with submaximal activity, indicating that the binding characteristics of these Opa proteins are actually slightly different. These data provide a first step toward resolving the structural requirements for Opa–CD66 interaction.

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