Guidelines for the assembly of novel coiled-coil structures: α-sheets and α-cylinders

2001 ◽  
Vol 68 ◽  
pp. 111-123 ◽  
Author(s):  
John Walshaw ◽  
Jennifer M. Shipway ◽  
Derek N. Woolfson
Keyword(s):  
Protein Design ◽  
Protein Interactions ◽  
Protein Structures ◽  
Coiled Coil ◽  
Data Bank ◽  
Coiled Coils ◽  
Heptad Repeat ◽  
Protein Protein Interactions ◽  
Helical Bundles ◽  
Heptad Repeats

The coiled coil is a ubiquitous motif that guides many different protein-protein interactions. The accepted hallmark of coiled coils is a seven-residue (heptad) sequence repeat. The positions of this repeat are labelled a-b-c-d-e-f-g, with residues at a and d tending to be hydrophobic. Such sequences form amphipathic α-helices, which assemble into helical bundles via knobs-into-holes interdigitation of residues from neighbouring helices. We wrote an algorithm, SOCKET, to identify this packing in protein structures, and used this to gather a database of coiled-coil structures from the Protein Data Bank. Surprisingly, in addition to commonly accepted structures with a single, contiguous heptad repeat, we identified sequences with multiple, offset heptad repeats. These 'new' sequence patterns help to explain oligomer-state specification in coiled coils. Here we focus on the structural consequences for sequences with two heptad repeats offset by two residues, i.e. a/f′-b/g′-c/a′-d/b′-e/c′-f/d′-g/e′. This sets up two hydrophobic seams on opposite sides of the helix formed. We describe how such helices may combine to bury these hydrophobic surfaces in two different ways and form two distinct structures: open 'α-sheets' and closed 'α-cylinders'. We highlight these with descriptions of natural structures and outline possibilities for protein design.

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 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 Socket2: A Program for Locating, Visualising, and Analysing Coiled-coil Interfaces in Protein Structures

2021 ◽  
Author(s):  
Prasun Kumar ◽  
Derek N Woolfson
Keyword(s):  
Protein Interactions ◽  
De Novo ◽  
Protein Structures ◽  
Coiled Coil ◽  
Biological Research ◽  
Biological Processes ◽  
Protein Protein Interactions ◽  
Wide Range ◽  
Core Packing ◽  
Natural Amino Acids

Abstract Motivation Protein-protein interactions are central to all biological processes. One frequently observed mode of such interactions is the α-helical coiled coil (CC). Thus, an ability to extract, visualise, and analyse CC interfaces quickly and without expert guidance would facilitate a wide range of biological research. In 2001, we reported Socket, which locates and characterises CCs in protein structures based on the knobs-into-holes (KIH) packing between helices in CCs. Since then, studies of natural and de novo designed CCs have boomed, and the number of CCs in the RCSB PDB has increased rapidly. Therefore, we have updated Socket and made it accessible to expert and non-expert users alike. Results The original Socket only classified CCs with up to 6 helices. Here, we report Socket2, which rectifies this oversight to identify CCs with any number of helices, and KIH interfaces with any of the 20 proteinogenic residues or incorporating non-natural amino acids. In addition, we have developed a new and easy-to-use web server with additional features. These include the use of NGL Viewer for instantly visualising CCs, and tabs for viewing the sequence repeats, helix-packing angles, and core-packing geometries of CCs identified and calculated by Socket2. Availability and implementation Socket2 has been tested on all modern browsers. It can be accessed freely at http://coiledcoils.chm.bris.ac.uk/socket2/home.html. The source code is distributed using an MIT license and available to download under the Downloads tab of the Socket2 home page.

scholarly journals Navigating the structural landscape of de novo α–helical bundles

2018 ◽  
Author(s):  
Guto G. Rhys ◽  
Christopher W. Wood ◽  
Joseph L. Beesley ◽  
Nathan R. Zaccai ◽  
Antony J. Burton ◽  
...  
Keyword(s):  
De Novo ◽  
Hydrophobic Effect ◽  
Protein Structures ◽  
Coiled Coil ◽  
Coiled Coils ◽  
Single Amino Acid ◽  
Helical Bundles ◽  
Helical Bundle ◽  
Peptide System ◽  
Antiparallel Coiled Coil

ABSTRACTThe association of amphipathic α helices in water leads to α-helical-bundle protein structures. However, the driving force for this—the hydrophobic effect—is not specific and does not define the number or the orientation of helices in the associated state. Rather, this is achieved through deeper sequence-to-structure relationships, which are increasingly being discerned. For example, for one structurally extreme but nevertheless ubiquitous class of bundle—the α-helical coiled coils—relationships have been established that discriminate between all-parallel dimers, trimers and tetramers. Association states above this are known, as are antiparallel and mixed arrangements of the helices. However, these alternative states are less-well understood. Here, we describe a synthetic-peptide system that switches between parallel hexamers and various up-down-up-down tetramers in response to single-amino-acid changes and solution conditions. The main accessible states of each peptide variant are characterized fully in solution and, in most cases, to high-resolution X-ray crystal structures. Analysis and inspection of these structures helps rationalize the different states formed. This navigation of the structural landscape of α-helical coiled coils above the dimers and trimers that dominate in nature has allowed us to design rationally a well-defined and hyperstable antiparallel coiled-coil tetramer (apCC-Tet). This robust de novo protein provides another scaffold for further structural and functional designs in protein engineering and synthetic biology.

scholarly journals The trans-Golgi network GRIP-domain proteins form α-helical homodimers

2005 ◽  
Vol 388 (3) ◽  
pp. 835-841 ◽  
Author(s):  
Michael R. LUKE ◽  
Fiona HOUGHTON ◽  
Matthew A. PERUGINI ◽  
Paul A. GLEESON
Keyword(s):  
Protein Interactions ◽  
Coiled Coil ◽  
Helical Structure ◽  
Cd Spectroscopy ◽  
Protein Protein Interactions ◽  
Yeast Two Hybrid ◽  
Trans Golgi Network ◽  
Heptad Repeats ◽  
Golgi Network ◽  
Two Hybrid

A recently described family of TGN (trans-Golgi network) proteins, all of which contain a GRIP domain targeting sequence, has been proposed to play a role in membrane transport. On the basis of the high content of heptad repeats, GRIP domain proteins are predicted to contain extensive coiled-coil regions that have the potential to mediate protein–protein interactions. Four mammalian GRIP domain proteins have been identified which are targeted to the TGN through their GRIP domains, namely p230, golgin-97, GCC88 and GCC185. In the present study, we have investigated the ability of the four mammalian GRIP domain proteins to interact. Using a combination of immunoprecipitation experiments of epitope-tagged GRIP domain proteins, cross-linking experiments and yeast two-hybrid interactions, we have established that the GRIP proteins can self-associate to form homodimers exclusively. Two-hybrid analysis indicated that the N- and C-terminal fragments of GCC88 can interact with themselves but not with each other, suggesting that the GRIP domain proteins form parallel coiled-coil dimers. Analysis of purified recombinant golgin-97 by CD spectroscopy indicated a 67% α-helical structure, consistent with a high content of coiled-coil sequences. These results support a model for GRIP domain proteins as extended rod-like homodimeric molecules. The formation of homodimers, but not heterodimers, indicates that each of the four mammalian TGN golgins has the potential to function independently.

scholarly journals How coiled-coil assemblies accommodate multiple aromatic residues

2021 ◽  
Author(s):  
Guto G. Rhys ◽  
William M. Dawson ◽  
Joseph L. Beesley ◽  
Freddie J. O. Martin ◽  
R. Leo Brady ◽  
...  
Keyword(s):  
Hydrogen Bond ◽  
Protein Design ◽  
De Novo ◽  
Complex Structure ◽  
Protein Structures ◽  
Coiled Coil ◽  
Coiled Coils ◽  
Aromatic Residues ◽  
Hydrogen Bond Networks ◽  
Rational Protein Design

ABSTRACTRational protein design requires understanding the contribution of each amino acid to a targeted protein fold. For a subset of protein structures, namely the α;-helical coiled coils (CCs), knowledge is sufficiently advanced to allow the rational de novo design of many structures, including entirely new protein folds. However, current CC design rules center on using aliphatic hydrophobic residues predominantly to drive the folding and assembly of amphipathic α helices. The consequences of using aromatic residues—which would be useful for introducing structural probes, and binding and catalytic functionalities—into these interfaces is not understood. There are specific examples of designed CCs containing such aromatic residues, e.g., phenylalanine-rich sequences, and the use of polar aromatic residues to make buried hydrogen-bond networks. However, it is not known generally if sequences rich in tyrosine can form CCs, or what CC assemblies these would lead to. Here we explore tyrosine-rich sequences in a general CC-forming background and resolve new CC structures. In one of these, an antiparallel tetramer, the tyrosine residues are solvent accessible and pack at the interface between the core and the surface. In the other more-complex structure, the residues are buried and form an extended hydrogen-bond network.

scholarly journals Membrane-spanning α-helical barrels as tractable protein-design targets

2017 ◽  
Vol 372 (1726) ◽  
pp. 20160213 ◽  
Author(s):  
Ai Niitsu ◽  
Jack W. Heal ◽  
Kerstin Fauland ◽  
Andrew R. Thomson ◽  
Derek N. Woolfson
Keyword(s):  
Protein Structure ◽  
Protein Design ◽  
De Novo ◽  
Protein Structures ◽  
Coiled Coil ◽  
Deep Understanding ◽  
Data Bank ◽  
Water Soluble ◽  
Limiting Factor ◽  
Membrane Spanning

The rational ( de novo ) design of membrane-spanning proteins lags behind that for water-soluble globular proteins. This is due to gaps in our knowledge of membrane-protein structure, and experimental difficulties in studying such proteins compared to water-soluble counterparts. One limiting factor is the small number of experimentally determined three-dimensional structures for transmembrane proteins. By contrast, many tens of thousands of globular protein structures provide a rich source of ‘scaffolds’ for protein design, and the means to garner sequence-to-structure relationships to guide the design process. The α-helical coiled coil is a protein-structure element found in both globular and membrane proteins, where it cements a variety of helix–helix interactions and helical bundles. Our deep understanding of coiled coils has enabled a large number of successful de novo designs. For one class, the α-helical barrels—that is, symmetric bundles of five or more helices with central accessible channels—there are both water-soluble and membrane-spanning examples. Recent computational designs of water-soluble α-helical barrels with five to seven helices have advanced the design field considerably. Here we identify and classify analogous and more complicated membrane-spanning α-helical barrels from the Protein Data Bank. These provide tantalizing but tractable targets for protein engineering and de novo protein design. This article is part of the themed issue ‘Membrane pores: from structure and assembly, to medicine and technology’.

scholarly journals Heptad repeat regions of coiled-coil domains of Mfn1 are crucial for Mfn1 mediated mitochondrial fusion

2018 ◽  
Author(s):  
Sansrity Sinha ◽  
Gopala Krishna Aradhyam
Keyword(s):  
Coiled Coil ◽  
Helical Structure ◽  
Heptad Repeat ◽  
Outer Mitochondrial Membrane ◽  
Mitochondrial Membranes ◽  
Gtpase Domain ◽  
Helical Bundles ◽  
Gtpase Effector Domain ◽  
Heptad Repeats

Mitofusin mediate fusion of outer mitochondrial membranes (OMM). Recent studies have explained the role of GTPase domain for Mfn1 dimerization and outer mitochondrial membrane (OMM) fusion. Coiled-coiled domains [namely, coiled-coil/Middle domains (CC1MD) and coiled-coil-2 GTPase effector domain (CC2/GED)] form helical bundles that mediate open-to-close conformations of Mfn1 upon GTP binding and have been previously reported to be important for OMM tethering and OMM fusion. To further decipher the significance of helical structure of MD, we functionally characterized the heptad repeat regions of MD. Consistent with previous studies, we show that MD consists of two heptad repeats (HR1, namely HR1a and HR1b) and both of these are crucial for Mfn1 mediated OMM fusion.

scholarly journals De novo design of a reversible phosphorylation-dependent switch for membrane targeting

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Leon Harrington ◽  
Jordan M. Fletcher ◽  
Tamara Heermann ◽  
Derek N. Woolfson ◽  
Petra Schwille
Keyword(s):  
Protein Interactions ◽  
Lipid Membrane ◽  
De Novo ◽  
Protein Localization ◽  
Protein Structures ◽  
Spatiotemporal Pattern ◽  
Membrane Targeting ◽  
Protein Protein Interactions ◽  
Reversible Phosphorylation ◽  
Potential Applications

AbstractModules that switch protein-protein interactions on and off are essential to develop synthetic biology; for example, to construct orthogonal signaling pathways, to control artificial protein structures dynamically, and for protein localization in cells or protocells. In nature, the E. coli MinCDE system couples nucleotide-dependent switching of MinD dimerization to membrane targeting to trigger spatiotemporal pattern formation. Here we present a de novo peptide-based molecular switch that toggles reversibly between monomer and dimer in response to phosphorylation and dephosphorylation. In combination with other modules, we construct fusion proteins that couple switching to lipid-membrane targeting by: (i) tethering a ‘cargo’ molecule reversibly to a permanent membrane ‘anchor’; and (ii) creating a ‘membrane-avidity switch’ that mimics the MinD system but operates by reversible phosphorylation. These minimal, de novo molecular switches have potential applications for introducing dynamic processes into designed and engineered proteins to augment functions in living cells and add functionality to protocells.

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