X-ray Crystallographic Structure and Solution Behavior of an Antiparallel Coiled-Coil Hexamer Formed by de Novo Peptides

ABSTRACTThe rational design of linear peptides that assemble controllably and predictably in water is challenging. Sequences must encode unique target structures and avoid alternative states. However, the stabilizing and discriminating non-covalent forces available are weak in water. Nonetheless, for α-helical coiled-coil assemblies considerable progress has been made in rational de novo design. In these, sequence repeats of nominally hydrophobic (h) and polar (p) residues, hpphppp, direct the assembly of amphipathic helices into dimeric to tetrameric bundles. Expanding this pattern to hpphhph can produce larger α-helical barrels. Here, we show that pentamers to nonamers are achieved simply by varying the residue at one of these h sites. In L/I-K-E-I-A-x-Z repeats, decreasing the size of Z from threonine to serine to alanine to glycine gives progressively larger oligomers. X-ray crystal structures of the resulting α-helical barrels rationalize this: side chains at Z point directly into the helical interfaces, and smaller residues allow closer helix contacts and larger assemblies.

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Navigating the structural landscape of de novo α–helical bundles

10.1101/503698 ◽

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.

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Faculty Opinions recommendation of De novo protein crystal structure determination from X-ray free-electron laser data.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.718185624.793487506 ◽

2013 ◽

Author(s):

Chantal Abergel

Keyword(s):

Crystal Structure ◽

Structure Determination ◽

Free Electron ◽

Free Electron Laser ◽

De Novo ◽

Crystal Structure Determination ◽

Protein Crystal ◽

X Ray ◽

Protein Crystal Structure ◽

Laser Data

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The X-Ray Crystallographic Structure of the Human Neonatal Fc Receptor at Acidic pH Gives Insights into pH-Dependent Conformational Changes

Protein and Peptide Letters ◽

10.2174/0929866523666160404125850 ◽

2016 ◽

Vol 23 (6) ◽

pp. 525-529 ◽

Cited By ~ 2

Author(s):

Mohammed Taha ◽

Sally E. Ward ◽

Hyun-Joo Nam

Keyword(s):

Conformational Changes ◽

Fc Receptor ◽

Crystallographic Structure ◽

Acidic Ph ◽

Neonatal Fc Receptor ◽

X Ray ◽

Ph Dependent

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The X-ray Crystallographic Structure of Human EAT2 (SH2D1B)

Protein and Peptide Letters ◽

10.2174/0929866523666160831162239 ◽

2016 ◽

Vol 23 (10) ◽

pp. 862-866 ◽

Cited By ~ 1

Author(s):

Mohammed Taha ◽

Eric Nezerwa ◽

Hyun-Joo Nam

Keyword(s):

Crystallographic Structure ◽

X Ray

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An enzymatic activation of formaldehyde for nucleotide methylation

Nature Communications ◽

10.1038/s41467-021-24756-8 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Charles Bou-Nader ◽

Frederick W. Stull ◽

Ludovic Pecqueur ◽

Philippe Simon ◽

Vincent Guérineau ◽

...

Keyword(s):

Amino Acids ◽

Thymidylate Synthase ◽

Active Site ◽

De Novo ◽

Crystallographic Structure ◽

De Novo Synthesis ◽

Active Site Residues ◽

Enzymatic Activation ◽

Enzyme Cofactors ◽

Unique Ability

AbstractFolate enzyme cofactors and their derivatives have the unique ability to provide a single carbon unit at different oxidation levels for the de novo synthesis of amino-acids, purines, or thymidylate, an essential DNA nucleotide. How these cofactors mediate methylene transfer is not fully settled yet, particularly with regard to how the methylene is transferred to the methylene acceptor. Here, we uncovered that the bacterial thymidylate synthase ThyX, which relies on both folate and flavin for activity, can also use a formaldehyde-shunt to directly synthesize thymidylate. Combining biochemical, spectroscopic and anaerobic crystallographic analyses, we showed that formaldehyde reacts with the reduced flavin coenzyme to form a carbinolamine intermediate used by ThyX for dUMP methylation. The crystallographic structure of this intermediate reveals how ThyX activates formaldehyde and uses it, with the assistance of active site residues, to methylate dUMP. Our results reveal that carbinolamine species promote methylene transfer and suggest that the use of a CH2O-shunt may be relevant in several other important folate-dependent reactions.

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Optimization in S-SAD phasing - difference between solved and unsolved structure

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314093863 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C613-C613

Author(s):

Jan Stránský ◽

Tomáš Kovaľ ◽

Lars Østergaard ◽

Jarmila Dušková ◽

Tereza Skálová ◽

...

Keyword(s):

Data Processing ◽

De Novo ◽

Protein Structures ◽

X Ray Diffraction ◽

X Ray ◽

Structure Solution ◽

Sad Phasing ◽

Optimal Values ◽

Grant Agency ◽

Intensity Reading

Development of X-ray diffraction technologies have made de novo phasing of protein structures by single-wavelength anomalous dispersion by sulphur (S-SAD) more common. As anomalous differences in the sulphur atomic factors are in the order of errors of measurement, careful intensity reading and data processing are crucial. S-SAD was used for de novo phasing of a small 12 kDa protein with 4 sulphur atoms per molecule at 2.3 Å, where the data did not enable a straightforward structure solution. Data processing was performed using XDS [1] and scaling using XSCALE. The sulphur substructure was determined by SHELXD [2] and phases were obtained from SHELXE [2]. Both algorithms strongly depend on input parameters and default values did not lead to the correct phases. Therefore a systematic search of optimal values of several parameters was used to find a solution. This method helped to confirm sulphur substructure and to differentiate the handedness of the solutions. Moreover, a script for comfortable conversion of SHELX outputs to MTZ format was developed, using programmes included in the CCP4 package [3]. The previously unsolvable protein structure was successfully resolved with the described procedure. This work was supported by the Grant Agency of the Czech Technical University in Prague, (SGS13/219/OHK4/3T/14), the Czech Science Foundation (P302/11/0855), project BIOCEV CZ.1.05/1.1.00/02.0109 from the ERDF.

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