Crystal structure of the C-terminal four-helix bundle of the potassium channel KCa3.1

The circadian clock found inSynechococcus elongatus, the most ancient circadian clock, is regulated by the interaction of three proteins, KaiA, KaiB, and KaiC. While the precise function of these proteins remains unclear, KaiA has been shown to be a positive regulator of the expression of KaiB and KaiC. The 2.0-Å structure of KaiA ofS. elongatusreported here shows that the protein is composed of two independently folded domains connected by a linker. The NH2-terminalpseudo-receiver domain has a similar fold with that of bacterial response regulators, whereas the COOH-terminal four-helix bundle domain is novel and forms the interface of the 2-fold-related homodimer. The COOH-terminal four-helix bundle domain has been shown to contain the KaiC binding site. The structure suggests that the KaiB binding site is covered in the dimer interface of the KaiA “closed” conformation, observed in the crystal structure, which suggests an allosteric regulation mechanism.

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Crystal structure of TTHA0303 (TT2238), a four‐helix bundle protein with an exposed histidine triad from Thermus thermophilus HB8 at 2.0 Å

Proteins Structure Function and Bioinformatics ◽

10.1002/prot.21765 ◽

2008 ◽

Vol 70 (3) ◽

pp. 1103-1107 ◽

Cited By ~ 5

Author(s):

Koji Nagata ◽

Jun Ohtsuka ◽

Mihoko Takahashi ◽

Atsuko Asano ◽

Hitoshi Iino ◽

...

Keyword(s):

Crystal Structure ◽

Thermus Thermophilus ◽

Thermus Thermophilus Hb8 ◽

Helix Bundle ◽

Four Helix Bundle ◽

Bundle Protein

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Rational thermostabilisation of four-helix bundle dimeric de novo proteins

Scientific Reports ◽

10.1038/s41598-021-86952-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Shin Irumagawa ◽

Kaito Kobayashi ◽

Yutaka Saito ◽

Takeshi Miyata ◽

Mitsuo Umetsu ◽

...

Keyword(s):

Protein Design ◽

De Novo ◽

Protein Complexes ◽

Salt Bridge ◽

Dynamics Simulation ◽

Saturation Mutagenesis ◽

Helix Bundle ◽

Stable Mutant ◽

Four Helix Bundle ◽

The Stability

AbstractThe stability of proteins is an important factor for industrial and medical applications. Improving protein stability is one of the main subjects in protein engineering. In a previous study, we improved the stability of a four-helix bundle dimeric de novo protein (WA20) by five mutations. The stabilised mutant (H26L/G28S/N34L/V71L/E78L, SUWA) showed an extremely high denaturation midpoint temperature (Tm). Although SUWA is a remarkably hyperstable protein, in protein design and engineering, it is an attractive challenge to rationally explore more stable mutants. In this study, we predicted stabilising mutations of WA20 by in silico saturation mutagenesis and molecular dynamics simulation, and experimentally confirmed three stabilising mutations of WA20 (N22A, N22E, and H86K). The stability of a double mutant (N22A/H86K, rationally optimised WA20, ROWA) was greatly improved compared with WA20 (ΔTm = 10.6 °C). The model structures suggested that N22A enhances the stability of the α-helices and N22E and H86K contribute to salt-bridge formation for protein stabilisation. These mutations were also added to SUWA and improved its Tm. Remarkably, the most stable mutant of SUWA (N22E/H86K, rationally optimised SUWA, ROSA) showed the highest Tm (129.0 °C). These new thermostable mutants will be useful as a component of protein nanobuilding blocks to construct supramolecular protein complexes.

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Self-Assembly of a Four-Helix Bundle on a DNA Quadruplex

Angewandte Chemie International Edition ◽

10.1002/anie.200804849 ◽

2009 ◽

Vol 48 (15) ◽

pp. 2749-2751 ◽

Cited By ~ 14

Author(s):

Brooke A. Rosenzweig ◽

Andrew D. Hamilton

Keyword(s):

Self Assembly ◽

Helix Bundle ◽

Dna Quadruplex ◽

Four Helix Bundle

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Structural basis for the alternating access mechanism of the cation diffusion facilitator YiiP

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1715051115 ◽

2018 ◽

Vol 115 (12) ◽

pp. 3042-3047 ◽

Cited By ~ 19

Author(s):

Maria Luisa Lopez-Redondo ◽

Nicolas Coudray ◽

Zhening Zhang ◽

John Alexopoulos ◽

David L. Stokes

Keyword(s):

Large Scale ◽

Structural Basis ◽

Membrane Domains ◽

Cation Diffusion ◽

Cation Diffusion Facilitator ◽

X Ray ◽

Bacterial Membranes ◽

Helix Bundle ◽

Four Helix Bundle ◽

Alternating Access

YiiP is a dimeric antiporter from the cation diffusion facilitator family that uses the proton motive force to transport Zn2+ across bacterial membranes. Previous work defined the atomic structure of an outward-facing conformation, the location of several Zn2+ binding sites, and hydrophobic residues that appear to control access to the transport sites from the cytoplasm. A low-resolution cryo-EM structure revealed changes within the membrane domain that were associated with the alternating access mechanism for transport. In the current work, the resolution of this cryo-EM structure has been extended to 4.1 Å. Comparison with the X-ray structure defines the differences between inward-facing and outward-facing conformations at an atomic level. These differences include rocking and twisting of a four-helix bundle that harbors the Zn2+ transport site and controls its accessibility within each monomer. As previously noted, membrane domains are closely associated in the dimeric structure from cryo-EM but dramatically splayed apart in the X-ray structure. Cysteine crosslinking was used to constrain these membrane domains and to show that this large-scale splaying was not necessary for transport activity. Furthermore, dimer stability was not compromised by mutagenesis of elements in the cytoplasmic domain, suggesting that the extensive interface between membrane domains is a strong determinant of dimerization. As with other secondary transporters, this interface could provide a stable scaffold for movements of the four-helix bundle that confers alternating access of these ions to opposite sides of the membrane.

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