Dataset Information

Controlling the dynamics of the Nek2 leucine zipper by engineering of "kinetic" disulphide bonds.

ABSTRACT: Nek2 is a dimeric serine/ threonine protein kinase that belongs to the family of NIMA-related kinases (Neks). Its N-terminal catalytic domain and its C-terminal regulatory region are bridged by a leucine zipper, which plays an important role in the activation of Nek2's catalytic activity. Unusual conformational dynamics on the intermediary/slow timescale has thwarted all attempts so far to determine the structure of the Nek2 leucine zipper by means of X-ray crystallography and Nuclear Magnetic Resonance (NMR). Disulfide engineering, the strategic placement of non-native disulfide bonds into flexible regions flanking the coiled coil, was used to modulate the conformational exchange dynamics of this important dimerization domain. The resulting reduction in exchange rate leads to substantial improvements of important features in NMR spectra, such as line width, coherence transfer leakage and relaxation. These effects were comprehensively analyzed for the wild type protein, two single disulfide bond-bearing mutants and another double disulfide bonds-carrying mutant. Furthermore, exchange kinetics were measured across a wide temperature range, allowing for a detailed analysis of activation energy (?G‡) and maximal rate constant (k'ex). For one mutant carrying a disulfide bond at its C-terminus, a full backbone NMR assignment could be obtained for both conformers, demonstrating the benefits of the disulfide engineering. Our study demonstrates the first successful application of 'kinetic' disulfide bonds for the purpose of controlling the adverse effects of protein dynamics. Firstly, this provides a promising, robust platform for the full structural and functional investigation of the Nek2 leucine zipper in the future. Secondly, this work broadens the toolbox of protein engineering by disulfide bonds through the addition of a kinetic option in addition to the well-established thermodynamic uses of disulfide bonds.

SUBMITTER: Gutmans DS

PROVIDER: S-EPMC6358272 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Controlling the dynamics of the Nek2 leucine zipper by engineering of "kinetic" disulphide bonds.

Gutmans Daniel S DS Whittaker Sara B-M SB Asiani Karishma K Atkinson R Andrew RA Oregioni Alain A Pfuhl Mark M

PloS one 20190201 2

Nek2 is a dimeric serine/ threonine protein kinase that belongs to the family of NIMA-related kinases (Neks). Its N-terminal catalytic domain and its C-terminal regulatory region are bridged by a leucine zipper, which plays an important role in the activation of Nek2's catalytic activity. Unusual conformational dynamics on the intermediary/slow timescale has thwarted all attempts so far to determine the structure of the Nek2 leucine zipper by means of X-ray crystallography and Nuclear Magnetic R ...[more]

PMID: 30707691

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Project description:Disulphide bonds are stabilizing crosslinks in proteins and serve to enhance their thermal stability. In proteins that are small and rich in disulphide bonds, they could be the major determining factor for the choice of conformational state since their constraints on appropriate backbone conformation can be substantial. Such crosslinks and their positional conservation could itself enable protein family and functional association. Despite the importance of the field, there is no comprehensive database on disulphide crosslinks that is available to the public. Herein we provide information on disulphides in DSDBASE2.0, an updated and significantly expanded database that is freely available, fully annotated and manually curated database on native and modelled disulphides. The web interface also provides several useful computational tools that have been specifically developed for proteins containing disulphide crosslinks. The modelling of disulphide crosslinks is performed using stereochemical criteria, coded within our Modelling of Disulphides in Proteins (MODIP) algorithm. The inclusion of modelled disulphides potentially enhances the loop database substantially, thereby permitting the recognition of compatible polypeptide segments that could serve as templates for immediate modelling. The DSDBASE2.0 database has been updated to include 153,944 PDB entries, 216,096 native and 20,153,850 modelled disulphide bond segments from PDB January 2021 release. The current database also provides a resource to user-friendly search for multiple disulphide bond containing loops, along with annotation of their function using GO and subcellular localization of the query. Furthermore, it is possible to obtain the three-dimensional models of disulphide-rich small proteins using an independent algorithm, RANMOD, that generates and examines random, but allowed backbone conformations of the polypeptide. DSDBASE2.0 still remains the largest open-access repository that organizes all disulphide bonds of proteins on a single platform. The database can be accessed from http://caps.ncbs.res.in/dsdbase2.

Project description:The cryptocephal (crc) mutation causes pleiotropic defects in ecdysone-regulated events during Drosophila molting and metamorphosis. Here we report that crc encodes a Drosophila homolog of vertebrate ATF4, a member of the CREB/ATF family of basic-leucine zipper (bZIP) transcription factors. We identified three putative protein isoforms. CRC-A and CRC-B contain the bZIP domain, and CRC-D is a C-terminally truncated form. We have generated seven new crc alleles. Consistent with the molecular diversity of crc, these alleles show that crc is a complex genetic locus with two overlapping lethal complementation groups. Alleles representing both groups were rescued by a cDNA encoding CRC-B. One lethal group (crc(1), crc(R6), and crc(Rev8)) consists of strong hypomorphic or null alleles that are associated with mutations of both CRC-A and CRC-B. These mutants display defects associated with larval molting and pupariation. In addition, they fail to evert the head and fail to elongate the imaginal discs during pupation, and they display variable defects in the subsequent differentiation of the adult abdomen. The other group (crc(R1), crc(R2), crc(E85), crc(E98), and crc(929)) is associated with disruptions of CRC-A and CRC-D; except for a failure to properly elongate the leg discs, these mutants initiate metamorphosis normally. Subsequently, they display a novel metamorphic phenotype, involving collapse of the head and abdomen toward the thorax. The crc gene is expressed throughout development and in many tissues. In third instar larvae, crc expression is high in targets of ecdysone signaling, such as the leg and wing imaginal discs, and in the ring gland, the source of ecdysone. Together, these findings implicate CREB/ATF proteins in essential functions during molting and metamorphosis. In addition, the similarities between the mutant phenotypes of crc and the ecdysone-responsive genes indicate that these genes are likely to be involved in common signaling pathways.

Dataset Information

Controlling the dynamics of the Nek2 leucine zipper by engineering of "kinetic" disulphide bonds.

Publications

Controlling the dynamics of the Nek2 leucine zipper by engineering of "kinetic" disulphide bonds.

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