The Additional Methionine Residue at the N-Terminus of Bacterially Expressed Human Interleukin-2 Affects the Interaction between the N- and C-Termini

Endo, Satoshi; Yamamoto, Yoshio; Sugawara, Tohru; Nishimura, Osamu; Fujino, Masahiko

doi:10.1021/bi001170r

Cited by 16 publications

(8 citation statements)

References 31 publications

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“…NMR chemical shift perturbations and isoelectric point changes upon addition of methionine to IL-2's N-terminus suggested a potential interaction between the N-and C-termini of apo IL-2 in solution. 82 Intriguingly, Thr3 is a site on human IL-2 that is variably glycosylated; 83 in mice, the N-terminus is longer and displays substantial sequence and glycosylation pattern variability, which in turn impacts IL-2's function in type I diabetes in mouse models. 84,85 An important caveat is that correlated motions are necessary but not sufficient for biologically relevant allostery.…”

Section: Discussionmentioning

confidence: 99%

Quantifying Correlations Between Allosteric Sites in Thermodynamic Ensembles

McClendon

Friedland

Mobley

et al. 2009

J. Chem. Theory Comput.

214

284

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Allostery describes altered protein function at one site due to a perturbation at another site. One mechanism of allostery involves correlated motions, which can occur even in the absence of substantial conformational change. We present a novel method, "MutInf", to identify statistically significant correlated motions from equilibrium molecular dynamics simulations. Our approach analyzes both backbone and sidechain motions using internal coordinates to account for the gearlike twists that can take place even in the absence of the large conformational changes typical of traditional allosteric proteins. We quantify correlated motions using a mutual information metric, which we extend to incorporate data from multiple short simulations and to filter out correlations that are not statistically significant. Applying our approach to uncover mechanisms of cooperative small molecule binding in human interleukin-2, we identify clusters of correlated residues from 50 ns of molecular dynamics simulations. Interestingly, two of the clusters with the strongest correlations highlight known cooperative small-molecule binding sites and show substantial correlations between these sites. These cooperative binding sites on interleukin-2 are correlated not only through the hydrophobic core of the protein but also through a dynamic polar network of hydrogen bonding and electrostatic interactions. Since this approach identifies correlated conformations in an unbiased, statistically robust manner, it should be a useful tool for finding novel or "orphan" allosteric sites in proteins of biological and therapeutic importance.

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Section: Discussionmentioning

confidence: 99%

Quantifying Correlations Between Allosteric Sites in Thermodynamic Ensembles

McClendon

Friedland

Mobley

et al. 2009

J. Chem. Theory Comput.

214

284

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“…On the basis of the recently resolved AQP1 structure (36), which does not cover the tails, it can be deduced that the distance between the NH 2 and COOH termini of an AQP1 monomer is about 25 Å, whereas that between the NH 2 and COOH termini of neighboring monomers is 12-15 Å, indicating that about 12 or 8 amino acids are needed to cross the distance, respectively. Because the NH 2 (16 amino acids) and COOH termini (46 amino acids) of AQP2 are large enough to cross these intraand intermolecular distances, both the NH 2 and COOH tails of AQP2 appeared to be required for trafficking of AQP1/2 proteins to intracellular vesicles, and interactions between NH 2 and COOH termini have shown to be important for the regulation of several proteins (12,18,19), we investigated whether the NH 2 and COOH tails of AQP2 could physically interact with each other using yeast two hybrid assays. As negative controls, L40 cells were transformed with AQP2 COOH tail or NH 2 tail constructs combined with empty bait or prey vectors.…”

Section: Analysis Of a Possible Interaction Between The Aqp2 Nh 2 Andmentioning

confidence: 99%

Role of cytoplasmic termini in sorting and shuttling of the aquaporin-2 water channel

Balkom¹,

Graat²,

Raak³

et al. 2004

American Journal of Physiology-Cell Physiology

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of cytoplasmic termini in sorting and shuttling of the aquaporin-2 water channel. Am J Physiol Cell Physiol 286: C372-C379, 2004. First published October 15, 2003 10.1152/ajpcell.00271.2003.-In mammals, the regulation of water homeostasis is mediated by the aquaporin-1 (AQP1) water channel, which localizes to the basolateral and apical membranes of the early nephron segment, and AQP2, which is translocated from intracellular vesicles to the apical membrane of collecting duct cells after vasopressin stimulation. Because a similar localization and regulation are observed in transfected Madin-Darby Canine Kidney (MDCK) cells, we investigated which segments of AQP2 are important for its routing to forskolin-sensitive vesicles and the apical membrane through analysis of AQP1-AQP2 chimeras. AQP1 with the entire COOH tail of AQP2 was constitutively localized in the apical membrane, whereas chimeras with shorter COOH tail segments of AQP2 were localized in the apical and basolateral membrane. AQP1 with the NH2 tail of AQP2 was constitutively localized in both plasma membranes, whereas AQP1 with the NH2 and COOH tail of AQP2 was sorted to intracellular vesicles and translocated to the apical membrane with forskolin. These data indicate that region N220-S229 is essential for localization of AQP2 in the apical membrane and that the NH2 and COOH tail of AQP2 are essential for trafficking of AQP2 to intracellular vesicles and its shuttling to and from the apical membrane. routing signals; chimera; Madin-Darby canine kidney cells; regulated trafficking TO MAINTAIN WATER AND OSMOLYTE BALANCE, the human kidney daily forms 180 l of pro-urine. The main portion of the water from the pro-urine is reabsorbed, which occurs mainly through aquaporin-1 (AQP1) and AQP2 (9, 10, 35). AQP1, responsible for 90% of the water reabsorption, is constitutively present in the apical and basolateral membrane of proximal tubules and the descending limbs of Henle (40). The fine-tuning of water reabsorption takes place in the renal collecting duct and is regulated by the antidiuretic hormone arginine vasopressin (AVP). After release of AVP by the pituitary gland and binding to its type-2 receptor (V2R) in the basolateral membrane of collecting duct principal cells, an intracellular cAMP signaling cascade is initiated, resulting in the phosphorylation of AQP2 and its redistribution from vesicles to the apical membrane. Then, driven by an osmotic gradient, collecting duct water uptake and urine concentration is initiated via AQP2 in the apical and AQP3/AQP4 in the basolateral membranes (11, 37). Removal of AVP reverses this translocation process, restoring the water-impermeable state of the apical membrane (7,23,26). A proper regulation of AQP2 sorting and translocation to the apical membrane is thus of critical importance for human water homeostasis. At present, however, it is unclear which protein segments of AQP2 are critical for this regulation.All AQPs are homotetrameric integral membrane proteins, consisting of subunits of about 30 kDa, which pas...

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“…Not all proteins naturally contain a methionine at their N-terminus and the cytoplasmic production of a protein in the cytoplasm does not guarantee the presence of an N-terminal methionine due to the action of methionine aminopeptidase ( Liao et al, 2004 ; Frottin et al, 2006 ). Removal of the N-formyl methionine can be critical for the proper folding, stability and function of a recombinant protein ( Endo et al, 2001 ; Liao et al, 2003 ).…”

Section: Introductionmentioning

confidence: 99%

Strategies to Enhance Periplasmic Recombinant Protein Production Yields in Escherichia coli

Karyolaimos

Gier

2021

Front. Bioeng. Biotechnol.

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Main reasons to produce recombinant proteins in the periplasm of E. coli rather than in its cytoplasm are to -i- enable disulfide bond formation, -ii- facilitate protein isolation, -iii- control the nature of the N-terminus of the mature protein, and -iv- minimize exposure to cytoplasmic proteases. However, hampered protein targeting, translocation and folding as well as protein instability can all negatively affect periplasmic protein production yields. Strategies to enhance periplasmic protein production yields have focused on harmonizing secretory recombinant protein production rates with the capacity of the secretory apparatus by transcriptional and translational tuning, signal peptide selection and engineering, increasing the targeting, translocation and periplasmic folding capacity of the production host, preventing proteolysis, and, finally, the natural and engineered adaptation of the production host to periplasmic protein production. Here, we discuss these strategies using notable examples as a thread.

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The Additional Methionine Residue at the N-Terminus of Bacterially Expressed Human Interleukin-2 Affects the Interaction between the N- and C-Termini

Cited by 16 publications

References 31 publications

Quantifying Correlations Between Allosteric Sites in Thermodynamic Ensembles

Quantifying Correlations Between Allosteric Sites in Thermodynamic Ensembles

Role of cytoplasmic termini in sorting and shuttling of the aquaporin-2 water channel

Strategies to Enhance Periplasmic Recombinant Protein Production Yields in Escherichia coli

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