International Journal of Molecular Sciences

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18 pages, 2608 KiB

Open AccessArticle

14-3-3 Proteins and Other Candidates form Protein-Protein Interactions with the Cytosolic C-terminal End of SOS1 Affecting Its Transport Activity

by Kerstin Duscha, Cristina Martins Rodrigues, Maria Müller, Ruth Wartenberg, Larry Fliegel, Joachim W. Deitmer, Martin Jung, Richard Zimmermann and H. Ekkehard Neuhaus

Int. J. Mol. Sci. 2020, 21(9), 3334; https://doi.org/10.3390/ijms21093334 - 8 May 2020

Cited by 14 | Viewed by 3769

Abstract

The plasma membrane transporter SOS1 (SALT-OVERLY SENSITIVE1) is vital for plant survival under salt stress. SOS1 activity is tightly regulated, but little is known about the underlying mechanism. SOS1 contains a cytosolic, autoinhibitory C-terminal tail (abbreviated as SOS1 C-term), which is targeted by [...] Read more.

The plasma membrane transporter SOS1 (SALT-OVERLY SENSITIVE1) is vital for plant survival under salt stress. SOS1 activity is tightly regulated, but little is known about the underlying mechanism. SOS1 contains a cytosolic, autoinhibitory C-terminal tail (abbreviated as SOS1 C-term), which is targeted by the protein kinase SOS2 to trigger its transport activity. Here, to identify additional binding proteins that regulate SOS1 activity, we synthesized the SOS1 C-term domain and used it as bait to probe Arabidopsis thaliana cell extracts. Several 14-3-3 proteins, which function in plant salt tolerance, specifically bound to and interacted with the SOS1 C-term. Compared to wild-type plants, when exposed to salt stress, Arabidopsis plants overexpressing SOS1 C-term showed improved salt tolerance, significantly reduced Na⁺ accumulation in leaves, reduced induction of the salt-responsive gene WRKY25, decreased soluble sugar, starch, and proline levels, less impaired inflorescence formation and increased biomass. It appears that overexpressing SOS1 C-term leads to the sequestration of inhibitory 14-3-3 proteins, allowing SOS1 to be more readily activated and leading to increased salt tolerance. We propose that the SOS1 C-term binds to previously unknown proteins such as 14-3-3 isoforms, thereby regulating salt tolerance. This finding uncovers another regulatory layer of the plant salt tolerance program. Full article

(This article belongs to the Special Issue Structure and Function of Membrane Proteins)

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18 pages, 4183 KiB

Open AccessArticle

Amino Acids 563–566 of the Na⁺/H⁺ Exchanger Isoform 1 C-Terminal Cytosolic Tail Prevent Protein Degradation and Stabilize Protein Expression and Activity

by Xiuju Li, Debajyoti Dutta, Martin Jung, Richard Zimmermann and Larry Fliegel

Int. J. Mol. Sci. 2020, 21(5), 1737; https://doi.org/10.3390/ijms21051737 - 3 Mar 2020

Cited by 6 | Viewed by 3024

Abstract

Isoform one of the mammalian Na⁺/H⁺ exchanger is a plasma membrane protein that is ubiquitously present in humans. It regulates intracellular pH through the removal of one intracellular proton in exchange for a single extracellular sodium. It consists of a [...] Read more.

Isoform one of the mammalian Na⁺/H⁺ exchanger is a plasma membrane protein that is ubiquitously present in humans. It regulates intracellular pH through the removal of one intracellular proton in exchange for a single extracellular sodium. It consists of a 500 amino acid membrane domain plus a 315 amino acid, C-terminal tail. We examined amino acids of the C-terminal tail that are important in the targeting and activity of the protein. A previous study demonstrated that stop codon polymorphisms can result in decreased activity, expression, targeting and enhanced protein degradation. Here, we determine elements that are critical in these anomalies. A series of progressive deletions of the C-terminal tail demonstrated a progressive decrease in activity and targeting, though these remained until a final drop off with the deletion of amino acids 563–566. The deletion of the ⁵⁶²LIAGERS⁵⁶⁸ sequence or the alteration to the ⁵⁶²LAAAARS⁵⁶⁸ sequence caused the decreased protein expression, aberrant targeting, reduced activity and enhanced degradation of the Na⁺/H⁺ exchanger (NHE1) protein. The ⁵⁶²LIAGERS⁵⁶⁸ sequence bound to other regions of the C-terminal cytosolic domain. We suggest this region is necessary for the activity, targeting, stability, and expression of the NHE1 protein. The results define a new sequence that is important in maintenance of NHE1 protein levels and activity. Full article

(This article belongs to the Special Issue Structure and Function of Membrane Proteins)

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27 pages, 14940 KiB

Open AccessArticle

Structural Insights into the Intracellular Region of the Human Magnesium Transport Mediator CNNM4

by Paula Giménez-Mascarell, Iker Oyenarte, Irene González-Recio, Carmen Fernández-Rodríguez, María Ángeles Corral-Rodríguez, Igone Campos-Zarraga, Jorge Simón, Elie Kostantin, Serge Hardy, Antonio Díaz Quintana, Mara Zubillaga Lizeaga, Nekane Merino, Tammo Diercks, Francisco J. Blanco, Irene Díaz Moreno, María Luz Martínez-Chantar, Michel L. Tremblay, Dominik Müller, Dritan Siliqi and Luis Alfonso Martínez-Cruz

Int. J. Mol. Sci. 2019, 20(24), 6279; https://doi.org/10.3390/ijms20246279 - 12 Dec 2019

Cited by 11 | Viewed by 4180

Abstract

The four member family of “Cyclin and Cystathionine β-synthase (CBS) domain divalent metal cation transport mediators”, CNNMs, are the least-studied mammalian magnesium transport mediators. CNNM4 is abundant in the brain and the intestinal tract, and its abnormal activity causes Jalili Syndrome. Recent findings [...] Read more.

The four member family of “Cyclin and Cystathionine β-synthase (CBS) domain divalent metal cation transport mediators”, CNNMs, are the least-studied mammalian magnesium transport mediators. CNNM4 is abundant in the brain and the intestinal tract, and its abnormal activity causes Jalili Syndrome. Recent findings show that suppression of CNNM4 in mice promotes malignant progression of intestinal polyps and is linked to infertility. The association of CNNM4 with phosphatases of the regenerating liver, PRLs, abrogates its Mg²⁺-efflux capacity, thus resulting in an increased intracellular Mg²⁺ concentration that favors tumor growth. Here we present the crystal structures of the two independent intracellular domains of human CNNM4, i.e., the Bateman module and the cyclic nucleotide binding-like domain (cNMP). We also derive a model structure for the full intracellular region in the absence and presence of MgATP and the oncogenic interacting partner, PRL-1. We find that only the Bateman module interacts with ATP and Mg²⁺, at non-overlapping sites facilitating their positive cooperativity. Furthermore, both domains dimerize autonomously, where the cNMP domain dimer forms a rigid cleft to restrict the Mg²⁺ induced sliding of the inserting CBS1 motives of the Bateman module, from a twisted to a flat disk shaped dimer. Full article

(This article belongs to the Special Issue Structure and Function of Membrane Proteins)

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14 pages, 1465 KiB

Open AccessArticle

Identifying Common Features in the Activation of Melanocortin-2 Receptors: Studies on the Xenopus tropicalis Melanocortin-2 Receptor

by Perry E. Davis, Emily C. Wilkinson and Robert M. Dores

Int. J. Mol. Sci. 2019, 20(17), 4166; https://doi.org/10.3390/ijms20174166 - 26 Aug 2019

Cited by 6 | Viewed by 2328

Abstract

The interaction between the pituitary hormone, adrenocorticotropin (ACTH), and melanocortin-2 receptor (MC2R) orthologs involves the H6 F7 R8 W9 and R/K15 K16 R17 R18 motifs in ACTH making contact with corresponding contact sites on MC2R. Earlier studies have localized the common HFRW binding [...] Read more.

The interaction between the pituitary hormone, adrenocorticotropin (ACTH), and melanocortin-2 receptor (MC2R) orthologs involves the H6 F7 R8 W9 and R/K15 K16 R17 R18 motifs in ACTH making contact with corresponding contact sites on MC2R. Earlier studies have localized the common HFRW binding site of all melanocortin receptors to residues in TM2, TM3, and TM6 that are located close to the extracellular space. The current study has identified residues in Xenopus tropicalis (xt) MC2R in TM4 (I158, F161), in EC2 (M166), and in TM5 (V172) that also are involved in activation of xtMC2R, and may be in the R/KKRR contact site of xtMC2R. These results are compared to earlier studies on the corresponding domains of human MC2R and rainbow trout MC2R in an effort to identify common features in the activation of teleost and tetrapod MC2R orthologs following stimulation with ACTH. Full article

(This article belongs to the Special Issue Structure and Function of Membrane Proteins)

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14 pages, 3157 KiB

Open AccessArticle

Simultaneous Ligand and Receptor Tracking through NMR Spectroscopy Enabled by Distinct ¹⁹F Labels

by Jeffrey R. Simmons, Alexandre Murza, Michael D. Lumsden, Calem Kenward, Éric Marsault and Jan K. Rainey

Int. J. Mol. Sci. 2019, 20(15), 3658; https://doi.org/10.3390/ijms20153658 - 26 Jul 2019

Cited by 7 | Viewed by 4291

Abstract

To probe ligand-receptor binding at the atomic-level, a frequent approach involves multidimensional nuclear magnetic resonance (NMR) spectroscopy experiments relying on ¹³C- and/or ¹⁵N-enrichment alongside ¹H. Alternatively, the lack of fluorine in biomolecules may be exploited through specific incorporation of ¹⁹ [...] Read more.

To probe ligand-receptor binding at the atomic-level, a frequent approach involves multidimensional nuclear magnetic resonance (NMR) spectroscopy experiments relying on ¹³C- and/or ¹⁵N-enrichment alongside ¹H. Alternatively, the lack of fluorine in biomolecules may be exploited through specific incorporation of ¹⁹F nuclei into a sample. The ¹⁹F nucleus is highly sensitive to environmental changes and allows for one-dimensional NMR spectroscopic study, with perturbation to chemical shift and spin dynamics diagnostic of structural change, ligand binding, and modified conformational sampling. This was applied to the apelinergic system, which comprises a rhodopsin-like G protein-coupled receptor (the apelin receptor (AR)/APJ) and two families of cognate ligands, the apelin and apela (ELABELA/toddler) peptides. Specifically, AR fragments consisting of either the N-terminal tail and first transmembrane (TM) α-helix (AR55) or the first three transmembrane α-helices (TM1-3) were prepared with biosynthetic fluorotryptophan incorporation. Interactions of each AR fragment with a high-affinity, 2,4,5-trifluorophenylalanine labeled apelin analogue were compared by ¹⁹F NMR. Distinct ranges of ¹⁹F chemical shifts for ligand and receptor provide unambiguous tracking of both species, with distinct binding behaviour observed for each AR fragment implying that AR55 is not sufficient to recapitulate the physiological binding event. Site-specific perturbation was also apparent for the apelin analogue as a function of substitution site, indicating an orientational binding preference. As a whole, this strategy of distinctive ¹⁹F labelling for ligand and receptor provides a relatively fast (i.e., employing 1D NMR experiments) and highly sensitive method to simultaneously and definitively track binding in both species. Full article

(This article belongs to the Special Issue Structure and Function of Membrane Proteins)

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Review

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15 pages, 3569 KiB

Open AccessReview

Structure and Stoichiometry of the Ton Molecular Motor

by Herve Celia, Nicholas Noinaj and Susan K Buchanan

Int. J. Mol. Sci. 2020, 21(2), 375; https://doi.org/10.3390/ijms21020375 - 7 Jan 2020

Cited by 31 | Viewed by 6444

Abstract

The Ton complex is a molecular motor that uses the proton gradient at the inner membrane of Gram-negative bacteria to generate force and movement, which are transmitted to transporters at the outer membrane, allowing the entry of nutrients into the periplasmic space. Despite [...] Read more.

The Ton complex is a molecular motor that uses the proton gradient at the inner membrane of Gram-negative bacteria to generate force and movement, which are transmitted to transporters at the outer membrane, allowing the entry of nutrients into the periplasmic space. Despite decades of investigation and the recent flurry of structures being reported by X-ray crystallography and cryoEM, the mode of action of the Ton molecular motor has remained elusive, and the precise stoichiometry of its subunits is still a matter of debate. This review summarizes the latest findings on the Ton system by presenting the recently reported structures and related reports on the stoichiometry of the fully assembled complex. Full article

(This article belongs to the Special Issue Structure and Function of Membrane Proteins)

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26 pages, 6056 KiB

Open AccessReview

Selection of Biophysical Methods for Characterisation of Membrane Proteins

by Tristan O. C. Kwan, Rosana Reis, Giuliano Siligardi, Rohanah Hussain, Harish Cheruvara and Isabel Moraes

Int. J. Mol. Sci. 2019, 20(10), 2605; https://doi.org/10.3390/ijms20102605 - 27 May 2019

Cited by 25 | Viewed by 9196

Abstract

Over the years, there have been many developments and advances in the field of integral membrane protein research. As important pharmaceutical targets, it is paramount to understand the mechanisms of action that govern their structure–function relationships. However, the study of integral membrane proteins [...] Read more.

Over the years, there have been many developments and advances in the field of integral membrane protein research. As important pharmaceutical targets, it is paramount to understand the mechanisms of action that govern their structure–function relationships. However, the study of integral membrane proteins is still incredibly challenging, mostly due to their low expression and instability once extracted from the native biological membrane. Nevertheless, milligrams of pure, stable, and functional protein are always required for biochemical and structural studies. Many modern biophysical tools are available today that provide critical information regarding to the characterisation and behaviour of integral membrane proteins in solution. These biophysical approaches play an important role in both basic research and in early-stage drug discovery processes. In this review, it is not our objective to present a comprehensive list of all existing biophysical methods, but a selection of the most useful and easily applied to basic integral membrane protein research. Full article

(This article belongs to the Special Issue Structure and Function of Membrane Proteins)

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13 pages, 1496 KiB

Open AccessReview

Physiological, Structural, and Functional Analysis of the Paralogous Cation–Proton Antiporters of NhaP Type from Vibrio cholerae

by Muntahi Mourin, Alvan Wai, Joe O’Neil, Georg Hausner and Pavel Dibrov

Int. J. Mol. Sci. 2019, 20(10), 2572; https://doi.org/10.3390/ijms20102572 - 25 May 2019

Cited by 9 | Viewed by 3442

Abstract

The transmembrane K⁺/H⁺ antiporters of NhaP type of Vibrio cholerae (Vc-NhaP1, 2, and 3) are critical for maintenance of K⁺ homeostasis in the cytoplasm. The entire functional NhaP group is indispensable for the survival of V. cholerae at low [...] Read more.

The transmembrane K⁺/H⁺ antiporters of NhaP type of Vibrio cholerae (Vc-NhaP1, 2, and 3) are critical for maintenance of K⁺ homeostasis in the cytoplasm. The entire functional NhaP group is indispensable for the survival of V. cholerae at low pHs suggesting their possible role in the acid tolerance response (ATR) of V. cholerae. Our findings suggest that the Vc-NhaP123 group, and especially its major component, Vc-NhaP2, might be a promising target for the development of novel antimicrobials by narrowly targeting V. cholerae and other NhaP-expressing pathogens. On the basis of Vc-NhaP2 in silico structure modeling, Molecular Dynamics Simulations, and extensive mutagenesis studies, we suggest that the ion-motive module of Vc-NhaP2 is comprised of two functional regions: (i) a putative cation-binding pocket that is formed by antiparallel unfolded regions of two transmembrane segments (TMSs V/XII) crossing each other in the middle of the membrane, known as the NhaA fold; and (ii) a cluster of amino acids determining the ion selectivity. Full article

(This article belongs to the Special Issue Structure and Function of Membrane Proteins)

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19 pages, 1881 KiB

Open AccessReview

Structural and Functional Changes in the Na⁺/H⁺ Exchanger Isoform 1, Induced by Erk1/2 Phosphorylation

by Larry Fliegel

Int. J. Mol. Sci. 2019, 20(10), 2378; https://doi.org/10.3390/ijms20102378 - 14 May 2019

Cited by 34 | Viewed by 7733

Abstract

The human Na⁺/H⁺ exchanger isoform 1 (NHE1) is a plasma membrane transport protein that plays an important role in pH regulation in mammalian cells. Because of the generation of protons by intermediary metabolism as well as the negative membrane potential, [...] Read more.

The human Na⁺/H⁺ exchanger isoform 1 (NHE1) is a plasma membrane transport protein that plays an important role in pH regulation in mammalian cells. Because of the generation of protons by intermediary metabolism as well as the negative membrane potential, protons accumulate within the cytosol. Extracellular signal-regulated kinase (ERK)-mediated regulation of NHE1 is important in several human pathologies including in the myocardium in heart disease, as well as in breast cancer as a trigger for growth and metastasis. NHE1 has a N-terminal, a 500 amino acid membrane domain, and a C-terminal 315 amino acid cytosolic domain. The C-terminal domain regulates the membrane domain and its effects on transport are modified by protein binding and phosphorylation. Here, we discuss the physiological regulation of NHE1 by ERK, with an emphasis on the critical effects on structure and function. ERK binds directly to the cytosolic domain at specific binding domains. ERK also phosphorylates NHE1 directly at multiple sites, which enhance NHE1 activity with subsequent downstream physiological effects. The NHE1 cytosolic regulatory tail possesses both ordered and disordered regions, and the disordered regions are stabilized by ERK-mediated phosphorylation at a phosphorylation motif. Overall, ERK pathway mediated phosphorylation modulates the NHE1 tail, and affects the activity, structure, and function of this membrane protein. Full article

(This article belongs to the Special Issue Structure and Function of Membrane Proteins)

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