The HAB1 PP2C is inhibited by ABA-dependent PYL10 interaction.

PYL10 is a monomeric abscisic acid (ABA) receptor that inhibits protein phosphatase 2C (PP2C) activity in Arabidopsis thaliana. Previous studies reported that the PP2C phosphatase inhibition by PYL10 was ABA-independent. Here, systematic PYL10 biochemical studies demonstrated that PYL10 activity was ABA-dependent, and the previously reported studies was interfered by the presence of BSA in the commercial kit. To investigate dynamic mechanism of how ABA binding to PYL10 induces PP2C phosphatase inhibiting activity, solution NMR relaxation analysis of apo-PYL10 and PYL10/ABA were conducted following backbone resonance assignments. Reduced spectrum density mapping of the backbone relaxation data revealed that PYL10 was more flexible in ABA bound form than apo-PYL10, indicating an increased conformational entropy upon ligand binding. Moreover, to illustrate conformation exchanges of PYL10 upon ABA binding, NMR line shape analysis was performed with increasing concentrations of ABA, and the results indicated that PYL10 backbone conformational changes occur at different time scales.

CW-EPR studies revealed different motional properties and oligomeric states of the integrin ß1a transmembrane domain in detergent micelles or liposomes.

Integrins are heterodimeric membrane proteins that regulate essential processes: cell migration, cell growth, extracellular matrix assembly and tumor metastasis. Each integrin α or ß subunit contains a large extracellular domain, a single transmembrane (TM) domain, and a short cytoplasmic tail. The integrin TM domains are important for heterodimeric association and dissociation during the conversion from inactive to active states. Moreover, integrin clustering occurs by homo-oligomeric interactions between the TM helices. Here, the transmembrane and cytoplasmic (TMC) domains of integrin ß1a were overexpressed, and the protein was purified in detergent micelles and/or reconstituted in liposomes. To investigate the TM domain conformational properties of integrin ß1a, 26 consecutive single cysteine mutants were generated for site-directed spin labeling and continuous-wave electron paramagnetic resonance (CW-EPR) mobility and accessibility analyses. The mobility analysis identified two integrin ß1a-TM regions with different motional properties in micelles and a non-continuous integrin ß1a-TM helix with high immobility in liposomes. The accessibility analysis verified the TM range (Val737-Lys752) of the integrin ß1a-TMC in micelles. Further mobility and accessibility comparisons of the integrin ß1a-TMC domains in micelles or liposomes identified distinctively different oligomeric states of integrin ß1a-TM, namely a monomer embedded in detergent micelles and leucine-zipper-like homo-oligomeric clusters in liposomes.

Solution NMR of MPS-1 reveals a random coil cytosolic domain structure.

Caenorhabditis elegans MPS1 is a single transmembrane helical auxiliary subunit that co-localizes with the voltage-gated potassium channel KVS1 in the nematode nervous system. MPS-1 shares high homology with KCNE (potassium voltage-gated channel subfamily E member) auxiliary subunits, and its cytosolic domain was reported to have a serine/threonine kinase activity that modulates KVS1 channel function via phosphorylation. In this study, NMR spectroscopy indicated that the full length and truncated MPS-1 cytosolic domain (134-256) in the presence or absence of n-dodecylphosphocholine detergent micelles adopted a highly flexible random coil secondary structure. In contrast, protein kinases usually adopt a stable folded conformation in order to implement substrate recognition and phosphoryl transfer. The highly flexible random coil secondary structure suggests that MPS-1 in the free state is unstructured but may require a substrate or binding partner to adopt stable structure required for serine/threonine kinase activity.

Differential modulations of KCNQ1 by auxiliary proteins KCNE1 and KCNE2.

KCNQ1 channels play vital roles in cardiovascular, gastric and other systems. The conductance and dynamics of KCNQ1 could be modulated by different single transmembrane helical auxiliary proteins (such as KCNE1, KCNE2 and others). In this study, detail KCNQ1 function modulations by different regions of KCNE1 or KCNE2 were examined using combinational methods of electrophysiology, immunofluorescence, solution NMR and related backbone flexibility analysis. In the presence of KCNE2 N-terminus, decreased surface expression and consequent low activities of KCNQ1 were observed. The transmembrane domains (TMDs) of KCNE1 and KCNE2 were illustrated to associate with the KCNQ1 channel in different modes: Ile64 in KCNE2-TMD interacting with Phe340 and Phe275 in KCNQ1, while two pairs of interacting residues (Phe340-Thr58 and Ala244-Tyr65) in the KCNQ1/KCNE1 complex. The KCNE1 C-terminus could modulate gating property of KCNQ1, whereas KCNE2 C-terminus had only minimal influences on KCNQ1. All of the results demonstrated different KCNQ1 function modulations by different regions of the two auxiliary proteins.

Intracellular segment between transmembrane helices S0 and S1 of BK channel α subunit contains two amphipathic helices connected by a flexible loop.

The BK channel, a tetrameric potassium channel with very high conductance, has a central role in numerous physiological functions. The BK channel can be activated by intracellular Ca(2+) and Mg(2+), as well as by membrane depolarization. Unlike other tetrameric potassium channels, the BK channel has seven transmembrane helices (S0-S6) including an extra helix S0. The intracellular segment between S0 and S1 (BK-IS1) is essential to BK channel functions and Asp99 in BK-IS1 is reported to be responsible for Mg(2+) coordination. In this study, BK-IS1 (44-113) was over-expressed using a bacterial system and purified in the presence of detergent micelles for multidimensional heteronuclear nuclear magnetic resonance (NMR) structural studies. Backbone resonance assignment and secondary structure analysis showed that BK-IS1 contains two amphipathic helices connected by a 36-residue loop. Amide (1)H-(15)N heteronuclear NOE analysis indicated that the loop is very flexible, while the two amphipathic helices are possibly stabilized through interaction with the membrane. A solution NMR-based titration assay of BK-IS1 was performed with various concentrations of Mg(2+). Two residues (Thr45 and Leu46) with chemical shift changes were observed but no, or very minor, chemical shift difference was observed for Asp99, indicating a possible site for binding divalent ions or other modulation partners.

Integrin α1 has a long helix, extending from the transmembrane region to the cytoplasmic tail in detergent micelles.

Integrin proteins are very important adhesion receptors that mediate cell-cell and cell-extracellular matrix interactions. They play essential roles in cell signaling and the regulation of cellular shape, motility, and the cell cycle. Here, the transmembrane and cytoplasmic (TMC) domains of integrin α1 and ß1 were over-expressed and purified in detergent micelles. The structure and backbone relaxations of α1-TMC in LDAO micelles were determined and analyzed using solution NMR. A long helix, extending from the transmembrane region to the cytoplasmic tail, was observed in α1-TMC. Structural comparisons of α1-TMC with reported αIIb-TMC domains indicated different conformations in the transmembrane regions and cytoplasmic tails. An NMR titration experiment indicated weak interactions between α1-TMC and ß1-TMC through several α1-TMC residues located at its N-terminal juxta-transmembrane region and C-terminal extended helix region.

Structural studies of Mycobacterium tuberculosis Rv0899 reveal a monomeric membrane-anchoring protein with two separate domains.

Rv0899 from Mycobacterium tuberculosis belongs to the OmpA (outer membrane protein A) family of outer membrane proteins. It functions as a pore-forming protein; the deletion of this gene impairs the uptake of some water-soluble substances, such as serine, glucose, and glycerol. Rv0899 has also been shown to play a part in low-pH environment adaption, which may play a part in pathogenic mycobacteria overcoming the host's defense mechanisms. Based on many bacterial physiological data and recent structural studies, it was proposed that Rv0899 forms an oligomeric channel to carry out such functions. In this work, biochemical and structural data obtained from solution NMR and EPR spectroscopy indicated that Rv0899 is a monomeric membrane-anchoring protein with two separate domains, rather than an oligomeric pore. Using NMR chemical shift perturbation and isothermal calorimetric titration assays, we show that Rv0899 was able to interact with Zn(2+) ions, which may indicate a role for Rv0899 in the process of Zn(2+) acquisition.

Expression, purification, detergent screening and solution NMR backbone assignment of the human potassium channel accessory subunit MiRP1.

MiRP1 (MinK related protein 1) is a membrane protein in the KCNE family. It can associate with and modulate various voltage gated potassium channels. Mutations in human MiRP1 have been found to cause many congenital and acquired long QT syndromes, which are potentially life-threatening cardiac arrhythmias. Here, human MiRP1 was over-expressed in Escherichia coli, purified and eluted into different detergents. Two dimensional (1)H-(15)N correlated solution nuclear magnetic resonance (NMR) spectra of the human MiRP1 in four different detergent micelles indicated that high resolution solution NMR spectrum can be obtained for human MiRP1 in detergent lyso-myristoylphosphatidylglycerol (LMPG). Circular dichroism (CD) spectroscopy of human MiRP1 indicated a high content of alpha-helical secondary structure in LMPG. Backbone assignments of most MiRP1 residues were achieved through a series of triple resonance NMR experiments. Secondary structure analysis based on backbone chemical shifts showed several stretches of alpha-helices along the primary sequence of MiRP1 in LMPG.

Solution NMR characterization of Sgf73(1-104) indicates that Zn ion is required to stabilize zinc finger motif.

Zinc finger motif contains a zinc ion coordinated by several conserved amino acid residues. Yeast Sgf73 protein was identified as a component of SAGA (Spt/Ada/Gcn5 acetyltransferase) multi-subunit complex and Sgf73 protein was known to contain two zinc finger motifs. Sgf73(1-104), containing the first zinc finger motif, was necessary to modulate the deubiquitinase activity of SAGA complex. Here, Sgf73(1-104) was over-expressed using bacterial expression system and purified for solution NMR (nuclear magnetic resonance) structural studies. Secondary structure and site-specific relaxation analysis of Sgf73(1-104) were achieved after solution NMR backbone assignment. Solution NMR and circular dichroism analysis of Sgf73(1-104) after zinc ion removal using chelation reagent EDTA (ethylene-diamine-tetraacetic acid) demonstrated that zinc ion was required to maintain stable conformation of the zinc finger motif.