Leucine-isoleucine Zipper Research Articles

Hendra virus fusion protein transmembrane domain contributes to pre-fusion protein stability

Enveloped viruses utilize fusion (F) proteins studding the surface of the virus to facilitate membrane fusion with a target cell membrane. Fusion of the viral envelope with a cellular membrane is required for release of viral genomic material, so the virus can ultimately reproduce and spread. To drive fusion, the F protein undergoes an irreversible conformational change, transitioning from a metastable pre-fusion conformation to a more thermodynamically stable post-fusion structure. Understanding the elements that control stability of the pre-fusion state and triggering to the post-fusion conformation is important for understanding F protein function. Mutations in F protein transmembrane (TM) domains implicated the TM domain in the fusion process, but the structural and molecular details in fusion remain unclear. Previously, analytical ultracentrifugation was utilized to demonstrate that isolated TM domains of Hendra virus F protein associate in a monomer-trimer equilibrium (Smith, E. C., Smith, S. E., Carter, J. R., Webb, S. R., Gibson, K. M., Hellman, L. M., Fried, M. G., and Dutch, R. E. (2013) J. Biol. Chem. 288, 35726-35735). To determine factors driving this association, 140 paramyxovirus F protein TM domain sequences were analyzed. A heptad repeat of β-branched residues was found, and analysis of the Hendra virus F TM domain revealed a heptad repeat leucine-isoleucine zipper motif (LIZ). Replacement of the LIZ with alanine resulted in dramatically reduced TM-TM association. Mutation of the LIZ in the whole protein resulted in decreased protein stability, including pre-fusion conformation stability. Together, our data suggest that the heptad repeat LIZ contributed to TM-TM association and is important for F protein function and pre-fusion stability.

Fusion Protein TM-TM Interactions: Modulators of Pre-Fusion Protein Stability

Membrane associated domains of proteins are often under-studied because of their lipid environment, however recent studies suggest that protein transmembrane domains (TMDs) are important for cell signaling events, and protein oligomerization. Enveloped viruses utilize fusion proteins (F) studding the envelope of the virus to promote fusion of the viral envelope with a target cell membrane. To drive fusion, the F proteins undergo large conformational changes, transitioning from a meta-stable pre-fusion conformation to a more thermodynamically stable post-fusion conformation; understanding the elements which control stability of the pre-fusion state and triggering to the post-fusion conformation is important for understanding F protein function. Mutations in F protein TMDs have implicated the TMD in the fusion process, but the structural and molecular details of the role of TMDs in fusion remains unclear. We previously utilized analytical ultracentrifugation to demonstrate that isolated TMDs of Hendra virus F associate in a monomer-trimer equilibrium. To determine elements critical for this association, we examined the Hendra virus F sequence. Directly upstream of the TMDs is a heptad repeat domain that contains a leucine-isoleucine zipper (LIZ) which continues in frame into the TMD. We found that a TMD replacing four L/I residues with alanine dramatically reduced TM-TM association, implicating the LIZ in TM-TM interactions. Studies with the LIZ mutations in the whole protein indicate decreased protein stability, suggesting TM-TM interactions promoted by the LIZ may be critical for pre-fusion stability. Previous studies demonstrated that the pre-fusion conformation of F can be thermally triggered to its post-fusion conformation. We used this characteristic of F to demonstrate that reduced TM-TM association in the LIZ F mutant results in reduced stability of F in its pre-fusion conformation. Together our data indicate that the TMD is important in F protein stability and triggering.

Asymmetric Methyl Group Labeling as a Probe of Membrane Protein Homo-oligomers by NMR Spectroscopy

We present an asymmetric isotopic labeling strategy for probing unambiguously membrane protein binding interfaces in homo-oligomers. This technique is highly sensitive, rapid, and requires only one 13C methyl-edited nuclear Overhauser effect spectroscopy (NOESY) spectrum. As a proof of principle, we apply this technique to the homo-pentameric membrane protein phospholamban, defining the leucine-isoleucine zipper interface between its monomers with atomistic resolution.

CGMP-dependent Protein Kinase Type I Inhibits TAB1-p38 Mitogen-activated Protein Kinase Apoptosis Signaling in Cardiac Myocytes

Cardiac myocyte apoptosis during ischemia and reperfusion (I/R) is tightly controlled by a complex network of stress-responsive signaling pathways. One pro-apoptotic pathway involves the interaction of the scaffold protein TAB1 with p38 mitogen-activated protein kinase (p38 MAPK) leading to the autophosphorylation and activation of p38 MAPK. Conversely, NO and its second messenger cGMP protect cardiac myocytes from apoptosis during I/R. We provide evidence that the cGMP target cGMP-dependent protein kinase type I (PKG I) interferes with TAB1-p38 MAPK signaling to protect cardiac myocytes from I/R injury. In isolated neonatal cardiac myocytes, activation of PKG I inhibited the interaction of TAB1 with p38 MAPK, p38 MAPK phosphorylation, and apoptosis induced by simulated I/R. During I/R in vivo, mice with a cardiac myocyte-restricted deletion of PKG I displayed a more pronounced interaction of TAB1 with p38 MAPK and a stronger phosphorylation of p38 MAPK in the myocardial area at risk during reperfusion and more apoptotic cardiac myocytes in the infarct border zone as compared with wild-type littermates. Notably, adenoviral expression of a constitutively active PKG I mutant truncated at the N terminus(PKGI-DeltaN1-92) did not inhibit p38 MAPK phosphorylation and apoptosis induced by simulated I/R in vitro, indicating that the N terminus of PKG I is required. As shown by co-immunoprecipitation experiments in HEK293 cells, cGMP-activated PKG I, but not constitutively active PKG I-DeltaN1-92 or PKG I mutants carrying point mutations in the N-terminal leucine-isoleucine zipper, interacted with p38 MAPK, and prevented the binding of TAB1 to p38 MAPK. Together, our data identify a novel interaction between the cGMP target PKG I and the TAB1-p38 MAPK signaling pathway that serves as a defense mechanism against myocardial I/R injury.

Association of Type 1 Inositol 1,4,5-Trisphosphate Receptor with AKAP9 (Yotiao) and Protein Kinase A

Inositol 1,4,5-trisphosphate receptors (InsP(3)R) play a key role in intracellular calcium (Ca(2+)) signaling. Three InsP(3)R isoforms are expressed in mammals. Type 1 InsP(3)R (InsP(3)R1) is a predominant neuronal isoform. Neuronal InsP(3)R1 is one of the major substrates of protein kinase A (PKA) phosphorylation. In our previous study (Tang, T. S., Tu, H., Wang, Z., and Bezprozvanny, I. (2003) J. Neurosci. 23, 403-415) we discovered a direct association between InsP(3)R1 and protein phosphatase 1 alpha (PP1 alpha). In functional experiments we demonstrated that phosphorylation by PKA activates InsP(3)R1 and that dephosphorylation by PP1 alpha inhibits InsP(3)R1. To extend these findings, here we investigated the possibility of InsP(3)R1-PKA association. In a series of biochemical experiments we demonstrate the following findings. 1) InsP(3)R1 and PKA associate in the brain. 2) InsP(3)R1-PKA association is mediated by the AKAP9 (Yotiao) multi-functional PKA anchoring protein. 3) InsP(3)R1-AKAP9 association is mediated via the leucine/isoleucine zipper (LIZ) motif in the InsP(3)R1 coupling domain and the fourth LIZ motif in AKAP9. 4) The InsP(3)R association with AKAP9 is specific for type 1 InsP(3)R. 5) Both the SII(+) and the SII(-) coupling domain splice variants of InsP(3)R1 bind to AKAP9. 6) Binding to AKAP9 promotes association of neuronal InsP(3)R1 with the NR1 NMDA receptor; and 7) neuronal InsP(3)R1 associate with PP1 directly via carboxy-terminus and indirectly via AKAP9. The obtained results advance our understanding of cross-talk between cAMP and InsP(3)/Ca(2+) signaling pathways in the brain.

Sequence, gene structure, and expression pattern of CTNNBL1, a minor-class intron-containing gene—evidence for a role in apoptosis

We have identified and characterized a cDNA designated CTNNBL1 (catenin (cadherin-associated protein), β-like 1) coding for a protein of 563 amino acids having predicted structural homology to β-catenin and other armadillo (arm) family proteins. CTNNBL1 is expressed in multiple human tissues, and its sequence is conserved across widely divergent species. The human CTNNBL1 gene on chromosome 20q11.2 contains 16 exons spanning > 178 kb. Intron 4 is a minor-class intron bearing AT at the 5′ splice site and AC at the 3′ splice site. An acidic domain, as well as a putative bipartite nuclear localization signal, a nuclear export signal, a leucine-isoleucine zipper, and phosphorylation motifs are present in the protein sequence. Transient expression of CTNNBL1 in CHO cells results in localization to the nucleus and apoptosis. The rate of cell death was higher when cells were transfected with a carboxy-terminal fragment of CTNNBL1, suggesting that the apoptosis-inducing activity is a function of this region.

Zipping in the Regulators

Marx et al. show that the leucine/isoleucine zipper (LZ) protein motif found in several ion channels appears to mediate interaction of the channels with key regulatory proteins. They characterized the role of LZ motifs in ryanodine receptors (RyRs), which are ligand-controlled ion channels essential for proper excitation-contraction coupling in muscle. The activity of RyRs is regulated by phosphorylation, which modulates the channel properties of these receptors, and increased phosphorylation of RyR2 (the cardiac form of RyRs) is associated with heart failure. RyR2 has three LZ domains, each of which interacted with a distinct cellular regulator: LZ1 conferred binding of protein phosphatase 1 (PP1) through interaction with the PP1-targeting protein spinophilin, LZ2 conferred binding of PP2A through the PP2A-targeting protein PR130, and LZ3 mediated binding of the cyclic AMP-dependent protein kinase through the targeting protein mAKAP (also known as A-kinase anchoring protein). Each of the three targeting proteins also has LZ domains, which are required for specific interactions with LZ domains of RyR2. Disruption of the LZ-mediated interactions by incubation of receptor complexes with fusion proteins containing the LZ domains of RyR2 resulted in changes in RyR channel activity and altered regulation by phosphorylation. The identification of LZ-mediated interactions enabled Marks et al. to predict kinase and phosphatase interactions with RyR1 and, thus, may facilitate identification of components in other channel complexes and, thus, may expose other potential targets for therapeutic intervention. S. O. Marx, S. Reiken, Y. Hisamatsu, M. Gaburjakova, J. Gaburjakova, Y.-M. Yang, N. Rosemblit, A. R. Marks, Phosphorylation-dependent regulation of ryanodine receptors: A novel role for leucine/isoleucine zippers. J. Cell Biol. 153 , 699-708 (2001). [Abstract] [Full Text]

Phosphorylation-dependent regulation of ryanodine receptors: a novel role for leucine/isoleucine zippers.

Ryanodine receptors (RyRs), intracellular calcium release channels required for cardiac and skeletal muscle contraction, are macromolecular complexes that include kinases and phosphatases. Phosphorylation/dephosphorylation plays a key role in regulating the function of many ion channels, including RyRs. However, the mechanism by which kinases and phosphatases are targeted to ion channels is not well understood. We have identified a novel mechanism involved in the formation of ion channel macromolecular complexes: kinase and phosphatase targeting proteins binding to ion channels via leucine/isoleucine zipper (LZ) motifs. Activation of kinases and phosphatases bound to RyR2 via LZs regulates phosphorylation of the channel, and disruption of kinase binding via LZ motifs prevents phosphorylation of RyR2. Elucidation of this new role for LZs in ion channel macromolecular complexes now permits: (a) rapid mapping of kinase and phosphatase targeting protein binding sites on ion channels; (b) predicting which kinases and phosphatases are likely to regulate a given ion channel; (c) rapid identification of novel kinase and phosphatase targeting proteins; and (d) tools for dissecting the role of kinases and phosphatases as modulators of ion channel function.

Structure of the 1–36 Amino-Terminal Fragment of Human Phospholamban by Nuclear Magnetic Resonance and Modeling of the Phospholamban Pentamer

The structure of a 36-amino-acid-long amino-terminal fragment of phospholamban (phospholamban[1–36]) in aqueous solution containing 30% trifluoroethanol was determined by nuclear magnetic resonance. The peptide, which comprises the cytoplasmic domain and six residues of the transmembrane domain of phospholamban, assumes a conformation characterized by two α-helices connected by a turn. The residues of the turn are Ile18, Glu19, Met20, and Pro21, which are adjacent to the two phosphorylation sites Ser16 and Thr17. The proline is in a trans conformation. The helix comprising amino acids 22–36 is well determined (the root mean square deviation for the backbone atoms, calculated for a family of 18 nuclear magnetic resonance structures is 0.57 Å). Recently, two molecular models of the transmembrane domain of phospholamban were proposed in which a symmetric homopentamer is composed of a left-handed coiled coil of α-helices. The two models differ by the relative orientation of the helices. The model proposed by Simmerman et al. (H.K. Simmerman, Y.M. Kobayashi, J.M. Autry, and L.R. Jones, 1996, J. Biol. Chem. 271:5941–5946), in which the coiled coil is stabilized by a leucine-isoleucine zipper, is similar to the transmembrane pentamer structure of the cartilage oligomeric membrane protein determined recently by x-ray (V. Malashkevich, R. Kammerer, V. Efimov, T. Schulthess, and J. Engel, 1996, Science 274:761–765). In the model proposed by Adams et al. (P.D. Adams, I.T. Arkin, D.M. Engelman, and A.T. Brunger, 1995, Nature Struct. Biol. 2:154–162), the helices in the coiled coil have a different relative orientation, i.e., are rotated clockwise by ∼50°. It was possible to overlap and connect the structure of phospholamban[1–36] derived in the present study to the two transmembrane pentamer models proposed. In this way two models of the whole phospholamban in its pentameric form were generated. When our structure was connected to the leucine-isoleucine zipper model, the inner side of the cytoplasmic domain of the pentamer (where the helices face one another) was lined by polar residues (Gln23, Gln26, and Asn30), whereas the five Arg25 side chains were on the outer side. On the contrary, when our structure was connected to the other transmembrane model, in the inner side of the cytoplasmic domain of the pentamer, the five Arg25 residues formed a highly charged cluster.

Dimerization via Tandem Leucine Zippers Is Essential for the Activation of the Mitogen-activated Protein Kinase Kinase Kinase, MLK-3

Mixed lineage kinase-3 (MLK-3) is a mitogen-activated kinase kinase kinase that mediates stress-activating protein kinase (SAPK)/c-Jun NH2-terminal kinase activation. MLK-3 and other MLK family kinases are characterized by the presence of multiple protein-protein interaction domains including a tandem leucine/isoleucine zipper (LZs) motif. Leucine zippers are known to mediate protein dimerization raising the possibility that the tandem leucine/isoleucine zippers may function as a dimerization motif of MLK-3. Using both co-immunoprecipitation and nonreducing SDS-polyacrylamide gel electrophoresis, we demonstrated that MLK-3 forms disulfide bridged homo-dimers and that the LZs motif is sufficient for MLK-3 homodimerization. We next asked whether MLK-3 utilizes a dimerization-based activation mechanism analogous to that of receptor tyrosine kinases. We found that dimerization via the LZs motif is a prerequisite for MLK-3 autophosphorylation. We then demonstrated that co-expression of Cdc42 lead to a substantial increase in MLK-3 dimerization, indicating that binding by this GTPase may induce MLK-3 dimerization. Moreover, the LZs minus form of MLK-3 failed to activate the downstream target SAPK, and expression of a MLK-3 LZs polypeptide was found to block SAPK activation by wild type MLK-3. Taken together, these findings indicate that dimerization plays a pivotal role in MLK-3 activation.

Direct spectroscopic detection of molecular dynamics and interactions of the calcium pump and phospholamban.

In order to test molecular models of cardiac calcium transport regulation, we have used spectroscopy to probe the structures, dynamics, and interactions of the Ca pump (Ca-ATPase) and phospholamban (PLB) in cardiac sarcoplasmic reticulum (SR) and in reconstituted membranes. Electron paramagnetic resonance (EPR) and phosphorescence of probes bound to the Ca pump show that the activity of the pump is quite sensitive to its oligomeric interactions. In cardiac SR, PLB aggregates and inhibits the pump, and both effects are reversed by PLB phosphorylation. Previous analyses of PLB's oligomeric state were only in detergent solutions, so we used EPR and fluorescence to determine the oligomeric structure of PLB in its native state in lipid bilayers. Wild-type PLB is primarily oligomeric in the membrane, while the mutant L37A-PLB is monomeric. For both proteins, phosphorylation shifts the dynamic monomer-oligomer equilibrium toward oligomers, and induces a similar structural change, as indicated by tyrosine fluorescence; yet L37A-PLB is more effective than wild-type PLB in inhibiting and aggregating the pump. Fluorescence energy transfer shows that the Ca pump increases the fraction of monomeric PLB, indicating that the pump preferentially binds monomeric PLB. These results support a reciprocal aggregation model for Ca pump regulation, in which the Ca pump is aggregated and inhibited by association with PLB monomers, and phosphorylation of PLB reverses these effects while decreasing the concentration of PLB monomers. To investigate the structure of the PLB pentamer in more detail, we measured the reactivities of cysteine residues in the transmembrane domain of PLB, and recorded EPR spectra of spin labels attached to these sites. These results support an atomic structural model, based on molecular dynamics simulations and mutagenesis studies, in which the PLB pentamer is stabilized by a leucine-isoleucine zipper within the transmembrane domain.

Cysteine reactivity and oligomeric structures of phospholamban and its mutants.

To test models for the pentameric structure of phospholamban (PLB) and study its structure and molecular dynamics in SDS solution, we characterized recombinant PLB and several of its mutants by (a) reactivity of cysteine residues toward DTNB [5, 5'-dithiobis(2-nitrobenzoic acid)] and a thiol-reactive spin label, (b) oligomeric state on SDS-PAGE, and (c) EPR of the spin-labeled proteins. WT-PLB has three cysteine residues (36, 41, and 46), all located in the hydrophobic C-terminal transmembrane region. In SDS at pH 7.5, exhaustive reaction with either sulfhydryl reagent resulted in essentially 2 mol of cysteine reacted/mol of WT-PLB, with only slight destabilization of the native pentameric structure. When WT-PLB was denatured in guanidine at pH 8.1, all three cysteines reacted, disrupting the pentamer, which was restored upon cleavage of the disulfide bonds with DTT. In the tetrameric mutant C41L-PLB, the two remaining cysteine residues reacted, reversibly destabilizing the tetramer. In the monomeric mutant L37A-PLB, all three cysteines reacted. The pentameric double cysteine replacement mutant C36,46A-PLB showed negligible reactivity. We conclude that Cys-41 is the unreactive cysteine in PLB and is located at a crucial site for the maintenance of the pentameric structure. EPR spectra in SDS of spin-labeled WT-PLB and mutants correlate with the oligomeric state on SDS-PAGE; oligomeric proteins show decreased spin-label mobility compared with monomers. Molecular dynamics calculations were used to construct an atomic model for the transmembrane region of the PLB pentamer, constrained by previous mutagenesis results and the results of the present study. We conclude that (a) the mobilities of spin-labels attached to PLB and its mutants are sensitive to oligomeric state and (b) the pattern of cysteine reactivity, spin-label mobility, and oligomeric state supports a structural model for the PLB pentamer in which interactions between each pair of subunits are stabilized by a leucine-isoleucine zipper.