A synthetic peptide reproducing the mitochondrial targeting motif of AKAP121: A conformational study (original) (raw)
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The Journal of biological chemistry, 1986
Anionic phospholipids induce a marked conformational change in a synthetic peptide corresponding to residues 1-27 of pre-ornithine carbamyltransferase. The peptide designated, pO-(1-27)-peptide amide, becomes more alpha-helical in the presence of cardiolipin or dimyristoylphosphatidylglycerol but not in the presence of dimyristoylphosphatidylcholine. The greater helix-promoting action of anionic versus zwitterionic lipids is predicted by helix-coil transition theory. This statistical mechanical theory also predicts that a shorter peptide, N-acetyl-pO-(16-27)-peptide amide, has less helix-forming tendency, even in the presence of sodium dodecyl sulfate, despite the fact that it has a comparable number of positive charges. The N-acetyl-pO-(16-27)-peptide amide has no helical structure in buffer with or without dimyristoylphosphatidylglycerol but it has a small (5%) helical content in methanol. Thus, the ability of anionic lipids to promote helix formation requires more than the presen...
Biochemical Journal, 2006
PKA (protein kinase A) is tethered to subcellular compartments by direct interaction of its regulatory subunits (RI or RII) with AKAPs (A kinase-anchoring proteins). AKAPs preferentially bind RII subunits via their RII-binding domains. RII-binding domains form structurally conserved amphipathic helices with unrelated sequences. Their binding affinities for RII subunits differ greatly within the AKAP family. Amongst the AKAPs that bind RIIα subunits with high affinity is AKAP7δ [AKAP18δ; K d (equilibrium dissociation constant) value of 31 nM]. An N-terminally truncated AKAP7δ mutant binds RIIα subunits with higher affinity than the full-length protein presumably due to loss of an inhibitory region [Henn, Edemir, Stefan, Wiesner, Lorenz, Theilig, Schmidtt, Vossebein, Tamma, Beyermann et al. (2004) J. Biol. Chem. 279, 26654-26665]. In the present study, we demonstrate that peptides (25 amino acid residues) derived from the RII-binding domain of AKAP7δ bind RIIα subunits with higher affinity (K d = 0.4 + − 0.3 nM) than either fulllength or N-terminally truncated AKAP7δ, or peptides derived from other RII binding domains. The AKAP7δ-derived peptides and stearate-coupled membrane-permeable mutants effectively disrupt AKAP-RII subunit interactions in vitro and in cell-based assays. Thus they are valuable novel tools for studying anchored PKA signalling. Molecular modelling indicated that the high affinity binding of the amphipathic helix, which forms the RIIbinding domain of AKAP7δ, with RII subunits involves both the hydrophobic and the hydrophilic faces of the helix. Alanine scanning (25 amino acid peptides, SPOT technology, combined with RII overlay assays) of the RII binding domain revealed that hydrophobic amino acid residues form the backbone of the interaction and that hydrogen bond-and salt-bridge-forming amino acid residues increase the affinity of the interaction.
Analysis and prediction of mitochondrial targeting peptides
2001
CHAPTER 10 Analysis and Prediction of Mitochondrial Targeting Peptides Olof Emanuelsson,* Gunnar von Heijne,* and G isbert S chneidert* Stockholm Bioinformatics Center, Stockholm University, S-10691 Stockholm, Sweden t E Hoffmann-La Roche Ltd., Pharmaceuticals Division CH-4070 Basel, Switzerland I. Introduction II. What Mitochondrial Targeting Peptides (mTPs) Look Like III. Automatic Classification of Known mTPs IV. Automatic Identification of Unknown mTPs A.
Activity and NMR structure of synthetic peptides of the bovine ATPase inhibitor protein, IF1
Peptides, 2002
The protein IF 1 is a natural inhibitor of the mitochondrial F o F 1 -ATPase. Many investigators have been prompted to identify the shortest segment of IF 1 , retaining its native activity, for use in biomedical applications. Here, the activity of the synthetic peptides IF 1 -(42-58) and IF 1 -(22-46) is correlated to their structure and conformational plasticity determined by CD and [ 1 H]-NMR spectroscopy. Among all the IF 1 segments tested, IF 1 -(42-58) exerts the most potent, pH and temperature dependent activity on the F o F 1 complex. The results suggest that, due to its flexible structure, it can fold in helical and/or -spiral arrangements that favor the binding to the F o F 1 complex, where the native IF 1 binds. IF 1 -(22-46), instead, as it adopts a rigid ␣-helical conformation, it inhibits ATP hydrolysis only in the soluble F 1 moiety.
Protein expression and …, 2009
Extensive X-ray crystallographic studies carried out on the catalytic-subunit of protein kinase A (PKA-C) enabled the atomic characterization of inhibitor and/or substrate peptide analogues trapped at its active site. Yet, the structural and dynamic transitions of these peptides from the free to the bound state are missing. These conformational transitions are central to understanding molecular recognition and the enzymatic cycle. NMR spectroscopy allows one to study these phenomena under functionally relevant conditions. However, the amounts of isotopically labeled peptides required for this technique present prohibitive costs for solid-phase peptide synthesis. To enable NMR studies, we have optimized both expression and purification of isotopically enriched substrate/inhibitor peptides using a recombinant fusion protein system. Three of these peptides correspond to the cytoplasmic regions of the wild-type and lethal mutants of the membrane protein phospholamban, while the fourth peptide correspond to the binding epitope of the heat-stable protein kinase inhibitor ). The target peptides were fused to the maltose binding protein (MBP), which is further purified using a His 6 tag approach. This convenient protocol allows for the purification of milligram amounts of peptides necessary for NMR analysis.
Journal of Molecular Biology, 2002
A-kinase anchor proteins (AKAPs) assemble multi-enzyme signaling complexes in proximity to substrate/effector proteins, thus directing and amplifying membrane-generated signals. S-AKAP84 and AKAP121 are alternative splicing products with identical NH 2 termini. These AKAPs bind and target protein kinase A (PKA) to the outer mitochondrial membrane. Tubulin was identified as a binding partner of S-AKAP84 in a yeast two-hybrid screen. Immunoprecipitation and co-sedimentation experiments in rat testis extracts confirmed the interaction between microtubules and S-AKAP84. In situ immunostaining of testicular germ cells (GC2) shows that AKAP121 concentrates on mitochondria in interphase and on mitotic spindles during M phase. Purified tubulin binds directly to S-AKAP84 but not to a deletion mutant lacking the mitochondrial targeting domain (MT) at residues 1 -30. The MT is predicted to form a highly hydrophobic a-helical wheel that might also mediate interaction with tubulin. Disruption of the wheel by site-directed mutagenesis abolished tubulin binding and reduced mitochondrial attachment of an MT-GFP fusion protein. Some MT mutants retain tubulin binding but do not localize to mitochondria. Thus, the tubulin-binding motif lies within the mitochondrial attachment motif. Our findings indicate that S-AKAP84/AKAP121 use overlapping targeting motifs to localize signaling enzymes to mitochondrial and cytoskeletal compartments.
Binding of a synthetic targeting peptide to a mitochondrial channel protein
Journal of Bioenergetics and Biomembranes, 1992
Membrane crystals of the mitochondrial outer membrane channel VDAC (porin) from Neurospora crassa were incubated with a 20-amino-acid synthetic peptide corresponding to the N-terminal targeting region of subunit IV of cytochrome oxidase. The peptide caused disordering and contraction of the crystal lattice of the membrane arrays. Also, new stain-excluding features were observed on the peptide-treated arrays which most likely correspond to sites at which the peptide accumulates. The stain exclusion zones associated with binding of the targeting peptide (and with binding of apocytochrome c in an earlier study) have been localized on a two-dimensional density map of frozen-hydrated, crystalline VDAC previously obtained by cryo-electron microscopy. The results indicate that both the peptide and cytochrome e bind to protein "arms" which extend laterally between the channel lumens. The finding that imported polypeptides bind to a specific region of the VDAC protein implicates this channel in the process by which precursor proteins are recognized at and translocated across the mitochondrial outer membrane.
The Journal of Peptide Research, 2009
Peptides derived from the inhibitor of CAMP-dependent protein kinase, PKI, have been studied by 2D 'H NMR techniques. These include the inhibitor PKI( 6-22), the substrate [Ala2"-Ser"]PKI( 5-24), and a phosphorylated form of the latter [Ala'o-Ser"P]PKI( 5-24). A homologous fold was found in the three peptides which consisted of an h'-terminal segment in helical conformation to residue 13 and a C-terminal segment poorly defined conformationally. A parallel study was carried out by molecular dynamics (MD) for the inhibitor peptide PKI(5-24). The N-terminal helix, as observed in the crystal structure of the catalytic subunit-PKI( 5-24) complex. was conserved in the M D simulations with the enzyme-free inhibitor. Similarly the Gly'J-Glyl' turn was apparent in all M D structures, whereas the C-terminal region, residues 18-24, was directed towards the N-terminal helix in contrast to the extended conformation of this segment pointing away from the A'-terminal helix in the crystal structure. This is primarily due to ionic interaction between Asp' and Arg". Indeed. a detailed analysis of the NOE contacts by NOESY at low temperature ( 2 -C ) shows the occurrence of pH-dependent contacts with Phe'". We conclude that the binding of short inhibitors, such as PKI( 5-24), to the enzyme involves a conformational rearrangement of the C-terminal region. The substrate [Ala'o-Ser"]PKI(5-24) and the product [Alazo-SerZ1P]PKI(5-24),
Cloning and mitochondrial localization of full-length D-AKAP2, a protein kinase A anchoring protein
Proceedings of the National Academy of Sciences, 2001
and the corresponding mouse protein less the first two exons (617 residues). Expression of D-AKAP2 was characterized by using mouse tissue extracts. Full-length D-AKAP2 from various tissues shows different molecular weights, possibly because of alternative splicing or posttranslational modifications. The cloned human gene product has a molecular weight similar to one of the prominent mouse proteins. In vivo association of D-AKAP2 with PKA in mouse brain was demonstrated by using cAMP agarose pull-down assay. Subcellular localization for endogenous mouse, rat, and human D-AKAP2 was determined by immunocytochemistry, immunohistochemistry, and tissue fractionation. D-AKAP2 from all three species is highly enriched in mitochondria. The mitochondrial localization and the presence of RGS domains in D-AKAP2 may have important implications for its function in PKA and G protein signal transduction.
Structure of D-AKAP2:PKA RI Complex: Insights into AKAP Specificity and Selectivity
Structure, 2010
A-kinase anchoring proteins (AKAPs) regulate cyclic AMP-dependent protein kinase (PKA) signaling in space and time. Dual-specific AKAP 2 (D-AKAP2) binds to the dimerization/docking (D/D) domain of both RI and RII regulatory subunits of PKA with high affinity. Here we have determined the structures of the RIa D/D domain alone and in complex with D-AKAP2. The D/D domain presents an extensive surface for binding through a well-formed N-terminal helix, and this surface restricts the diversity of AKAPs that can interact. The structures also underscore the importance of a redox-sensitive disulfide in affecting AKAP binding. An unexpected shift in the helical register of D-AKAP2 compared to the RIIa:D-AKAP2 complex structure makes the mode of binding to RIa novel. Finally, the comparison allows us to deduce a molecular explanation for the sequence and spatial determinants of AKAP specificity.