An undecided coiled coil: the leucine zipper of Nek2 kinase exhibits atypical conformational exchange dynamics.
ABSTRACT: Leucine zippers are oligomerization domains used in a wide range of proteins. Their structure is based on a highly conserved heptad repeat sequence in which two key positions are occupied by leucines. The leucine zipper of the cell cycle-regulated Nek2 kinase is important for its dimerization and activation. However, the sequence of this leucine zipper is most unusual in that leucines occupy only one of the two hydrophobic positions. The other position, depending on the register of the heptad repeat, is occupied by either acidic or basic residues. Using NMR spectroscopy, we show that this leucine zipper exists in two conformations of almost equal population that exchange with a rate of 17 s(-1). We propose that the two conformations correspond to the two possible registers of the heptad repeat. This hypothesis is supported by a cysteine mutant that locks the protein in one of the two conformations. NMR spectra of this mutant showed the predicted 2-fold reduction of peaks in the (15)N HSQC spectrum and the complete removal of cross peaks in exchange spectra. It is possible that interconversion of these two conformations may be triggered by external signals in a manner similar to that proposed recently for the microtubule binding domain of dynein and the HAMP domain. As a result, the leucine zipper of Nek2 kinase is the first example where the frameshift of coiled-coil heptad repeats has been directly observed experimentally.
Project description:BACKGROUND: Severe acute respiratory syndrome (SARS) is a febrile respiratory illness. The disease has been etiologically linked to a novel coronavirus that has been named the SARS-associated coronavirus (SARS-CoV), whose genome was recently sequenced. Since it is a member of the Coronaviridae, its spike protein (S2) is believed to play a central role in viral entry by facilitating fusion between the viral and host cell membranes. The protein responsible for viral-induced membrane fusion of HIV-1 (gp41) differs in length, and has no sequence homology with S2. RESULTS: Sequence analysis reveals that the two viral proteins share the sequence motifs that construct their active conformation. These include (1) an N-terminal leucine/isoleucine zipper-like sequence, and (2) a C-terminal heptad repeat located upstream of (3) an aromatic residue-rich region juxtaposed to the (4) transmembrane segment. CONCLUSIONS: This study points to a similar mode of action for the two viral proteins, suggesting that anti-viral strategy that targets the viral-induced membrane fusion step can be adopted from HIV-1 to SARS-CoV. Recently the FDA approved Enfuvirtide, a synthetic peptide corresponding to the C-terminal heptad repeat of HIV-1 gp41, as an anti-AIDS agent. Enfuvirtide and C34, another anti HIV-1 peptide, exert their inhibitory activity by binding to a leucine/isoleucine zipper-like sequence in gp41, thus inhibiting a conformational change of gp41 required for its activation. We suggest that peptides corresponding to the C-terminal heptad repeat of the S2 protein may serve as inhibitors for SARS-CoV entry.
Project description:The Escherichia coli metR gene has been sequenced. The sequence predicts a protein of 317 amino acids and a calculated molecular weight of 35,628. This is about 15% larger than the protein from Salmonella typhimurium reported previously [Plamann, L.S. & Stauffer, G.V. (1987) J. Bacteriol. 169, 3932-3937]. The protein is a homodimer and contains a leucine zipper motif characteristic of many eukaryotic DNA-binding proteins. Replacement of two of the leucines in the leucine zipper region of the MetR protein, or substitution of proline for one of the leucines, results in loss of biological activity of the protein. In addition, truncation studies have identified a region on MetR that may be involved in the homocysteine activation of metE expression.
Project description:Proteins of the tetraspanin family contain four transmembrane domains (TM1-4) linked by two extracellular loops and a short intracellular loop, and have short intracellular N- and C-termini. While structure and function analysis of the larger extracellular loop has been performed, the organization and role of transmembrane domains have not been systematically assessed.Among 28 human tetraspanin proteins, the TM1-3 sequences display a distinct heptad repeat motif (abcdefg)n. In TM1, position a is occupied by structurally conserved bulky residues and position d contains highly conserved Asn and Gly residues. In TM2, position a is occupied by conserved small residues (Gly/Ala/Thr), and position d has a conserved Gly and two bulky aliphatic residues. In TM3, three a positions of the heptad repeat are filled by two leucines and a glutamate/glutamine residue, and two d positions are occupied by either Phe/Tyr or Val/Ile/Leu residues. No heptad motif is apparent in TM4 sequences. Mutations of conserved glycines in human CD9 (Gly25 and Gly32 in TM1; Gly67 and Gly74 in TM2) caused aggregation of mutant proteins inside the cell. Modeling of the TM1-TM2 interface in CD9, using a novel algorithm, predicts tight packing of conserved bulky residues against conserved Gly residues along the two helices. The homodimeric interface of CD9 was mapped, by disulfide cross-linking of single-cysteine mutants, to the vicinity of residues Leu14 and Phe17 in TM1 (positions g and c) and Gly77, Gly80 and Ala81 in TM2 (positions d, g and a, respectively). Mutations of a and d residues in both TM1 and TM2 (Gly25, Gly32, Gly67 and Gly74), involved in intramolecular TM1-TM2 interaction, also strongly diminished intermolecular interaction, as assessed by cross-linking of Cys80.Our results suggest that tetraspanin intra- and intermolecular interactions are mediated by conserved residues in adjacent, but distinct regions of TM1 and TM2. A key structural element that defines TM1-TM2 interaction in tetraspanins is the specific packing of bulky residues against small residues.
Project description:Basic leucine zipper (bZIP) transcription factors (TFs) govern diverse cellular processes and cell fate decisions. The hallmark of the leucine zipper domain is the heptad repeat, with leucine residues at every seventh position in the domain. These leucine residues enable homo- and heterodimerization between ZIP domain ?-helices, generating coiled-coil structures that stabilize interactions between adjacent DNA-binding domains and target DNA substrates. Several cancer-causing viruses encode viral bZIP TFs, including human T-cell leukemia virus (HTLV), hepatitis C virus (HCV) and the herpesviruses Marek's disease virus (MDV), Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV). Here, we provide a comprehensive review of these viral bZIP TFs and their impact on viral replication, host cell responses and cell fate.
Project description:The hydrophobic core of the GCN4 leucine-zipper dimerization domain is formed by a parallel helical association between nonpolar side chains at the a and d positions of the heptad repeat. Here we report a self-assembling coiled-coil array formed by the GCN4-pAe peptide that differs from the wild-type GCN4 leucine zipper by alanine substitutions at three charged e positions. GCN4-pAe is incompletely folded in normal solution conditions yet self-assembles into an antiparallel tetraplex in crystals by formation of unanticipated hydrophobic seams linking the last two heptads of two parallel double-stranded coiled coils. The GCN4-pAe tetramers in the lattice associate laterally through the identical interactions to those in the intramolecular dimer-dimer interface. The van der Waals packing interaction in the solid state controls extended supramolecular assembly of the protein, providing an unusual atomic scale view of a mesostructure.
Project description:Nek2 is a dimeric serine/ threonine protein kinase that belongs to the family of NIMA-related kinases (Neks). Its N-terminal catalytic domain and its C-terminal regulatory region are bridged by a leucine zipper, which plays an important role in the activation of Nek2's catalytic activity. Unusual conformational dynamics on the intermediary/slow timescale has thwarted all attempts so far to determine the structure of the Nek2 leucine zipper by means of X-ray crystallography and Nuclear Magnetic Resonance (NMR). Disulfide engineering, the strategic placement of non-native disulfide bonds into flexible regions flanking the coiled coil, was used to modulate the conformational exchange dynamics of this important dimerization domain. The resulting reduction in exchange rate leads to substantial improvements of important features in NMR spectra, such as line width, coherence transfer leakage and relaxation. These effects were comprehensively analyzed for the wild type protein, two single disulfide bond-bearing mutants and another double disulfide bonds-carrying mutant. Furthermore, exchange kinetics were measured across a wide temperature range, allowing for a detailed analysis of activation energy (?G‡) and maximal rate constant (k'ex). For one mutant carrying a disulfide bond at its C-terminus, a full backbone NMR assignment could be obtained for both conformers, demonstrating the benefits of the disulfide engineering. Our study demonstrates the first successful application of 'kinetic' disulfide bonds for the purpose of controlling the adverse effects of protein dynamics. Firstly, this provides a promising, robust platform for the full structural and functional investigation of the Nek2 leucine zipper in the future. Secondly, this work broadens the toolbox of protein engineering by disulfide bonds through the addition of a kinetic option in addition to the well-established thermodynamic uses of disulfide bonds.
Project description:Coiled-coil sequences in proteins commonly share a seven-amino acid repeat with nonpolar side chains at the first (a) and fourth (d) positions. We investigate here the role of a 3-3-1 hydrophobic repeat containing nonpolar amino acids at the a, d, and g positions in determining the structures of coiled coils using mutants of the GCN4 leucine zipper dimerization domain. When three charged residues at the g positions in the parental sequence are replaced by nonpolar alanine or valine side chains, stable four-helix structures result. The X-ray crystal structures of the tetramers reveal antiparallel, four-stranded coiled coils in which the a, d, and g side chains interlock in a combination of knobs-into-knobs and knobs-into-holes packing. Interfacial interactions in a coiled coil can therefore be prescribed by hydrophobic-polar patterns beyond the canonical 3-4 heptad repeat. The results suggest that the conserved, charged residues at the g positions in the GCN4 leucine zipper can impart a negative design element to disfavor thermodynamically more stable, antiparallel tetramers.
Project description:RPP13, a member of the cytoplasmic class of disease resistance genes, encodes one of the most variable Arabidopsis proteins so far identified. This variability is matched in ATR13, the protein from the oomycete downy mildew pathogen Hyaloperonospora parasitica recognized by RPP13, suggesting that these proteins are involved in tight reciprocal coevolution. ATR13 exhibits five domains: an N-terminal signal peptide, an RXLR motif, a heptad leucine/isoleucine repeat, an 11-amino-acid repeated sequence and a C-terminal domain. We show that the conserved RXLR-containing domain is dispensable for ATR13-mediated recognition, consistent with its role in transport into the plant cytoplasm. Sequencing ATR13 from 16 isolates of H. parasitica revealed high levels of amino acid diversity across the entire protein. The leucines/isoleucines of the heptad leucine repeat were conserved, and mutation of particular leucine or isoleucine residues altered recognition by RPP13. Natural variation has not exploited this route to detection avoidance, suggesting a key role of this domain in pathogenicity. The extensive variation in the 11-amino-acid repeat units did not affect RPP13 recognition. Domain swap analysis showed that recognition specificity lay in the C-terminal domain of ATR13. Variation analyses combined with functional assays allowed the identification of four amino acid positions that may play a role in recognition specificity. Site-directed mutagenesis confirmed that a threonine residue is absolutely required for RPP13 recognition and that recognition can be modulated by the presence of either an arginine or glutamic acid at other sites. Mutations in these three amino acids had no effect on the interaction of ATR13 with a resistance gene unlinked to RPP13, consistent with their critical role in determining RPP13-Nd recognition specificity.
Project description:We have isolated cDNA clones for a Drosophila poly(ADP-ribose) polymerase (PARP; EC 188.8.131.52) by screening a lambda gt11 cDNA library with a Drosophila partial cDNA fragment. The Drosophila PARP probe was obtained by the polymerase chain reaction with heterologous primers deduced from conserved amino acids in the mammalian, chicken, amphibian, and fish sequences. The Drosophila PARP mRNA is 3.2 kb in length and is expressed in the early stages of development. The PARP protein of 994 amino acids contains two zinc-finger motifs and an NAD-binding motif, which are conserved among different species. Interestingly, the heptad leucine repeat in an alpha-helix was found in Drosophila PARP. Alignments of the auto-modification domains of various species showed the repeated hydrophobic amino acids on the same face of the helix that make the coiled-coil configuration in the mammalian and chicken sequences. The presence of a leucine-zipper motif in the auto-modification domain suggests that this motif might be responsible for protein-protein interaction between PARP and physiological acceptors. PARP may have novel functions, possibly involving its homo- and/or heterodimerization with other nuclear leucine-zipper proteins and its regulation by ADP-ribosylation.
Project description:Na,K-ATPase is a hetero-oligomer of alpha and beta-subunits. The Na,K-ATPase beta-subunit (Na,K-beta) is involved in both the regulation of ion transport activity, and in cell-cell adhesion. By structure prediction and evolutionary analysis, we identified two distinct faces on the Na,K-beta transmembrane domain (TMD) that could mediate protein-protein interactions: a glycine zipper motif and a conserved heptad repeat. Here, we show that the heptad repeat face is involved in the hetero-oligomeric interaction of Na,K-beta with Na,K-alpha, and the glycine zipper face is involved in the homo-oligomerization of Na,K-beta. Point mutations in the heptad repeat motif reduced Na,K-beta binding to Na,K-alpha, and Na,K-ATPase activity. Na,K-beta TMD homo-oligomerized in biological membranes, and mutation of the glycine zipper motif affected oligomerization and cell-cell adhesion. These results provide a structural basis for understanding how Na,K-beta links ion transport and cell-cell adhesion.