The retro-GCN4 leucine zipper sequence forms a stable three-dimensional structure.
Ontology highlight
ABSTRACT:
SUBMITTER: Mittl PR
PROVIDER: S-EPMC15968 | biostudies-literature | 2000 Mar
REPOSITORIES: biostudies-literature
Publications
The retro-GCN4 leucine zipper sequence forms a stable three-dimensional structure.
Mittl P R PR Deillon C C Sargent D D Liu N N Klauser S S Thomas R M RM Gutte B B Grütter M G MG
Proceedings of the National Academy of Sciences of the United States of America 20000301 6
The question of whether a protein whose natural sequence is inverted adopts a stable fold is still under debate. We have determined the 2. 1-A crystal structure of the retro-GCN4 leucine zipper. In contrast to the two-stranded helical coiled-coil GCN4 leucine zipper, the retro-leucine zipper formed a very stable, parallel four-helix bundle, which now lends itself to further structural and functional studies. ...[more]
Similar Datasets
Project description:Ion pairs are ubiquitous in X-ray structures of coiled coils, and mutagenesis of charged residues can result in large stability losses. By contrast, pK(a) values determined by NMR in solution often predict only small contributions to stability from charge interactions. To help reconcile these results we used triple-resonance NMR to determine pK(a) values for all groups that ionize between pH 1 and 13 in the 33 residue leucine zipper fragment, GCN4p. In addition to the native state we also determined comprehensive pK(a) values for two models of the GCN4p denatured state: the protein in 6 M urea, and unfolded peptide fragments of the protein in water. Only residues that form ion pairs in multiple X-ray structures of GCN4p gave large pK(a) differences between the native and denatured states. Moreover, electrostatic contributions to stability were not equivalent for oppositely charged partners in ion pairs, suggesting that the interactions between a charge and its environment are as important as those within the ion pair. The pH dependence of protein stability calculated from NMR-derived pK(a) values agreed with the stability profile measured from equilibrium urea-unfolding experiments as a function of pH. The stability profile was also reproduced with structure-based continuum electrostatic calculations, although contributions to stability were overestimated at the extremes of pH. We consider potential sources of errors in the calculations, and how pK(a) predictions could be improved. Our results show that although hydrophobic packing and hydrogen bonding have dominant roles, electrostatic interactions also make significant contributions to the stability of the coiled coil.
| S-EPMC2105789 | biostudies-literature
Project description:Recently, we showed that leucine zipper (LZ) motifs of basic leucine zipper (bZIP) transcription factors GCN4 and c-Jun are capable of catalyzing degradation of RNA (Nikolaev et al., PLoS ONE 2010; 5:e10765). This observation is intriguing given the tight regulation of RNA turnover control and the antiquity of bZIP transcription factors. To support further mechanistic studies, herein, we elucidated RNA binding interface of the GCN4 leucine zipper motif from yeast. Solution NMR experiments showed that the LZ-RNA interaction interface is located in the first two heptads of LZ moiety, and that only the dimeric (coiled coil) LZ conformation is capable of binding RNA. Site-directed mutagenesis of the LZ-GCN4 RNA binding interface showed that substrate binding is facilitated by lysine and arginine side chains, and that at least one nucleophilic residue is located in proximity to the RNA phosphate backbone. Further studies in the context of full-length bZIP factors are envisaged to address the biological relevance of LZ RNase activity.
| S-EPMC3403464 | biostudies-literature
Project description:The dimerization domain of the yeast transcription factor GCN4, one of the first coiled-coil proteins to be structurally characterized at high resolution, has served as the basis for numerous fundamental studies on α-helical folding. Mutations in the GCN4 leucine zipper are known to change its preferred oligomerization state from dimeric to trimeric or tetrameric; however, the wild-type sequence has been assumed to encode a two-chain assembly exclusively. Here we demonstrate that the GCN4 coiled-coil domain can populate either a dimer or trimer fold, depending on environment. We report high-resolution crystal structures of the wild-type sequence in dimeric and trimeric assemblies. Biophysical measurements suggest populations of both oligomerization states under certain experimental conditions in solution. We use parallel tempering molecular dynamics simulations on the microsecond time scale to compare the stability of the dimer and trimer folded states in isolation. In total, our results suggest that the folding behavior of the well-studied GCN4 leucine-zipper domain is more complex than was previously appreciated. Our results have implications in ongoing efforts to establish predictive algorithms for coiled-coil folds and the selection of coiled-coil model systems for design and mutational studies where oligomerization state specificity is an important consideration.
| S-EPMC3535301 | biostudies-literature
Project description:A three-dimensional model of the leucine zipper GCN4 built from its amino acid sequence had been reported previously by us. When the two alternative x-ray structures of the GCN4 dimer became available, the root mean square (r.m.s.) shifts between our model and the structures were determined as approximately 2.7 A on all atoms. These values are similar to the r.m.s. shift of 2.8 A between the two GCN4 structures in the different crystal forms (C2 and P2(1)2(1)2(1)). CONGEN conformational searches were run to better understand the conditions that may determine the preference of different conformers in different environments and to test the sensitivity of our current modeling techniques. With a judicious choice of CONGEN search parameters, the backbone r.m.s. deviation improved to 0.8 A and 2.5 A on all atoms. The side-chain conformations of Val and Leu at the helical interface were well reproduced (1.2 A r.m.s.), and the large side-chain misplacements occurred with only a small number of charged amino acids and a tyrosine. Inclusion of the crystal environment (C2 symmetry), as a passive background, into the side-chain conformational search further improved the accuracy of the model to an r.m.s. deviation of 2.1 A. Conformational searches carried out in the two different crystal environments and employing the AMBER protein/DNA forcefield, as implemented in CONGEN, gave the r.m.s. values of 2.2 A (for the C2 symmetry) and 2.5 A (for the P2(1)2(1)2(1) symmetry). In the C2 symmetry crystal, as much as 40% of the surface of each dimer was involved in crystal contacts with other dimers, and the charged residues on the surface often interacted with immobilized water molecules. Thus, occasional large r.m.s. deviations between the model and the x-ray side chains were due to specific conditions that did not occur in solution.
| S-EPMC1225039 | biostudies-other
Project description:Basic-region leucine zipper (bZIP) proteins are one of the largest transcription factor families that regulate a wide range of cellular functions. Owing to the stability of their coiled coil structure leucine zipper (LZ) domains of bZIP factors are widely employed as dimerization motifs in protein engineering studies. In the course of one such study, the X-ray structure of the retro-version of the LZ moiety of yeast transcriptional activator GCN4 suggested that this retro-LZ may have ribonuclease activity. Here we show that not only the retro-LZ but also the authentic LZ of GCN4 has weak but distinct ribonuclease activity. The observed cleavage of RNA is unspecific, it is not suppressed by the ribonuclease A inhibitor RNasin and involves the breakage of 3',5'-phosphodiester bonds with formation of 2',3'-cyclic phosphates as the final products as demonstrated by HPLC/electrospray ionization mass spectrometry. Several mutants of the GCN4 leucine zipper are catalytically inactive, providing important negative controls and unequivocally associating the enzymatic activity with the peptide under study. The leucine zipper moiety of the human factor c-Jun as well as the entire c-Jun protein are also shown to catalyze degradation of RNA. The presented data, which was obtained in the test-tube experiments, adds GCN4 and c-Jun to the pool of proteins with multiple functions (also known as moonlighting proteins). If expressed in vivo, the endoribonuclease activity of these bZIP-containing factors may represent a direct coupling between transcription activation and controlled RNA turnover. As an additional result of this work, the retro-leucine zipper of GCN4 can be added to the list of functional retro-peptides.
| S-EPMC2874015 | biostudies-literature
Project description:The GCN4 leucine zipper is a peptide homodimer that has been the subject of a number of experimental and theoretical investigations into the determinants of affinity and specificity. Here, we utilize this model system to investigate electrostatic effects in protein binding using continuum calculations. A particularly novel feature of the computations made here is that they provide an interaction-by-interaction breakdown of the electrostatic contributions to the free energy of docking that includes changes in the interaction of each functional group with solvent and changes in interactions between all pairs of functional groups on binding. The results show that (1) electrostatic effects disfavor binding by roughly 15 kcal/mol due to desolvation effects that are incompletely compensated in the bound state, (2) while no groups strongly stabilize binding, the groups that are most destabilizing are charged and polar side chains at the interface that have been implicated in determining binding specificity, and (3) attractive intramolecular interactions (e.g., backbone hydrogen bonds) that are enhanced on binding due to reduced solvent screening in the bound state contribute significantly to affinity and are likely to be a general effect in other complexes. A comparison is made between the results obtained in an electrostatic analysis carried out calculationally and simulated results corresponding to idealized data from a scanning mutagenesis experiment. It is shown that scanning experiments provide incomplete information on interactions and, if overinterpreted, tend to overestimate the energetic effect of individual side chains that make attractive interactions. Finally, a comparison is made between the results available from a continuum electrostatic model and from a simpler surface-area dependent solvation model. In this case, although the simpler model neglects certain interactions, on average it performs rather well.
| S-EPMC2144375 | biostudies-other
Project description:Leucine zipper-EF-hand-containing transmembrane protein1 (LETM1) is located in the mitochondrial inner membrane and is defective in Wolf-Hirschhorn syndrome. LETM1 contains only one transmembrane helix, but it behaves as a putative transporter. Our data shows that LETM1 knockdown or overexpression robustly increases or decreases mitochondrial Ca(2+) level in HeLa cells, respectively. Also the residue Glu221 of mouse LETM1 is identified to be necessary for Ca(2+) flux. The mutation of Glu221 to glutamine abolishes the Ca(2+)-transport activity of LETM1 in cells. Furthermore, the purified LETM1 exhibits Ca(2+)/H(+) anti-transport activity, and the activity is enhanced as the proton gradient is increased. More importantly, electron microscopy studies reveal a hexameric LETM1 with a central cavity, and also, observe two different conformational states under alkaline and acidic conditions, respectively. Our results indicate that LETM1 is a Ca(2+)/H(+) antiporter and most likely responsible for mitochondrial Ca(2+) output.
| S-EPMC5037442 | biostudies-literature
Project description:Protein-protein interactions play an essential role in the assembly of the macromolecular complexes that form functional networks and control cellular behavior. Elucidating principles of molecular recognition governing potentially complex interfaces is a challenging goal for structural and systems biology. Extensive studies of alpha-helical coiled coils have provided fundamental insight into the determinants of one seemingly tractable class of oligomeric protein interfaces. We report here that two different valine-containing mutants of the GCN4 leucine zipper that fold individually as four-stranded coiled coils associate preferentially in mixtures to form an antiparallel, heterotetrameric structure. X-ray crystallographic analysis reveals that the coinciding hydrophobic interfaces of the hetero- and homotetramers differ in detail, thereby controlling their partnering and structural specificity. Equilibrium disulfide exchange and thermal denaturation experiments show that the 50-fold preference for heterospecificity is determined by interfacial van der Waals interactions and hydrophobicity. Parallel studies of two alanine-containing variants confirm the above-mentioned interpretation of the basis and mechanism of this heterospecificity. Our results suggest that coiled-coil recognition is an inherently geometric process in which heterotypic interaction specificity derives from a complementarity of both shape and chemistry.
| S-EPMC7111190 | biostudies-literature
Project description:Zipper-interacting protein kinase (ZIP kinase) has been thought to be involved in apoptosis and the C-terminal leucine zipper motif is important for its function. Recent studies have revealed that ZIP kinase also plays a role in regulating myosin phosphorylation. Here, we found novel ZIP kinase isoform in which the C-terminal non-kinase domain containing a leucine zipper is eliminated (hZIPK-S). hZIPK-S binds to myosin phosphatase targeting subunit 1(MYPT1) similar to the long isoform (hZIPK-L). In addition, we found that hZIPK-S as well as hZIPK-L bind to myosin. These results indicate that a leucine zipper is not critical for the binding of ZIP kinase to MYPT1 and myosin. Consistently, hZIPK-S localized with stress-fibers where they co-localized with myosin. The residues 278-311, the C-terminal side of the kinase domain common to the both isoforms, is involved in the binding to MYPT1, while the myosin binding domain is within the kinase domain. These results suggest that the newly found hZIPK-S as well as the long isoform play an important role in the regulation of myosin phosphorylation.
| S-EPMC2758612 | biostudies-literature
Project description:Snake venom phospholipases B (SVPLBs) are the least studied enzymes. They constitute about 1% of Bothrops crude venoms, however, in other snake venoms, it is present in less than 1%. These enzymes are considered the most potent hemolytic agent in the venom. Currently, no structural information is available about these enzymes from snake venom. To better understand its three-dimensional structure and mechanisms of envenomation, the current work describes the first model-based structure report of this enzyme from Bothrops moojeni venom named as B. moojeni phospholipase B (PLB_Bm). The structure model of PLB_Bm was generated using model building software like I-TESSER, MODELLER 9v19, and Swiss-Model. The build PLB_Bm model was validated using validation tools (PROCHECK, ERRAT, and Verif3D). The analysis of the PLB_Bm modeled structure indicates that it contains 491 amino acid residues that form a well-defined four-layer αββα sandwich core and has a typical fold of the N-terminal nucleophile aminohydrolase (Ntn-hydrolase). The overall structure of PLB_Bm contains 18 β-strands and 17 α-helices with many connecting loops. The structure divides into two chains (A and B) after maturation. The A chain is smaller and contains 207 amino acid residues, whereas the B chain is larger and contains 266 amino acid residues. The sequence and structural comparison among homologous snake venom, bacterial, and mammals PLBs indicate that differences in the length and sequence composition may confer variable substrate specificity to these enzymes. Moreover, the surface charge distribution, average volume, and depth of the active site cavity also vary in these enzymes. The present work will provide more information about the structure-function relationship and mechanism of action of these enzymes in snakebite envenomation.
| S-EPMC7419708 | biostudies-literature