A Switch Between Two-, Three-, and Four-stranded Coiled Coils in GCN4 Leucine Zipper Mutants

Harbury, Pehr B.; Zhang, Tao; Kim, Peter; Alber, Tom

doi:10.1126/science.8248779

Cited by 1,475 publications

(1,770 citation statements)

References 53 publications

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“…Here we describe the crystal structure of GCN4-pLI in a different spacegroup than originally reported (14) (Figure 1a), and report structures of 11 peptides in the parallel configuration and one peptide in the antiparallel configuration containing hydrophobic core substitutions ( Figure 2, Table 3). All crystals in which peptides adopted the parallel configuration were processed in the P4 1 32 spacegroup, where the four-helix bundle is generated by a crystallographic two-fold symmetry axis acting on the two-strand asymmetric unit, so that 12 four-helix bundles comprise the unit cell contents.…”

Section: Crystal Structuresmentioning

confidence: 95%

“…Apparent aggregation states (N agg ) were calculated from the elution volume by first using the calibration plot to determine the apparent MW of the bundle, and then dividing this value by the calculated MW of an individual peptide. A control peptide reported to form a coiled-coil trimer in solution (14) had an observed N agg of 3.2.…”

Section: Size Exclusion Chromatographymentioning

confidence: 99%

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Structure-Based Engineering of Internal Cavities in Coiled-Coil Peptides^,

et al. 2005

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Cavities and clefts are frequently important sites of interaction between natural enzymes or receptors with their corresponding substrate or ligand molecules and exemplify the types of molecular surfaces that would facilitate engineering artificial catalysts and receptors. Even so, structural characterizations of designed cavities are rare. To address this issue, we performed a systematic study of the structural effects of single amino acid substitutions within the hydrophobic cores of tetrameric coiled-coil peptides. Peptides containing single glycine, serine, alanine, or threonine amino acid substitutions at the buried L9, L16, L23, and I26 hydrophobic core positions of a GCN4-based sequence were synthesized and studied by solution-phase and crystallographic techniques. All peptides adopt the expected tetrameric state and contain tunnels or internal cavities ranging in size from 80 Å 3 to 370 Å 3 . Two closely-related sequences containing an L16G substitution, one of which adopts an antiparallel configuration and one of which adopts a parallel configuration, illustrate that cavities of different volumes and shapes can be engineered from identical core substitutions. Finally, we demonstrate that two of the peptides (L9G and L9A) bind the small molecule iodobenzene when present during crystallization, leaving the general peptide quaternary structure intact but altering the local peptide conformation and certain superhelical parameters. These high-resolution descriptions of varied molecular surfaces within solvent-occluded internal cavities illustrate the breadth of design space available in even closely-related peptides and offer valuable models for the engineering of de novo helical proteins.A significant challenge frustrating the de novo design of functional molecules is that of precisely constructing molecular surfaces able to suitably participate in desired molecular recognition events. Internal cavities, relatively buried clefts, and tunnels are frequently important sites of interaction between natural enzymes or receptors with their corresponding substrate or ligand molecules, and the ability to engineer such surfaces would facilitate the successful design of highly-selective artificial catalysts and receptors. Mutagenesis of natural proteins to create or alter internal cavities has been used for such purposes as redesigning † We thank the National Institutes of Health for financial support (GM52190). JER, JMAG, and LJL thank the Wellcome Trust (061454/ Z/00/Z), the Spanish MCYT, and NSF, respectively, for fellowships. § Coordinates have been deposited in the RCSB Protein Data Bank: GCN4-pLI, 1UO2; L9G, 1UO3; L9S, 1UNU; L9A, 1UNT; L9T, 1UNV; L9G+IB, 1UO4; L9A+IB, 1UO5; L23G, 1UNW; L23S 1UNX; I26G, 1UNY; I26S, 1UNZ; I26A, 1UN0; I26T, 1UN1; L16G E20C, 1W5I; L16G E20C Y17H, 2BNI.* Address correspondence to this author. (858) 784-2700 (phone); (858) 784-2798 (fax); ghadiri@scripps.edu (e-mail).. 1 Abbreviations: CD, circular dichroism; SEC, size exclusion chromatography; ABA, acetamidobenzoic acid; MW,...

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Section: Crystal Structuresmentioning

confidence: 95%

Section: Size Exclusion Chromatographymentioning

confidence: 99%

Structure-Based Engineering of Internal Cavities in Coiled-Coil Peptides^,

et al. 2005

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“…Size exclusion chromatography (SEC) showed that all the peptides were dimers except for 6F, the all-Ala surface sequence, which migrated as a tetramer. These data show that surface redesign did not change the tertiary structure of these peptides, in convast to some core redesigns (Harbury et al, 1993). In addition, nuclear magnetic resonance (NMR) spectra of the peptides at -1 mM showed chemical shift dispersion similar to GCN4-pI (data not shown).…”

Section: Resultsmentioning

confidence: 75%

“…GCN4-pl and p-LI (Harbury et al, 1993) were used as size standards for dimer and tetramer, respectively. Five-pL injections of -1 mM peptide solution were chromatographed at 0.50 mL/min and monitored at 214 nm.…”

Section: Size Exclusion Chromatographymentioning

confidence: 99%

Automated design of the surface positions of protein helices

Dahiyat¹,

Gordon²,

Mayo³

1997

Protein Science

223

233

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Using a protein design algorithm that quantitatively considers side-chain interactions, the design of surface residues of (Y helices was examined. Three scoring functions were tested: a hydrogen-bond potential, a hydrogen-bond potential in conjunction with a penalty for uncompensated burial of polar hydrogens, and a hydrogen-bond potential in combination with helix propensity. The solvent exposed residues of a homodimeric coiled coil based on GCN4pI were designed by using the Dead-End Elimination Theorem to find the optimal amino acid sequence for each scoring function. The corresponding peptides were synthesized and characterized by circular dichroism spectroscopy and size exclusion chromatography. The designed peptides were dimeric and nearly 100% helical at 1 "C, with melting temperatures from 69-72 "C, over 12 "C higher than GCNCpl, whereas a random hydrophilic sequence at the surface positions produced a peptide that melted at 15 "C. Analysis of the designed sequences suggests that helix propensity is the key factor in sequence design for surface helical positions.Keywords: coiled coils; helix propensities; protein design; surface residuesSeveral groups have proposed and tested systematic, quantitative methods for protein design that screen possible sequences for compatibility with the desired protein fold (Hellinga et al., 1991;Hurley et al., 1992;Desjarlais & Handel, 1995;Harbury et al., 1995;Klemba et al., 1995;Nautiyal et al., 1995;Betz & Degrado, 1996;Dahiyat & Mayo, 1996). These algorithms consider the spatial positioning and steric complementarity of side chains by explicitly modeling the atoms of sequences under consideration. To date, such techniques have typically focused on designing the cores of proteins and have scored sequences with van der Waals and sometimes hydrophobic solvation potentials. We seek to extend this sequence selection approach to the design of the solvent-exposed residues of proteins as part of an effort to develop a complete de novo design algorithm. In this study, we consider the design of the surface positions of (Y helices.Although mutagenesis studies suggest that surface positions are more tolerant of substitutions than core positions (Reidhaar-Olson & Sauer, 1988;Bowie et al., 1990), surface residues can still have a significant effect on protein structure and stability. To assess the importance of surface residue selection for protein design, we used several scoring functions to compute sequences for the surface positions of our model helical protein, the coiled coil GCN4-pl (O'Shea et al., 1991). By experimentally characterizing the result-

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“…18,19 Leucine zippers are short coiled-coil domains that serve to dimerize or oligomerize protein subunits. 20,21 Crystal structural analysis indicates that an ILZ forms trimers through self-assembly. 22 Soluble TNF/ TNF receptor modified by an ILZ motif can form a stable trimeric or multiple oligomerization states.…”

Section: Introductionmentioning

confidence: 99%

An isoleucine-zipper motif enhances costimulation of human soluble trimeric GITR ligand

et al. 2010

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Glucocorticoid-induced tumor-necrosis factor receptor (GITR) and its ligand, GITRL, play significant roles in regulating immune responses. It is clear that human soluble GITRL (hsGITRL) transduces signal activity through multiple oligomerization states. To develop human soluble trimeric GITRL protein as a potential therapeutic target, we explored the link of the isoleucine-zipper (ILZ) motif to the N-terminus of the human soluble GITRL with two leucine sequences. hsGITRL, with the ILZ motif (ILZ-hsGITRL), was firstly expressed in Escherichia coli, which exhibited a predominant trimer when identified by Sephadex G-100 filtration and non-reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The significantly higher biological activity of the ILZ-hsGITRL compared with hsGITRL was confirmed by CD4 1 T proliferation, interferon-c (IFN-c) secretion and binding activity assay. To reveal and compare the underlying mechanisms, the level of extracellular signal-regulated kinase-1/2 (ERK1/2) phosphorylation was examined, indicating that ILZ-hsGITRL induced more persistent and stronger ERK1/2 activation than hsGITRL. In conclusion, the incorporation of an ILZ motif could markedly improve the costimulation of hsGITRL.

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A Switch Between Two-, Three-, and Four-stranded Coiled Coils in GCN4 Leucine Zipper Mutants

Cited by 1,475 publications

References 53 publications

Structure-Based Engineering of Internal Cavities in Coiled-Coil Peptides^,

Structure-Based Engineering of Internal Cavities in Coiled-Coil Peptides^,

Automated design of the surface positions of protein helices

An isoleucine-zipper motif enhances costimulation of human soluble trimeric GITR ligand

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A Switch Between Two-, Three-, and Four-stranded Coiled Coils in GCN4 Leucine Zipper Mutants

Cited by 1,475 publications

References 53 publications

Structure-Based Engineering of Internal Cavities in Coiled-Coil Peptides,

Structure-Based Engineering of Internal Cavities in Coiled-Coil Peptides,

Automated design of the surface positions of protein helices

An isoleucine-zipper motif enhances costimulation of human soluble trimeric GITR ligand

Contact Info

Product

Resources

About

Structure-Based Engineering of Internal Cavities in Coiled-Coil Peptides^,

Structure-Based Engineering of Internal Cavities in Coiled-Coil Peptides^,