The contribution of buried polar groups to the conformational stability of the GCN4 coiled coil11Edited by C. R. Matthews

Zhu, Hai‐Liang; Sa, Celinski; Jm, Scholtz; Jc, Hu

doi:10.1006/jmbi.2000.3936

Cited by 39 publications

(47 citation statements)

References 37 publications

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“…60 For instance, the well-known, and aforementioned example of the N@a residue Asn16 in the leucine-zipper region of the transcriptional activator GCN4, which appears vital to maintaining a parallel dimeric oligomeric state. 35 Regarding this natural GCN4 background, our observations appear to be at odds with the studies of Hu and colleagues, 37,38 who mutated the a positions of GCN4-p1 to combinations of Ile and Asn and concluded: "the effect of an Ile to Asn mutation on the free energy of unfolding is largest at the N terminus of the peptide and decreases almost twofold as we move the substitution from the N to C-terminal heptads". There are, however, several differences between our systems and GCN4, and between the two studies.…”

Section: Discussioncontrasting

confidence: 89%

“…13 From considerable work, the generally accepted view is that, at a sites in coiled-coil dimers, Asn residues make side chain-side chain hydrogen bonds to partly offset the cost of burying a polar group, but that this is not possible in other oligomers and topologies. [35][36][37][38] Studies have also been performed to quantify the contribution of these Asn-Asn pairs to dimer stability at least. [39][40][41] To summarize a large body of literature, N@a is destabilising, but it specifies parallel dimer over alternative states such as antiparallel dimer and parallel trimer.…”

Section: Introductionmentioning

confidence: 99%

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N@a and N@d: Oligomer and Partner Specification by Asparagine in Coiled-Coil Interfaces

Fletcher¹,

Bartlett²,

Boyle³

et al. 2017

ACS Chem. Biol.

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The α-helical coiled coil is one of the best-studied protein–protein interaction motifs. As a result, sequence-to-structure relationships are available for the prediction of natural coiled-coil sequences and the de novo design of new ones. However, coiled coils adopt a wide range of oligomeric states and topologies, and our understanding of the specification of these and the discrimination between them remains incomplete. Gaps in our knowledge assume more importance as coiled coils are used increasingly to construct biomimetic systems of higher complexity; for this, coiled-coil components need to be robust, orthogonal, and transferable between contexts. Here, we explore how the polar side chain asparagine (Asn, N) is tolerated within otherwise hydrophobic helix–helix interfaces of coiled coils. The long-held view is that Asn placed at certain sites of the coiled-coil sequence repeat selects one oligomer state over others, which is rationalized by the ability of the side chain to make hydrogen bonds, or interactions with chelated ions within the coiled-coil interior of the favored state. We test this with experiments on de novo peptide sequences traditionally considered as directing parallel dimers and trimers, and more widely through bioinformatics analysis of natural coiled-coil sequences and structures. We find that when located centrally, rather than near the termini of such coiled-coil sequences, Asn does exert the anticipated oligomer-specifying influence. However, outside of these bounds, Asn is observed less frequently in the natural sequences, and the synthetic peptides are hyperthermostable and lose oligomer-state specificity. These findings highlight that not all regions of coiled-coil repeat sequences are equivalent, and that care is needed when designing coiled-coil interfaces.

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Section: Discussioncontrasting

confidence: 89%

Section: Introductionmentioning

confidence: 99%

N@a and N@d: Oligomer and Partner Specification by Asparagine in Coiled-Coil Interfaces

Fletcher¹,

Bartlett²,

Boyle³

et al. 2017

ACS Chem. Biol.

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“…8A). The predicted coiled coil would contain four heptad repeats that resemble a leucine zipper motif (70). Coiled-coil domains were also predicted for all members of the TadA subfamily of NTPases present in gram-negative bacteria and several of the TadA homologs in Archaea (data not shown).…”

Section: Discussionmentioning

confidence: 93%

Nonspecific Adherence and Fibril Biogenesis by Actinobacillus actinomycetemcomitans : TadA Protein Is an ATPase

et al. 2001

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Cells of Actinobacillus actinomycetemcomitans, a gram-negative pathogen responsible for an aggressive form of juvenile periodontitis, form tenaciously adherent biofilms on solid surfaces. The bacteria produce long fibrils of bundled pili, which are required for adherence. Mutations in flp-1, which encodes the major subunit of the pili, or any of seven downstream tad genes (tadABCDEFG) cause defects in fibril production, autoaggregation, and tenacious adherence. We proposed that the tad genes specify part of a novel secretion system for the assembly and transport of Flp pili. The predicted amino acid sequence of TadA (426 amino acids, 47,140 Da) contains motifs for nucleotide binding and hydrolysis common among secretion NTP hydrolase (NTPase) proteins. In addition, the tadA gene is the first representative of a distinct subfamily of potential type IV secretion NTPase genes. Here we report studies on the function of TadA. The tadA gene was altered to express a modified version of TadA that has the 11-residue epitope (T7-TAG) fused to its C terminus. The TadA-T7 protein was indistinguishable from the wild type in its ability to complement the fibril and adherence defects of A. actinomycetemcomitans tadA mutants. Although TadA is not predicted to have a transmembrane domain, the protein was localized to the inner membrane and cytoplasmic fractions of A. actinomycetemcomitans cells, indicating a possible peripheral association with the inner membrane. TadA-T7 was purified and found to hydrolyze ATP in vitro. The ATPase activity is stimulated by Triton X-100, with maximal stimulation at the critical micellar concentration. TadA-T7 forms multimers that are stable during sodium dodecyl sulfatepolyacrylamide gel electrophoresis in nonreducing conditions, and electron microscopy revealed that TadA-T7 can form structures closely resembling the hexameric rings of other type IV secretion NTPases. Site-directed mutagenesis was used to substitute Ala and Gln residues for the conserved Lys residue of the Walker A box for nucleotide binding. Both mutants were found to be defective in their ability to complement tadA mutants. We suggest that the ATPase activity of TadA is required to energize the assembly or secretion of Flp pili for tight adherence of A. actinomycetemcomitans.

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“…The hydrophobic effect drives the burial of nonpolar residues at the helix-pairing interface and influences geometric details of resulting structures (4). Electrostatic and polar residues at buried or solvent-exposed locations provide additional means to manipulate structural features by stabilization or destabilization (negative design) of key interactions (5)(6)(7)(8).…”

mentioning

confidence: 99%

Toward the development of peptide nanofilaments and nanoropes as smart materials

Wagner

Phillips

Ali

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

118

117

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Protein design studies using coiled coils have illustrated the potential of engineering simple peptides to self-associate into polymers and networks. Although basic aspects of self-assembly in protein systems have been demonstrated, it remains a major challenge to create materials whose large-scale structures are well determined from design of local protein-protein interactions. Here, we show the design and characterization of a helical peptide, which uses phased hydrophobic interactions to drive assembly into nanofilaments and fibrils (''nanoropes''). Using the hydrophobic effect to drive self-assembly circumvents problems of uncontrolled self-assembly seen in previous approaches that used electrostatics as a mode for self-assembly. The nanostructures designed here are characterized by biophysical methods including analytical ultracentrifugation, dynamic light scattering, and circular dichroism to measure their solution properties, and atomic force microscopy to study their behavior on surfaces. Additionally, the assembly of such structures can be predictably regulated by using various environmental factors, such as pH, salt, other molecular crowding reagents, and specifically designed ''capping'' peptides. This ability to regulate self-assembly is a critical feature in creating smart peptide biomaterials.biomaterial ͉ coiled coil ͉ protein design ͉ circular dichroism ͉ atomic force microscopy D esigned proteins are valuable paradigms for engineering at the nanoscale, offering favorable properties such as atomiclevel precision and tight regulation of self-assembly by using a variety of environmental cues (i.e., pH, ionic strength, temperature) (1-3). As one of the most well studied and naturally abundant structural motifs in proteins, coiled coils are particularly suited to protein design. Their basic sequence feature, the heptad repeat, is a seven-residue pattern (abcdefg) n of nonpolar and polar residues that gives rise to amphipathic ␣-helices. The hydrophobic effect drives the burial of nonpolar residues at the helix-pairing interface and influences geometric details of resulting structures (4). Electrostatic and polar residues at buried or solvent-exposed locations provide additional means to manipulate structural features by stabilization or destabilization (negative design) of key interactions (5-8).Recent attempts to design self-assembling protein networks by using coiled coils have focused on simple systems, such as linear and branched fibrils (9-14), planar assemblies (15), and hydrogels (16-18). Specifically, filament formation has been achieved by stabilization of intermediate structures that foster intermolecular coiled-coil interactions (11)(12)(13)(14). Previous studies have reported the design of short helical peptides, which interact via a dimeric coiled-coil motif and are stabilized by a combination of overlapping hydrophobic and electrostatic intermolecular interactions (9, 13, 14). These peptides do indeed self-assemble into filaments; however, these filaments also show evidence of extensive...

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The contribution of buried polar groups to the conformational stability of the GCN4 coiled coil11Edited by C. R. Matthews

Cited by 39 publications

References 37 publications

N@a and N@d: Oligomer and Partner Specification by Asparagine in Coiled-Coil Interfaces

N@a and N@d: Oligomer and Partner Specification by Asparagine in Coiled-Coil Interfaces

Nonspecific Adherence and Fibril Biogenesis by Actinobacillus actinomycetemcomitans : TadA Protein Is an ATPase

Toward the development of peptide nanofilaments and nanoropes as smart materials

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