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A coiled-coil motif that sequesters ions to the hydrophobic core.

ABSTRACT: Most core residues of coiled coils are hydrophobic. Occasional polar residues are thought to lower stability, but impart structural specificity. The coiled coils of trimeric autotransporter adhesins (TAAs) are conspicuous for their large number of polar residues in position d of the core, which often leads to their prediction as natively unstructured regions. The most frequent residue, asparagine (N@d), can occur in runs of up to 19 consecutive heptads, frequently in the motif [I/V]xxNTxx. In the Salmonella TAA, SadA, the core asparagines form rings of interacting residues with the following threonines, grouped around a central anion. This conformation is observed generally in N@d layers from trimeric coiled coils of known structure. Attempts to impose a different register on the motif show that the asparagines orient themselves specifically into the core, even against conflicting information from flanking domains. When engineered into the GCN4 leucine zipper, N@d layers progressively destabilized the structure, but zippers with 3 N@d layers still folded at high concentration. We propose that N@d layers maintain the coiled coils of TAAs in a soluble, export-competent state during autotransport through the outer membrane. More generally, we think that polar motifs that are both periodic and conserved may often reflect special folding requirements, rather than an unstructured state of the mature proteins.

SUBMITTER: Hartmann MD

PROVIDER: S-EPMC2749845 | biostudies-literature |

REPOSITORIES: biostudies-literature

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A Hendecad Motif Is Preferred for Heterochiral Coiled-Coil Formation.

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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.

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Project description:Polar residues lying between adjacent ?-helical chains of coiled-coils often contribute to coiled-coil curvature and flexibility, while more typical core hydrophobic residues anneal the chains together. In tropomyosins, ranging from smooth and skeletal muscle to cytoplasmic isoforms, a highly conserved Asp at residue 137 places negative charges within the tropomyosin coiled-coil core in a position which may affect the conformation needed for tropomyosin binding and regulatory movements on actin. Proteolytic susceptibility suggested that substituting a canonical Leu for the naturally occurring Asp at residue 137 increases inter-chain rigidity by stabilizing the tropomyosin coiled-coil. Using molecular dynamics, we now directly assess changes in coiled-coil curvature and flexibility caused by such mutants. Although the coiled-coil flexibility is modestly diminished near the residue 137 mutation site, as expected, a delocalized increase in flexibility along the overall coiled-coil is observed. Even though the average shape of the D137L tropomyosin is straighter than that of wild-type tropomyosin, it is still capable of binding actin due to this increase in flexibility. We conclude that the conserved, non-canonical Asp-137 destabilizes the local structure resulting in a local flexible region in the middle of tropomyosin that normally is important for tropomyosin steady-state equilibrium position on actin.

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In vivo bypass of chaperone by extended coiled-coil motif in T4 tail fiber.

Project description:The distal-half tail fiber of bacteriophage T4 is made of three gene products: trimeric gp36 and gp37 and monomeric gp35. Chaperone P38 is normally required for folding gp37 peptides into a P37 trimer; however, a temperature-sensitive mutation in T4 (ts3813) that suppresses this requirement at 30 degrees C but not at 42 degrees C was found in gene 37 (R. J. Bishop and W. B. Wood, Virology 72:244-254, 1976). Sequencing of the temperature-sensitive mutant revealed a 21-bp duplication of wild-type gene 37 inserted into its C-terminal portion (S. Hashemolhosseini et al., J. Mol. Biol. 241:524-533, 1994). We noticed that the 21-amino-acid segment encompassing this duplication in the ts3813 mutant has a sequence typical of a coiled coil and hypothesized that its extension would relieve the temperature sensitivity of the ts3813 mutation. To test our hypothesis, we crossed the T4 ts3813 mutant with a plasmid encoding an engineered pentaheptad coiled coil. Each of the six mutants that we examined retained two amber mutations in gene 38 and had a different coiled-coil sequence varying from three to five heptads. While the sequences varied, all maintained the heptad-repeating coiled-coil motif and produced plaques at up to 50 degrees C. This finding strongly suggests that the coiled-coil motif is a critical factor in the folding of gp37. The presence of a terminal coiled-coil-like sequence in the tail fiber genes of 17 additional T-even phages implies the conservation of this mechanism. The increased melting temperature should be useful for "clamps" to initiate the folding of trimeric beta-helices in vitro and as an in vivo screen to identify, sequence, and characterize trimeric coiled coils.

| S-EPMC532435 | biostudies-literature

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