Crystal structure of a trimeric form of the K(V)7.1 (KCNQ1) A-domain tail coiled-coil reveals structural plasticity and context dependent changes in a putative coiled-coil trimerization motif.
ABSTRACT Coiled-coils are widespread protein-protein interaction motifs typified by the heptad repeat (abcdefg)(n) in which "a" and "d" positions are hydrophobic residues. Although identification of likely coiled-coil sequences is robust, prediction of strand order remains elusive. We present the X-ray crystal structure of a short form (residues 583-611), "Q1-short," of the coiled-coil assembly specificity domain from the voltage-gated potassium channel Kv7.1 (KCNQ1) determined at 1.7 A resolution. Q1-short lacks one and half heptads present in a previously studied tetrameric coiled-coil construct, Kv7.1 585-621, "Q1-long." Surprisingly, Q1-short crystallizes as a trimer. In solution, Q1-short self-assembles more poorly than Q1-long and depends on an R-h-x-x-h-E motif common to trimeric coiled-coils. Addition of native sequences that include "a" and "d" positions C-terminal to Q1-short overrides the R-h-x-x-h-E motif influence and changes assembly state from a weakly associated trimer to a strongly associated tetramer. These data provide a striking example of a naturally occurring amino sequence that exhibits context-dependent folding into different oligomerization states, a three-stranded versus a four-stranded coiled-coil. The results emphasize the degenerate nature of coiled-coil energy landscapes in which small changes can have drastic effects on oligomerization. Discovery of these properties in an ion channel assembly domain and prevalence of the R-h-x-x-h-E motif in coiled-coil assembly domains of a number of different channels that are thought to function as tetrameric assemblies raises the possibility that such sequence features may be important for facilitating the assembly of intermediates en route to the final native state.
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ABSTRACT: A conserved asparagine (Asn 16) buried in the interface of the GCN4 leucine zipper selectively favours the parallel, dimeric, coiled-coil structure. To test if other polar residues confer oligomerization specificity, the structural effects of Gln and Lys substitutions for Asn 16 were characterized. Like the wild-type peptide, the Asn 16Lys mutant formed exclusively dimers. In contrast, Gln 16, despite its chemical similarity to Asn, allowed the peptide to form both dimers and trimers. The Gln 16 side chain was accommodated by qualitatively different interactions in the dimer and trimer crystal structures. These findings demonstrate that the structural selectivity of polar residues results not only from the burial of polar atoms, but also depends on the complementarity of the side-chain stereochemistry with the surrounding structural environment.Natural Structural Biology 01/1997; 3(12):1011-8.
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ABSTRACT: Recurrent vaginal candidiasis (RVC) is an important health problem with unknown pathogenesis. Although impairment of the T-cell response is associated with persistent or recurrent candidiasis, data on immunologic responses in patients with RVC are controversial. To evaluate the T-cell response in patients with RVC and the ability of cytokines and cytokine antagonists to modulate IFN-gamma production in cultures stimulated with Candida albicans antigens. Participants in the study included 13 patients with RVC and 7 control women with sporadic candidiasis. Cytokines were determined by ELISA in supernatants of mononuclear cells with C albicans, purified protein derivative, or tetanus toxoid antigen. IFN-gamma production was absent or low in 11 of 13 women (84.6%) with RVC. Absent or low IFN-gamma production was specific to C albicans antigens (189 +/- 389 pg/mL), because high IFN-gamma levels were found in cultures stimulated with purified protein derivative (739 +/- 774 pg/mL) or tetanus toxoid antigens (1085 +/- 546 pg/mL). Monoclonal antibody anti-IL-10 enhanced IFN-gamma levels (750 +/- 753 pg/mL), and IL-10 suppressed this cytokine production in patients with sporadic candidiasis. Mononuclear cells from patients with RVC stimulated with C albicans antigen have low or absent IFN-gamma production. IL-10 plays an important role in downregulation of the T-cell response in these patients.Journal of Allergy and Clinical Immunology 02/2002; 109(1):102-5. · 11.00 Impact Factor
Article: Trafficking of the Ca2+-activated K+ channel, hIK1, is dependent upon a C-terminal leucine zipper.[show abstract] [hide abstract]
ABSTRACT: We demonstrate that the C-terminal truncation of hIK1 results in a loss of functional channels. This could be caused by either (i) a failure of the channel to traffic to the plasma membrane or (ii) the expression of non-functional channels. To delineate among these possibilities, a hemagglutinin epitope was inserted into the extracellular loop between transmembrane domains S3 and S4. Surface expression and channel function were measured by immunofluorescence, cell surface immunoprecipitation, and whole-cell patch clamp techniques. Although deletion of the last 14 amino acids of hIK1 (L414STOP) had no effect on plasma membrane expression and function, deletion of the last 26 amino acids (K402STOP) resulted in a complete loss of membrane expression. Mutation of the leucine heptad repeat ending at Leu(406) (L399A/L406A) completely abrogated membrane localization. Additional mutations within the heptad repeat (L385A/L392A, L392A/L406A) or of the a positions (I396A/L403A) resulted in a near-complete loss of membrane-localized channel. In contrast, mutating individual leucines did not compromise channel trafficking or function. Both membrane localization and function of L399A/L406A could be partially restored by incubation at 27 degrees C. Co-immunoprecipitation studies demonstrated that leucine zipper mutations do not compromise multimer formation. In contrast, we demonstrated that the leucine zipper region of hIK1 is capable of co-assembly and that this is dependent upon an intact leucine zipper. Finally, this leucine zipper is conserved in another member of the gene family, SK3. However, mutation of the leucine zipper in SK3 had no effect on plasma membrane localization or function. In conclusion, we demonstrate that the C-terminal leucine zipper is critical to facilitate correct folding and plasma membrane trafficking of hIK1, whereas this function is not conserved in other gene family members.Journal of Biological Chemistry 04/2003; 278(10):8476-86. · 4.77 Impact Factor