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A small surface hydrophobic stripe in the coiled-coil domain of type I keratins mediates tetramer stability.

Bernot KM, Lee CH, Coulombe PA - J. Cell Biol. (2005)

Bottom Line: Through molecular modeling and site-directed mutagenesis, we document a hitherto unnoticed hydrophobic stripe exposed at the surface of coiled-coil keratin heterodimers that contributes to the extraordinary stability of heterotetramers.The inability of K16 to form urea-stable tetramers in vitro correlates with an increase in its turnover rate in vivo.The data presented support a specific conformation for the assembly competent IF tetramer, provide a molecular basis for their differential stability in vitro, and point to the physiological relevance associated with this property in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

ABSTRACT
Intermediate filaments (IFs) are fibrous polymers encoded by a large family of differentially expressed genes that provide crucial structural support in the cytoplasm and nucleus in higher eukaryotes. The mechanisms involved in bringing together approximately 16 elongated coiled-coil dimers to form an IF are poorly defined. Available evidence suggests that tetramer subunits play a key role during IF assembly and regulation. Through molecular modeling and site-directed mutagenesis, we document a hitherto unnoticed hydrophobic stripe exposed at the surface of coiled-coil keratin heterodimers that contributes to the extraordinary stability of heterotetramers. The inability of K16 to form urea-stable tetramers in vitro correlates with an increase in its turnover rate in vivo. The data presented support a specific conformation for the assembly competent IF tetramer, provide a molecular basis for their differential stability in vitro, and point to the physiological relevance associated with this property in vivo.

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Mutation of surface hydrophobic residues in type I keratins results in destabilization of tetramers. (a) Anion-exchange chromatography of types I and II keratin complexes, followed by cross-linking with BS3 in the presence of 4, 6, or 8 M urea was performed as in Fig. 1. Cross-linked products (4 μg of proteins) were analyzed by SDS-PAGE on 4–12% gradient gels. Molecular mass standards are indicated on the left, whereas oligomeric complex positions of tetramer (T), dimer (D) type II (II M) and type I (I M) monomer are indicated on the right. Dashed boxes identify the region quantitated in b. (b) Densitometry was performed on the SDS-PAGE gel in panel a and in replicate experiments. The intensities of monomer, dimer, and tetramer were summed to form 100%, and the percentages of tetramers at 4 M (light gray box), 6 M (dark gray box), or 8 M (black box) urea were graphed (mean ± SEM). (c–g) Heterotypic complexes isolated by anion-exchange chromatography were subjected to standard filament assembly conditions (see Materials and methods). (c) Assembly efficiency was analyzed by a high speed sedimentation assay. Supernatant (S) and pellet (P) were loaded onto an 8.5% polyacrylamide gel, followed by staining with Coomassie blue. White lines indicate that intervening lanes have been spliced out. (d–g) Filaments were negatively stained and visualized with transmission electron microscopy. Type I/type II keratins are listed in the bottom left-hand corner of each image. Bar, 500 nm.
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fig3: Mutation of surface hydrophobic residues in type I keratins results in destabilization of tetramers. (a) Anion-exchange chromatography of types I and II keratin complexes, followed by cross-linking with BS3 in the presence of 4, 6, or 8 M urea was performed as in Fig. 1. Cross-linked products (4 μg of proteins) were analyzed by SDS-PAGE on 4–12% gradient gels. Molecular mass standards are indicated on the left, whereas oligomeric complex positions of tetramer (T), dimer (D) type II (II M) and type I (I M) monomer are indicated on the right. Dashed boxes identify the region quantitated in b. (b) Densitometry was performed on the SDS-PAGE gel in panel a and in replicate experiments. The intensities of monomer, dimer, and tetramer were summed to form 100%, and the percentages of tetramers at 4 M (light gray box), 6 M (dark gray box), or 8 M (black box) urea were graphed (mean ± SEM). (c–g) Heterotypic complexes isolated by anion-exchange chromatography were subjected to standard filament assembly conditions (see Materials and methods). (c) Assembly efficiency was analyzed by a high speed sedimentation assay. Supernatant (S) and pellet (P) were loaded onto an 8.5% polyacrylamide gel, followed by staining with Coomassie blue. White lines indicate that intervening lanes have been spliced out. (d–g) Filaments were negatively stained and visualized with transmission electron microscopy. Type I/type II keratins are listed in the bottom left-hand corner of each image. Bar, 500 nm.

Mentions: When assessed by anion exchange chromatography and chemical cross-linking, all three mutant K17/K6 complexes formed unstable tetramers in a similar manner to mK16/K6 (<15% tetramers in 8 M urea). In stark contrast, the mK16VSIL/K6 formed stable tetramers similar to mK17/K6 (>35% tetramers in 8 M urea; Fig. 3, a and b). Of note, mK16 Gln179 (the second amino acid in the hydrophobic stripe; Fig. 2, d and e) is conserved in rat K16 (not depicted). Next, we determined that all mutants formed filaments with the same efficiency as wild-type proteins based on a pelleting assay with high speed centrifugation (Fig. 3 c). Analysis of filament morphology using negative staining and electron microscopy revealed that all mutants formed smooth-surfaced, long filaments that appeared similar to their respective wild-type filaments (Fig. 3, d–g). In particular, the propensity of hK16 (Wawersik et al., 1997) and mK16 (Fig. 3 e) to form small bundles containing two to three filaments was unaltered in mK16VSIL (Fig. 3 g). Thus, the residues forming the hydrophobic stripe on type I keratins are key determinants of the ability to form urea-stable heterotetramers, but do not otherwise significantly affect the potential to form mature filaments in vitro.


A small surface hydrophobic stripe in the coiled-coil domain of type I keratins mediates tetramer stability.

Bernot KM, Lee CH, Coulombe PA - J. Cell Biol. (2005)

Mutation of surface hydrophobic residues in type I keratins results in destabilization of tetramers. (a) Anion-exchange chromatography of types I and II keratin complexes, followed by cross-linking with BS3 in the presence of 4, 6, or 8 M urea was performed as in Fig. 1. Cross-linked products (4 μg of proteins) were analyzed by SDS-PAGE on 4–12% gradient gels. Molecular mass standards are indicated on the left, whereas oligomeric complex positions of tetramer (T), dimer (D) type II (II M) and type I (I M) monomer are indicated on the right. Dashed boxes identify the region quantitated in b. (b) Densitometry was performed on the SDS-PAGE gel in panel a and in replicate experiments. The intensities of monomer, dimer, and tetramer were summed to form 100%, and the percentages of tetramers at 4 M (light gray box), 6 M (dark gray box), or 8 M (black box) urea were graphed (mean ± SEM). (c–g) Heterotypic complexes isolated by anion-exchange chromatography were subjected to standard filament assembly conditions (see Materials and methods). (c) Assembly efficiency was analyzed by a high speed sedimentation assay. Supernatant (S) and pellet (P) were loaded onto an 8.5% polyacrylamide gel, followed by staining with Coomassie blue. White lines indicate that intervening lanes have been spliced out. (d–g) Filaments were negatively stained and visualized with transmission electron microscopy. Type I/type II keratins are listed in the bottom left-hand corner of each image. Bar, 500 nm.
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fig3: Mutation of surface hydrophobic residues in type I keratins results in destabilization of tetramers. (a) Anion-exchange chromatography of types I and II keratin complexes, followed by cross-linking with BS3 in the presence of 4, 6, or 8 M urea was performed as in Fig. 1. Cross-linked products (4 μg of proteins) were analyzed by SDS-PAGE on 4–12% gradient gels. Molecular mass standards are indicated on the left, whereas oligomeric complex positions of tetramer (T), dimer (D) type II (II M) and type I (I M) monomer are indicated on the right. Dashed boxes identify the region quantitated in b. (b) Densitometry was performed on the SDS-PAGE gel in panel a and in replicate experiments. The intensities of monomer, dimer, and tetramer were summed to form 100%, and the percentages of tetramers at 4 M (light gray box), 6 M (dark gray box), or 8 M (black box) urea were graphed (mean ± SEM). (c–g) Heterotypic complexes isolated by anion-exchange chromatography were subjected to standard filament assembly conditions (see Materials and methods). (c) Assembly efficiency was analyzed by a high speed sedimentation assay. Supernatant (S) and pellet (P) were loaded onto an 8.5% polyacrylamide gel, followed by staining with Coomassie blue. White lines indicate that intervening lanes have been spliced out. (d–g) Filaments were negatively stained and visualized with transmission electron microscopy. Type I/type II keratins are listed in the bottom left-hand corner of each image. Bar, 500 nm.
Mentions: When assessed by anion exchange chromatography and chemical cross-linking, all three mutant K17/K6 complexes formed unstable tetramers in a similar manner to mK16/K6 (<15% tetramers in 8 M urea). In stark contrast, the mK16VSIL/K6 formed stable tetramers similar to mK17/K6 (>35% tetramers in 8 M urea; Fig. 3, a and b). Of note, mK16 Gln179 (the second amino acid in the hydrophobic stripe; Fig. 2, d and e) is conserved in rat K16 (not depicted). Next, we determined that all mutants formed filaments with the same efficiency as wild-type proteins based on a pelleting assay with high speed centrifugation (Fig. 3 c). Analysis of filament morphology using negative staining and electron microscopy revealed that all mutants formed smooth-surfaced, long filaments that appeared similar to their respective wild-type filaments (Fig. 3, d–g). In particular, the propensity of hK16 (Wawersik et al., 1997) and mK16 (Fig. 3 e) to form small bundles containing two to three filaments was unaltered in mK16VSIL (Fig. 3 g). Thus, the residues forming the hydrophobic stripe on type I keratins are key determinants of the ability to form urea-stable heterotetramers, but do not otherwise significantly affect the potential to form mature filaments in vitro.

Bottom Line: Through molecular modeling and site-directed mutagenesis, we document a hitherto unnoticed hydrophobic stripe exposed at the surface of coiled-coil keratin heterodimers that contributes to the extraordinary stability of heterotetramers.The inability of K16 to form urea-stable tetramers in vitro correlates with an increase in its turnover rate in vivo.The data presented support a specific conformation for the assembly competent IF tetramer, provide a molecular basis for their differential stability in vitro, and point to the physiological relevance associated with this property in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

ABSTRACT
Intermediate filaments (IFs) are fibrous polymers encoded by a large family of differentially expressed genes that provide crucial structural support in the cytoplasm and nucleus in higher eukaryotes. The mechanisms involved in bringing together approximately 16 elongated coiled-coil dimers to form an IF are poorly defined. Available evidence suggests that tetramer subunits play a key role during IF assembly and regulation. Through molecular modeling and site-directed mutagenesis, we document a hitherto unnoticed hydrophobic stripe exposed at the surface of coiled-coil keratin heterodimers that contributes to the extraordinary stability of heterotetramers. The inability of K16 to form urea-stable tetramers in vitro correlates with an increase in its turnover rate in vivo. The data presented support a specific conformation for the assembly competent IF tetramer, provide a molecular basis for their differential stability in vitro, and point to the physiological relevance associated with this property in vivo.

Show MeSH
Related in: MedlinePlus