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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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Related in: MedlinePlus

Modeling hydrophobic interactions leading to keratin tetramer formation and/or stability. (a) Surface contour of the modeled K17 (blue)/K6α (green) heterodimer. Hydrophobic residues are highlighted in yellow; alanine residues are white. Arrows point to the main hydrophobic stripe. As shown in Fig. 2 e, an additional f-position leucine (#) may be part of the main hydrophobic stripe. (b) Same as a, except the model has been flipped 180° along a horizontal axis to show the back side of the coiled-coil. (c and d) Electrostatic surface contour of the K17/K6α heterodimer. The hydrophobic stripe is outlined with dashes. Negative charges are depicted by red; positive charges are depicted by blue. (e) Potential interactions between the main hydrophobic stripe and other shorter stripes present in the 1B domain of both types I and II keratins are depicted by dotted lines between two adjacent dimers. The face of interaction between two dimers within a tetramer is unknown. Also, it is unknown whether two dimers supercoil around each other, or exist as straight coils (Er Rafik et al., 2004). Depending on the axial alignment of antiparallel dimers, the hydrophobic stripe may interact with self (1—1′) or with the shorter strips (1—2, 1—3, 1—4). Because the azimuthal alignment is also unknown, the top and bottom models depict front and back views of the same dimer, oriented in the same direction (C–N), so that all hydrophobic stripes and potential tetrameric interactions can be viewed. The three dimers shown are not to be confused as a potential hexamer; rather the image shows two views of the same tetramer, with the central dimer present in both views. To simplify, we have depicted interactions with only the main hydrophobic stripe in the central dimer (1′). Additional interactions could occur between the shorter stripes 2′, 3′, and 4′ (not depicted) in the center dimer and those already denoted in the top and bottom dimers.
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fig5: Modeling hydrophobic interactions leading to keratin tetramer formation and/or stability. (a) Surface contour of the modeled K17 (blue)/K6α (green) heterodimer. Hydrophobic residues are highlighted in yellow; alanine residues are white. Arrows point to the main hydrophobic stripe. As shown in Fig. 2 e, an additional f-position leucine (#) may be part of the main hydrophobic stripe. (b) Same as a, except the model has been flipped 180° along a horizontal axis to show the back side of the coiled-coil. (c and d) Electrostatic surface contour of the K17/K6α heterodimer. The hydrophobic stripe is outlined with dashes. Negative charges are depicted by red; positive charges are depicted by blue. (e) Potential interactions between the main hydrophobic stripe and other shorter stripes present in the 1B domain of both types I and II keratins are depicted by dotted lines between two adjacent dimers. The face of interaction between two dimers within a tetramer is unknown. Also, it is unknown whether two dimers supercoil around each other, or exist as straight coils (Er Rafik et al., 2004). Depending on the axial alignment of antiparallel dimers, the hydrophobic stripe may interact with self (1—1′) or with the shorter strips (1—2, 1—3, 1—4). Because the azimuthal alignment is also unknown, the top and bottom models depict front and back views of the same dimer, oriented in the same direction (C–N), so that all hydrophobic stripes and potential tetrameric interactions can be viewed. The three dimers shown are not to be confused as a potential hexamer; rather the image shows two views of the same tetramer, with the central dimer present in both views. To simplify, we have depicted interactions with only the main hydrophobic stripe in the central dimer (1′). Additional interactions could occur between the shorter stripes 2′, 3′, and 4′ (not depicted) in the center dimer and those already denoted in the top and bottom dimers.

Mentions: The ability to obtain high yields of type I/II keratin heterotetramers in the presence of 8 M urea underscores the unusual stability of these complexes, and hints at a key role for hydrophobic interactions during their formation. We identified a novel determinant–a short stripe of four hydrophobic residues aligned on the surface of coil 1B in type I keratins engaged in heterodimers–that underlies this property. This stripe is specific to type I keratins capable of forming urea-stable heterotetramers, such as K14, K17 (Wawersik et al., 1997) and K18 (Yamada et al., 2002), and is imperfectly conserved in K19 (Fradette et al., 1998), K10 (unpublished data), and K16 (Wawersik et al., 1997; this study), all unable to form urea-stable tetramers. Three different assays (anion exchange chromatography, cross-linking, and blue native gel electrophoresis) have shown consistently that K16 forms less stable tetramers than related type I keratins. For mK16, this property is likely due to the discontinuity of the hydrophobic stripe in coil 1B. The duplication of these observations with both purified, recombinant proteins in vitro and native keratin complexes in the context of total protein lysates from cultured primary keratinocytes supports a likely physiological role for this newly defined determinant. The ability to interconvert between urea-stable and urea-unstable heterotetramers through site-directed mutagenesis suggests that this hydrophobic stripe is a key determinant of the differential stability of keratin tetramers under denaturing conditions. Other IFs, including the type III vimentin, are missing this stripe, correlating with the inability of vimentin to form stable tetramers in 6 M urea (Coulombe and Fuchs, 1990). Given that in vitro assembly buffer conditions differ between IF types, it is likely that the driving forces for tetramerization (and further assembly) also differ between IF types. Our report extends others focused on the role of charged residues (Meng et al., 1994; Mehrani et al., 2001), which are periodically distributed in the rod domain (Parry et al., 1977; McLachlan and Stewart, 1982). Charge interactions that strongly influence keratin tetramer stability (without disrupting filament formation) in vitro have been identified in coils 1A, 2A, and 2B, but not in coil 1B (Mehrani et al., 2001). Also, charge interactions between the relatively basic rod-proximal head domain and the relatively acidic rod domain have been shown to influence tetramerization (Hatzfeld and Burba, 1994; Mucke et al., 2004). Comparison of the presence of hydrophobic stripe identified in this work with other hydrophobic and charged residues in the 1B domain reveals an overall negative charge on the COOH-terminal portion of the 1B domain, a more negative charge on the NH2-terminal portion, with the hydrophobic stripe present near the junction of these two domains (Fig. 5, a–d). Here, we provide data that hydrophobic interactions in subdomain 1B are a contributing force in stabilizing keratin tetramers.


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)

Modeling hydrophobic interactions leading to keratin tetramer formation and/or stability. (a) Surface contour of the modeled K17 (blue)/K6α (green) heterodimer. Hydrophobic residues are highlighted in yellow; alanine residues are white. Arrows point to the main hydrophobic stripe. As shown in Fig. 2 e, an additional f-position leucine (#) may be part of the main hydrophobic stripe. (b) Same as a, except the model has been flipped 180° along a horizontal axis to show the back side of the coiled-coil. (c and d) Electrostatic surface contour of the K17/K6α heterodimer. The hydrophobic stripe is outlined with dashes. Negative charges are depicted by red; positive charges are depicted by blue. (e) Potential interactions between the main hydrophobic stripe and other shorter stripes present in the 1B domain of both types I and II keratins are depicted by dotted lines between two adjacent dimers. The face of interaction between two dimers within a tetramer is unknown. Also, it is unknown whether two dimers supercoil around each other, or exist as straight coils (Er Rafik et al., 2004). Depending on the axial alignment of antiparallel dimers, the hydrophobic stripe may interact with self (1—1′) or with the shorter strips (1—2, 1—3, 1—4). Because the azimuthal alignment is also unknown, the top and bottom models depict front and back views of the same dimer, oriented in the same direction (C–N), so that all hydrophobic stripes and potential tetrameric interactions can be viewed. The three dimers shown are not to be confused as a potential hexamer; rather the image shows two views of the same tetramer, with the central dimer present in both views. To simplify, we have depicted interactions with only the main hydrophobic stripe in the central dimer (1′). Additional interactions could occur between the shorter stripes 2′, 3′, and 4′ (not depicted) in the center dimer and those already denoted in the top and bottom dimers.
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Related In: Results  -  Collection

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fig5: Modeling hydrophobic interactions leading to keratin tetramer formation and/or stability. (a) Surface contour of the modeled K17 (blue)/K6α (green) heterodimer. Hydrophobic residues are highlighted in yellow; alanine residues are white. Arrows point to the main hydrophobic stripe. As shown in Fig. 2 e, an additional f-position leucine (#) may be part of the main hydrophobic stripe. (b) Same as a, except the model has been flipped 180° along a horizontal axis to show the back side of the coiled-coil. (c and d) Electrostatic surface contour of the K17/K6α heterodimer. The hydrophobic stripe is outlined with dashes. Negative charges are depicted by red; positive charges are depicted by blue. (e) Potential interactions between the main hydrophobic stripe and other shorter stripes present in the 1B domain of both types I and II keratins are depicted by dotted lines between two adjacent dimers. The face of interaction between two dimers within a tetramer is unknown. Also, it is unknown whether two dimers supercoil around each other, or exist as straight coils (Er Rafik et al., 2004). Depending on the axial alignment of antiparallel dimers, the hydrophobic stripe may interact with self (1—1′) or with the shorter strips (1—2, 1—3, 1—4). Because the azimuthal alignment is also unknown, the top and bottom models depict front and back views of the same dimer, oriented in the same direction (C–N), so that all hydrophobic stripes and potential tetrameric interactions can be viewed. The three dimers shown are not to be confused as a potential hexamer; rather the image shows two views of the same tetramer, with the central dimer present in both views. To simplify, we have depicted interactions with only the main hydrophobic stripe in the central dimer (1′). Additional interactions could occur between the shorter stripes 2′, 3′, and 4′ (not depicted) in the center dimer and those already denoted in the top and bottom dimers.
Mentions: The ability to obtain high yields of type I/II keratin heterotetramers in the presence of 8 M urea underscores the unusual stability of these complexes, and hints at a key role for hydrophobic interactions during their formation. We identified a novel determinant–a short stripe of four hydrophobic residues aligned on the surface of coil 1B in type I keratins engaged in heterodimers–that underlies this property. This stripe is specific to type I keratins capable of forming urea-stable heterotetramers, such as K14, K17 (Wawersik et al., 1997) and K18 (Yamada et al., 2002), and is imperfectly conserved in K19 (Fradette et al., 1998), K10 (unpublished data), and K16 (Wawersik et al., 1997; this study), all unable to form urea-stable tetramers. Three different assays (anion exchange chromatography, cross-linking, and blue native gel electrophoresis) have shown consistently that K16 forms less stable tetramers than related type I keratins. For mK16, this property is likely due to the discontinuity of the hydrophobic stripe in coil 1B. The duplication of these observations with both purified, recombinant proteins in vitro and native keratin complexes in the context of total protein lysates from cultured primary keratinocytes supports a likely physiological role for this newly defined determinant. The ability to interconvert between urea-stable and urea-unstable heterotetramers through site-directed mutagenesis suggests that this hydrophobic stripe is a key determinant of the differential stability of keratin tetramers under denaturing conditions. Other IFs, including the type III vimentin, are missing this stripe, correlating with the inability of vimentin to form stable tetramers in 6 M urea (Coulombe and Fuchs, 1990). Given that in vitro assembly buffer conditions differ between IF types, it is likely that the driving forces for tetramerization (and further assembly) also differ between IF types. Our report extends others focused on the role of charged residues (Meng et al., 1994; Mehrani et al., 2001), which are periodically distributed in the rod domain (Parry et al., 1977; McLachlan and Stewart, 1982). Charge interactions that strongly influence keratin tetramer stability (without disrupting filament formation) in vitro have been identified in coils 1A, 2A, and 2B, but not in coil 1B (Mehrani et al., 2001). Also, charge interactions between the relatively basic rod-proximal head domain and the relatively acidic rod domain have been shown to influence tetramerization (Hatzfeld and Burba, 1994; Mucke et al., 2004). Comparison of the presence of hydrophobic stripe identified in this work with other hydrophobic and charged residues in the 1B domain reveals an overall negative charge on the COOH-terminal portion of the 1B domain, a more negative charge on the NH2-terminal portion, with the hydrophobic stripe present near the junction of these two domains (Fig. 5, a–d). Here, we provide data that hydrophobic interactions in subdomain 1B are a contributing force in stabilizing keratin tetramers.

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