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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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Type I keratins display a stripe of hydrophobic amino acids on the surface of the type I/type II heterodimer. Human K16/mK6α, mK17/mK6α, and mK16/mK6α were modeled onto Cortexillin I with Modeller6 (Sali and Blundell, 1993). Figures were generated using POVScript (Fenn et al., 2003) and rendered with Raster3D (Merritt and Bacon, 1997). The carbon backbone coiled-coils were overlaid for comparison. (a) hK16/mK6α (yellow) and mK16/mK6α (pink). (b) mK17/mK6α (purple) and mK16/mK6α (pink). No significant differences in the backbone were observed. (c) mK17 (blue)/K6α (green) dimer. The carbon backbone is represented by the ribbon. The NH2 and COOH termini are left and right, respectively. A stripe of hydrophobic surface residues on K17 are highlighted in orange below their single-letter amino acid codes. (d) mK16 (blue)/K6α (green) dimer. A glutamine residue (red) occurs in the midst of the hydrophobic stripe. (e) Amino acid sequence alignment of type I keratins and consensus sequences of the residues corresponding to the hydrophobic stripe visualized in K17 (see c). The asterisk denotes the presence of an additional hydrophobic, f-position leucine at the COOH terminus of the hydrophobic stripe. Type I consensus sequence is taken from Branchiostoma Lancelet type I YI, Branchiostoma Floridae type I EI, hK14, hK17, hK18, hK12, and several hard α type I keratins. Consensus sequences for various other IFs (types III–V) are also listed. The heptad repeat (abcdefg) is listed at the top starting from the first amino acid of subdomain 1B. Highlighted residues indicate the positions of the four adjacent hydrophobic amino acids visualized in K17 (see c). Hydrophobic/nonpolar residues (blue), hydrophilic residues (green), and charged residues (red) are colored. Pro 188 in hK16 is coded orange. (f) Four mutant proteins were created, with the mutated amino acids listed below the residues that were changed in each mutant.
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fig2: Type I keratins display a stripe of hydrophobic amino acids on the surface of the type I/type II heterodimer. Human K16/mK6α, mK17/mK6α, and mK16/mK6α were modeled onto Cortexillin I with Modeller6 (Sali and Blundell, 1993). Figures were generated using POVScript (Fenn et al., 2003) and rendered with Raster3D (Merritt and Bacon, 1997). The carbon backbone coiled-coils were overlaid for comparison. (a) hK16/mK6α (yellow) and mK16/mK6α (pink). (b) mK17/mK6α (purple) and mK16/mK6α (pink). No significant differences in the backbone were observed. (c) mK17 (blue)/K6α (green) dimer. The carbon backbone is represented by the ribbon. The NH2 and COOH termini are left and right, respectively. A stripe of hydrophobic surface residues on K17 are highlighted in orange below their single-letter amino acid codes. (d) mK16 (blue)/K6α (green) dimer. A glutamine residue (red) occurs in the midst of the hydrophobic stripe. (e) Amino acid sequence alignment of type I keratins and consensus sequences of the residues corresponding to the hydrophobic stripe visualized in K17 (see c). The asterisk denotes the presence of an additional hydrophobic, f-position leucine at the COOH terminus of the hydrophobic stripe. Type I consensus sequence is taken from Branchiostoma Lancelet type I YI, Branchiostoma Floridae type I EI, hK14, hK17, hK18, hK12, and several hard α type I keratins. Consensus sequences for various other IFs (types III–V) are also listed. The heptad repeat (abcdefg) is listed at the top starting from the first amino acid of subdomain 1B. Highlighted residues indicate the positions of the four adjacent hydrophobic amino acids visualized in K17 (see c). Hydrophobic/nonpolar residues (blue), hydrophilic residues (green), and charged residues (red) are colored. Pro 188 in hK16 is coded orange. (f) Four mutant proteins were created, with the mutated amino acids listed below the residues that were changed in each mutant.

Mentions: We expected that Pro188 in hK16 would kink the α-helical backbone and create a local disturbance at surface of the dimer (Wawersik et al., 1997). Models of the hK16/K6 dimer indeed exhibited a turn in the backbone at Pro188; however, the α-helix surrounding the proline was not severely disrupted (Fig. 2 a). The mK16/K6 backbone proved similar to that of hK16/K6 (Fig. 2 a), with a RMSD of 1.14Å over the entire 1B subdomain (14 heptads), compared with a RMSD of 2.07Å when overlaid with mK17/K6 (Fig. 2 b). RMSD values decreased to 0.68Å when five heptads centered around Pro188 were compared in these dimers, implying that the peptide backbones of the helices are structurally very similar. These modeling efforts nevertheless proved useful in that they exposed an intriguing difference when comparing the side chains of hK16/K6, mK17/K6, and mK16/K6 dimers. In mK17, a hydrophobic stripe of four apolar amino acids, located in b and f positions of consecutive heptad repeats, was exposed to the surface of the dimer, rather than buried within (Fig. 2 c). This hydrophobic stripe spans the region where Pro188 is located in hK16. Moreover, a hydrophilic residue, Gln, replaces one of these hydrophobic residues in mK16 (Fig. 2 d). Otherwise, the hydrophobic stripe is evolutionarily conserved in many type I keratins, including vertebrates and cephalochordates (e.g., Branchiostoma; Fig. 2 e). The hydrophobic stripe is not conserved in any other IF sequence type, including type II keratins (Fig. 2 e).


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)

Type I keratins display a stripe of hydrophobic amino acids on the surface of the type I/type II heterodimer. Human K16/mK6α, mK17/mK6α, and mK16/mK6α were modeled onto Cortexillin I with Modeller6 (Sali and Blundell, 1993). Figures were generated using POVScript (Fenn et al., 2003) and rendered with Raster3D (Merritt and Bacon, 1997). The carbon backbone coiled-coils were overlaid for comparison. (a) hK16/mK6α (yellow) and mK16/mK6α (pink). (b) mK17/mK6α (purple) and mK16/mK6α (pink). No significant differences in the backbone were observed. (c) mK17 (blue)/K6α (green) dimer. The carbon backbone is represented by the ribbon. The NH2 and COOH termini are left and right, respectively. A stripe of hydrophobic surface residues on K17 are highlighted in orange below their single-letter amino acid codes. (d) mK16 (blue)/K6α (green) dimer. A glutamine residue (red) occurs in the midst of the hydrophobic stripe. (e) Amino acid sequence alignment of type I keratins and consensus sequences of the residues corresponding to the hydrophobic stripe visualized in K17 (see c). The asterisk denotes the presence of an additional hydrophobic, f-position leucine at the COOH terminus of the hydrophobic stripe. Type I consensus sequence is taken from Branchiostoma Lancelet type I YI, Branchiostoma Floridae type I EI, hK14, hK17, hK18, hK12, and several hard α type I keratins. Consensus sequences for various other IFs (types III–V) are also listed. The heptad repeat (abcdefg) is listed at the top starting from the first amino acid of subdomain 1B. Highlighted residues indicate the positions of the four adjacent hydrophobic amino acids visualized in K17 (see c). Hydrophobic/nonpolar residues (blue), hydrophilic residues (green), and charged residues (red) are colored. Pro 188 in hK16 is coded orange. (f) Four mutant proteins were created, with the mutated amino acids listed below the residues that were changed in each mutant.
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fig2: Type I keratins display a stripe of hydrophobic amino acids on the surface of the type I/type II heterodimer. Human K16/mK6α, mK17/mK6α, and mK16/mK6α were modeled onto Cortexillin I with Modeller6 (Sali and Blundell, 1993). Figures were generated using POVScript (Fenn et al., 2003) and rendered with Raster3D (Merritt and Bacon, 1997). The carbon backbone coiled-coils were overlaid for comparison. (a) hK16/mK6α (yellow) and mK16/mK6α (pink). (b) mK17/mK6α (purple) and mK16/mK6α (pink). No significant differences in the backbone were observed. (c) mK17 (blue)/K6α (green) dimer. The carbon backbone is represented by the ribbon. The NH2 and COOH termini are left and right, respectively. A stripe of hydrophobic surface residues on K17 are highlighted in orange below their single-letter amino acid codes. (d) mK16 (blue)/K6α (green) dimer. A glutamine residue (red) occurs in the midst of the hydrophobic stripe. (e) Amino acid sequence alignment of type I keratins and consensus sequences of the residues corresponding to the hydrophobic stripe visualized in K17 (see c). The asterisk denotes the presence of an additional hydrophobic, f-position leucine at the COOH terminus of the hydrophobic stripe. Type I consensus sequence is taken from Branchiostoma Lancelet type I YI, Branchiostoma Floridae type I EI, hK14, hK17, hK18, hK12, and several hard α type I keratins. Consensus sequences for various other IFs (types III–V) are also listed. The heptad repeat (abcdefg) is listed at the top starting from the first amino acid of subdomain 1B. Highlighted residues indicate the positions of the four adjacent hydrophobic amino acids visualized in K17 (see c). Hydrophobic/nonpolar residues (blue), hydrophilic residues (green), and charged residues (red) are colored. Pro 188 in hK16 is coded orange. (f) Four mutant proteins were created, with the mutated amino acids listed below the residues that were changed in each mutant.
Mentions: We expected that Pro188 in hK16 would kink the α-helical backbone and create a local disturbance at surface of the dimer (Wawersik et al., 1997). Models of the hK16/K6 dimer indeed exhibited a turn in the backbone at Pro188; however, the α-helix surrounding the proline was not severely disrupted (Fig. 2 a). The mK16/K6 backbone proved similar to that of hK16/K6 (Fig. 2 a), with a RMSD of 1.14Å over the entire 1B subdomain (14 heptads), compared with a RMSD of 2.07Å when overlaid with mK17/K6 (Fig. 2 b). RMSD values decreased to 0.68Å when five heptads centered around Pro188 were compared in these dimers, implying that the peptide backbones of the helices are structurally very similar. These modeling efforts nevertheless proved useful in that they exposed an intriguing difference when comparing the side chains of hK16/K6, mK17/K6, and mK16/K6 dimers. In mK17, a hydrophobic stripe of four apolar amino acids, located in b and f positions of consecutive heptad repeats, was exposed to the surface of the dimer, rather than buried within (Fig. 2 c). This hydrophobic stripe spans the region where Pro188 is located in hK16. Moreover, a hydrophilic residue, Gln, replaces one of these hydrophobic residues in mK16 (Fig. 2 d). Otherwise, the hydrophobic stripe is evolutionarily conserved in many type I keratins, including vertebrates and cephalochordates (e.g., Branchiostoma; Fig. 2 e). The hydrophobic stripe is not conserved in any other IF sequence type, including type II keratins (Fig. 2 e).

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