Correspondence to Pierre A. Coulombe: coulombe{at}jhmi.edu
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Introduction |
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The human genome contains at least 67 distinct genes encoding proteins able to self-polymerize into 10-12-nm-wide IFs that are regulated in a tissue-, cell type, and differentiation-specific fashion (Hesse et al., 2001). All IF proteins (Mr 40240 kD) share a tripartite domain organization consisting of a central -helical "rod" flanked by nonhelical head and tail domains. In cytoplasmic IF proteins, the rod domain is 310 aa residues long and features long-range heptad repeats (abcdefg)n in which amino acid residues located in the first a and fourth d positions are hydrophobic or apolar, leading to the "knob and hole" packing of two
-helices into a stable coiled-coil dimer (Crick, 1952; Cohen and Parry, 1990). The heptad repeats are interrupted by short linker sequences at three conserved locations, segmenting the rod domain into coils 1A, 1B, 2A, and 2B (35, 101, 19, and 121 resides long, respectively; Parry and Steinert, 1999). The head and tail domains exhibit variable primary and secondary structure and are substrates for phosphorylation and other modifications that regulate IF polymer assembly, dynamics, and interactions with other proteins (Coulombe and Omary, 2002).
Resolving the high resolution structure of F-actin and microtubule fibrous polymers has catapulted these research fields to new heights. Such a detailed understanding is lacking for IFs, owing to the elongated shape and polymerization-prone nature of their constituent proteins, and structural polymorphism (Strelkov et al., 2001). Scanning transmission electron microscopy has shown that on average the IF polymer backbone consists of 16 coiled-coil dimers in cross section (Herrmann et al., 1999). Dimers are structurally elongated (46-nm length; 23-nm width; Quinlan et al., 1984) and polar, because of the parallel and in-register alignment of -helices. Packing 16 dimers into a smooth surfaced, 1012-nm filament represents a tour de force that remains poorly understood. For cytoplasmic IFs, dimers interact along their lateral surfaces with an antiparallel orientation to form apolar tetramers. In vitro, further lateral interactions between tetramers yield unit length filaments (ULFs, 16-nm width; 60-nm length), which then anneal and compact to give rise to mature IFs (Strelkov et al., 2003). Beyond dimer formation, the interactions presiding over the polymerization of IF proteins are less understood.
Tetramers correspond to the oligomeric state of cytoplasmic IF subunits in the soluble pool in vivo (Soellner et al., 1985; Chou et al., 1993), and represent the major subunit that exists below the critical concentration for assembly in vitro (40 µg/ml; Steinert, 1991). Through biophysical techniques, modeling, and biochemical studies, several alignment modes of the two antiparallel dimers within a tetramer have been suggested (Herrmann and Aebi, 1998). The two favored models for the assembly competent tetramer subunit consist of an antiparallel overlap in which either the 1B or the 2B coiled-coil domains of participating dimers are facing one another (designated A11 and A22, respectively; Geisler et al., 1992; Steinert et al., 1993a,b,c; Mucke et al., 2004). Resolving this issue is crucial for furthering our understanding of the assembly, structure, and regulation of IFs in vivo.
Keratin polymerization is initiated through the formation of heterodimers involving types I and II IF proteins. We previously reported that unlike other type I keratins to which it is highly related in primary structure, including K14 and K17, human keratin 16 (hK16) cannot form urea-stable heterotetramers with various type II partners (Paladini et al., 1996). This property is conferred by a single amino acid, proline 188 (Pro 188), occurring in a "d" position of a heptad repeat within subdomain 1B (Wawersik et al., 1997), which is occupied by hydrophobic amino acids (e.g., Val, Ile) in hK14, hK17, and several other human type I keratins. Surprisingly, the corresponding position is occupied by a Phe residue in the mouse orthologue, raising doubts as to its significance in human K16 (Porter et al., 1998). While characterizing the tetramer-forming properties of mK16, we uncovered a hitherto unnoticed hydrophobic stripe exposed on the surface of type I keratins, in a region encompassing Pro188 in human K16, that accounts for the unusual stability of keratin heterotetramers in vitro. We also show that mK16 protein turns over faster in the absence of its polymerization partner mK6 in keratinocytes in primary cultures, correlating tetramer stability in vitro with keratin turnover rate in vivo. We discuss the implication of our results for keratin tetramer structure and keratin regulation.
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Results |
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Given that the previous experiments were performed on recombinant protein, we sought to validate these findings in vivo. We solubilized cultured mouse primary keratinocytes in either 8 M or 6 M urea and separated the lysate on a blue native acrylamide gel, which resolves protein complexes in their native form according to mass. Western blotting of the resolved keratinocyte lysate revealed that K16-containing complexes were smaller than complexes containing the related type I keratins K14 or K17 (Fig. 1 d). Aside from monomers, all bands contain both types I and II keratins (K6 shown, K5 not shown). In another experiment, primary keratinocyte total protein lysates were separated by anion exchange chromatography as in Fig. 1 a. Here, K16 eluted predominantly in the type I monomer and heterodimer peaks, whereas K14 eluted predominantly in the heterotetramer peak (unpublished data). Together, these results suggest that native keratins present in total protein extracts prepared from cells in primary culture behave similarly to purified recombinant proteins.
Type I keratins exhibit a hydrophobic stripe at the surface of coiled-coil heterodimers
Inspection of the 1B subdomain sequence in mK16 does not reveal obvious amino acid candidates likely to disrupt an -helix and thus explain its inability to form urea-stable tetramers, unlike the prediction for Pro188 in hK16 (Wawersik et al., 1997). We applied structural modeling with the aim of identifying features that would be unique to subdomain 1B in mK16, relative to other type I keratins. We used the structure of cortexillin I as a template for modeling (PDB 1D7M; Burkhard et al., 2000) as done before (Briki et al., 2002; Hess et al., 2002), given that it consists of a 18-heptad repeat-long, uninterrupted, coiled-coil-forming domain. After aligning the target and cortexillin I sequences with ClustalX (Thompson et al., 1997), homology model building was performed using default parameters of Modeller6 (Sali and Blundell, 1993). We created several models of the in-register, parallel 1B domain of vimentin and various keratin pairs. All models aligned well with the overall geometry of cortexillin I and yielded satisfactory evaluations (see Materials and methods), except mK17/mK6
, which showed a slight out of range value for surface hydrophilicity.
We expected that Pro188 in hK16 would kink the -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
-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).
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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, dg). 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.
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Probing the significance of keratin tetramer stability
We previously showed that K16 could not compete effectively with K14 in the formation of stable heterotypic complexes in the presence of substoichiometric amounts of type II binding partners in vitro (K5 or K6; Paladini et al., 1996). The availability of suitable mouse models created an opportunity to monitor the fate of K16 protein under conditions of limited partner availability (K6 null mice; Wong et al., 2000) or loss of a major type I keratin competitor (K17 null mice; McGowan et al., 2002). Relative to wild-type control, the steady-state levels of K16 protein (but not mRNA) are much decreased in K6 null skin keratinocytes in primary culture, in which K5 is the only type II keratin left and whose protein level does not become elevated (Fig. 4 a; Wong et al., 2000; Wong and Coulombe, 2003). In contrast, the levels of K14 and K17 proteins are not altered in these cells (Wong et al., 2000; Wong and Coulombe, 2003). Conversely, the levels of K16 protein, but not K14, are increased in K17 null keratinocytes compared with control (Fig. 4 a). These data show that in a living cell context in which K5, K6, K6ß, K14, K15, K16, and K17 all coexist, K16 is uniquely sensitive to perturbations of the balance between types I and II keratins.
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Discussion |
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The paucity of knowledge regarding IF assembly in vivo complicates the determination of the significance of differential tetramer stability, observed in an artificial setting in vitro. By the criteria of mild detergent extraction and high speed centrifugation, the soluble pool of IFs in the cytoplasm is small (<1% in keratinocytes) and consists mostly of tetramers (Soellner et al., 1985; Chou et al., 1993). The amount of hK16 retrieved from the soluble pool does not increase when overexpressed in transgenic mouse skin (Paladini and Coulombe, 1999), suggesting that Pro188 does not affect partitioning to the "soluble pool" in living keratinocytes. Tetramer instability could, as shown here, influence protein half-life under specific circumstances defined in part by assembly partner availability. Another possible outcome is an influence on the size or composition of keratin heterotetramers. Our studies in skin keratinocytes in primary culture revealed at least two types of keratin complexes: those rich in K14 and K17; and those rich in K14 and K16 (Fig. 4 b, 0.5-h time point). Monomeric composition can influence the micromechanical properties of keratin filaments in vitro (Yamada et al., 2002). The physiological relevance of these and other possibilities can be addressed in future studies.
Implications for the structure of the polymerization-competent IF tetramer
The discovery of the hydrophobic stripe in the coil 1B domain of type I keratins creates an obvious question: To what does it bind as antiparallel dimers dock alongside one another to form the tetramer? In the answer lies key insight into the axial alignment of antiparallel dimers within the keratin tetramer subunit. Substoichiometric cross-linking of large oligomers of either type I/II or type III IF proteins identified several tetramer conformations by "nearest neighbor analyses" (Geisler et al., 1992; Steinert et al., 1993a,b,c; Mucke et al., 2004). A recent study (Hess et al., 2004) in which site-directed spin labeling and electron paramagnetic resonance (EPR) were used to analyze the interactions occurring as vimentin transitions from a monomeric state to large oligomers in vitro provided direct, site-specific evidence that the A11 intermediate, which places coil 1B of antiparallel dimers en face, corresponds to the earliest tetramer intermediate. The A22 conformation, which places coil 2B of antiparallel dimers en face, occurs concomitant with the formation of larger oligomers (Hess et al., 2004). On one hand, the EPR findings of Hess et al. (2004) yield spatial constraints that are not compatible with the hydrophobic stripe interacting with self in the context of a keratin A11 tetramer. On the other hand, comparative studies based on cross-linking (Steinert et al., 1993c) and mass-per-unit length determination (Herrmann et al., 1999) yielded strong evidence that precise axial stagger of antiparallel dimers differs for keratin and vimentin tetramers.
Higher resolution analyses are needed to identify the site(s) interacting with the newly defined hydrophobic stripe on type I keratins, and the resulting structure of the keratin tetramer subunit. There are 20 hydrophobic amino acids in coil 1B domain of both types I and II keratins that are not in "a" or "d" heptad positions, and potentially exposed to the surface of the dimer. Short stripes of less than three hydrophobic amino acids exist in the COOH-terminal half of coil 1B in both types I and II keratins (Fig. 5 e) which, depending on their orientation in space, could interact with the hydrophobic stripe identified in the NH2-terminal half of 1B. Whether coiled-coil dimers are supercoiled within tetramers or not (Er Rafik et al., 2004) is an important consideration. As an additional possibility, the hydrophobic stripe could sequentially interact with multiple regions within coil 1B through "axial slippage", which is believed to occur in several instances. For example, as part of conformational changes within a filament as it reaches its final, most energetically stable structure (Mucke et al., 2004), in response to mechanical stretching (Kreplak et al., 2002) or association with other filaments or interacting proteins (Aebi et al., 1988), or as the basis for the IF-dependent mechanotransduction of signals (Mucke et al., 2004).
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Materials and methods |
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Recombinant protein purification, anion exchange chromatography, and cross-linking assay
pET plasmids were transformed into BL21(DE3) E. coli for recombinant protein expression. Proteins were extracted from inclusion bodies (Paladini et al., 1996) and solubilized in buffer A containing 6.5 M urea, 50 mM Tris-HCl, pH 8.0, 1 mM EGTA, 2 mM DTT, and 30 µg/ml PMSF. Proteins were purified to near homogeneity via anion exchange chromatography with a High-Trap Q column (Amersham Biosciences; Wawersik et al., 1997). Purified types I and II keratins (0.5 mg/ml) were mixed (45:55 M ratio) for 1 h, loaded onto a Mono Q column (Amersham Biosciences), and eluted with linear gradients of 0125 mM and 125300 mM guanidine-HCl in buffer A (Coulombe and Fuchs, 1990; Wawersik et al., 1997). Fractions were analyzed by SDS-PAGE. Heterotypic complexes (200 µg/ml) containing types I and II keratin in a 1:1 M ratio were dialyzed overnight into 25 mM sodium-phosphate buffer, pH 7.4, containing 4, 6, 6.5, 8, or 9 M urea plus 10 mM ß-mercaptoethanol. The cross-linker BS3 (Pierce Chemical Co.) was added for 1 h, and products analyzed by SDS-PAGE and gel scanning densitometry.
Blue native gel electrophoresis
A detailed description of blue native gel electrophoresis was provided previously (Schagger, 2001). Primary keratinocytes were cultured and lysed with 8 M or 6 M urea in buffer A (see above). After centrifugation to remove debris, 10 µg lysate was mixed with 5% Coomassie G250 in 500 mM 6-aminohexanoic acid and loaded onto a 513% acrylamide gradient gel (48 acrylamide:1.5 bisacrylamide; 25 mM imidazole; 500 mM 6-aminohexanoic acid). After gel electrophoresis, the protein was then transferred onto PVDF membrane (Bio-Rad Laboratories), destained in 30% methanol, 10% acetic acid), and conventional Western analysis was performed.
Filament assembly and pelleting assay
Heterotypic complexes (200 µg/ml) obtained from Mono Q fractionation were denatured in 9 M urea, 1 mM DTT, 25 mM Tris-HCl, pH 7.5, for 4 h. Keratin polymerization was induced by dialysis in 5 mM Tris-HCl, pH 7.5, 1 mM DTT, containing 4 M urea (2 h), 2 M urea (2 h), then no urea for >2 h, all at RT. Polymerized filaments were viewed by negative staining (1% uranyl acetate) on a carbon-coated 400 mesh grid (Ted Pella) with a transmission electron microscope (model CM120; Philips) operated at 60 kV. For the pelleting assay, 50 µl of assembled filaments (10 µg protein) was subjected to centrifugation in an airfuge at 28 psi for 30 min. The supernatant and pellet were analyzed by SDS-PAGE and gel-scanning densitometry.
Modeling keratin 1B dimers
The crystal structure of Cortexillin I (PCB 1D7M) was chosen as a template for the coiled coil 1B dimer. Alignment of Cortexillin I, vimentin, and keratin sequences was performed using default parameters by CLUSTALX (Thompson et al., 1997). Homology model building was performed using the default parameters for energy minimization of Modeller6 (Sali and Blundell, 1993). No further energy minimization was performed. After a minimum of 10 model-building runs, models of individual keratin heterodimers or vimentin homodimers were almost identical. Models with the lowest energy states were chosen for further analysis. Manual inspection was performed using the Swiss pdb viewer (Guex and Peitsch, 1997), and further evaluation was performed through analyses of 3D profiles (Eisenberg method), atomic interactions (ERRAT), and Ramachandran plots (PROCHECK, all available at UCLA-DOE Institute for Genomics and Proteomics, http://www.doe-mbi.ucla.edu/Services/SV/). Variability was assessed by superimposing C- traces and backbones of select models onto the template and calculating RMSD values for positional differences between equivalent atoms. The protein structures were visualized and analyzed on MolMol (Koradi et al., 1996).
Pulse-chase labeling experiments and immunoprecipitations
Skin keratinocytes were isolated from 3-d-old 129SvJ wild-type or K6/ (Wong et al., 2000) pups and seeded in primary culture in 35- or 60-mm tissue culture plates. After 3 d, or when the plates were 90% confluent, cells were starved with DME medium lacking Met and Cys for 30 min. Cells were then pulse labeled with 0.1 mCi/ml [35S]Met/Cys (Easy Tag Express Protein Labeling Mix; PerkinElmer) for 20 min. After washing the cells with PBS, labeled cells were chased with normal mKer medium. After 0, 24, or 48 h, the cells were washed and collected in the presence of protease inhibitors, and stored (80°C). For immunoprecipitation, cells were solubilized with 2% Empigen BB (Calbiochem) in PBS supplemented with 5 mM EDTA and protease inhibitors. Lysates were analyzed by SDS-PAGE and Western blotting for actin (AC40; Sigma-Aldrich) or ß-tubulin (DM1A; Sigma-Aldrich). Protein A Sepharose beads (Amersham Biosciences) were washed with PBS and then bound to rabbit polyclonal antibodies directed against K16 (Bernot et al., 2002), K17 (McGowan and Coulombe, 1998), and K14 (AF 64; Covance). Equal protein amounts from lysates (based on actin or ß-tubulin load) were added to conjugated beads and incubated overnight at 4°C. Beads were washed with 0.2% Empigen BB. Bound protein was eluted with 5x gel sample buffer containing ß-mercaptoethanol. A second immunoprecipitation was performed on the same lysate to ensure complete epitope(s) depletion. Eluted proteins were subjected to SDS-PAGE and Coomassie staining. The gel was incubated for 20 min in ENLIGHTENING autoradiography enhancer (PerkinElmer), dried, and exposed to BiomaxMR film (Kodak), and analyzed by densitometry.
Online supplemental material
Online supplemental material includes the generation of chimeric proteins in which the 1B subdomain between K17 and K16 has been swapped as well as anion exchange chromatography and cross-linking data obtained using these chimeras (Fig. S1). Also shown is spectroscopy analysis of ANS binding to wild-type and mutant dimers (Fig. S2), which provides direct support for the existence of a hydrophobic stripe contributed by type I keratins at the surface of keratin heterodimers. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb. 200408116/DC1.
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Acknowledgments |
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This work was supported by grants AR44232 and AR42047 from the National Institutes of Health to P.A. Coulombe.
Submitted: 20 August 2004
Accepted: 28 January 2005
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References |
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