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• W2943595942 abstract "Article29 April 2019free access Transparent process Human keratin 1/10-1B tetramer structures reveal a knob-pocket mechanism in intermediate filament assembly Sherif A Eldirany Department of Dermatology, Yale University, New Haven, CT, USA Search for more papers by this author Minh Ho Department of Dermatology, Yale University, New Haven, CT, USA Search for more papers by this author Alexander J Hinbest Department of Dermatology, Yale University, New Haven, CT, USA Search for more papers by this author Ivan B Lomakin Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA Search for more papers by this author Christopher G Bunick Corresponding Author [email protected] orcid.org/0000-0002-4011-8308 Department of Dermatology, Yale University, New Haven, CT, USA Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA Search for more papers by this author Sherif A Eldirany Department of Dermatology, Yale University, New Haven, CT, USA Search for more papers by this author Minh Ho Department of Dermatology, Yale University, New Haven, CT, USA Search for more papers by this author Alexander J Hinbest Department of Dermatology, Yale University, New Haven, CT, USA Search for more papers by this author Ivan B Lomakin Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA Search for more papers by this author Christopher G Bunick Corresponding Author [email protected]ale.edu orcid.org/0000-0002-4011-8308 Department of Dermatology, Yale University, New Haven, CT, USA Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA Search for more papers by this author Author Information Sherif A Eldirany1,‡, Minh Ho1,‡, Alexander J Hinbest1, Ivan B Lomakin2 and Christopher G Bunick *,1,2 1Department of Dermatology, Yale University, New Haven, CT, USA 2Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA ‡These authors contributed equally to this work *Corresponding author. Tel: +1 203-785-4092; Fax: +1 203-785-7637; E-mail: [email protected] EMBO J (2019)38:e100741https://doi.org/10.15252/embj.2018100741 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract To characterize keratin intermediate filament assembly mechanisms at atomic resolution, we determined the crystal structure of wild-type human keratin-1/keratin-10 helix 1B heterotetramer at 3.0 Å resolution. It revealed biochemical determinants for the A11 mode of axial alignment in keratin filaments. Four regions on a hydrophobic face of the K1/K10-1B heterodimer dictated tetramer assembly: the N-terminal hydrophobic pocket (defined by L227K1, Y230K1, F231K1, and F234K1), the K10 hydrophobic stripe, K1 interaction residues, and the C-terminal anchoring knob (formed by F314K1 and L318K1). Mutation of both knob residues to alanine disrupted keratin 1B tetramer and full-length filament assembly. Individual knob residue mutant F314AK1, but not L318AK1, abolished 1B tetramer formation. The K1-1B knob/pocket mechanism is conserved across keratins and many non-keratin intermediate filaments. To demonstrate how pathogenic mutations cause skin disease by altering filament assembly, we additionally determined the 2.39 Å structure of K1/10-1B containing a S233LK1 mutation linked to epidermolytic palmoplantar keratoderma. Light scattering and circular dichroism measurements demonstrated enhanced aggregation of K1S233L/K10-1B in solution without affecting secondary structure. The K1S233L/K10-1B octamer structure revealed S233LK1 causes aberrant hydrophobic interactions between 1B tetramers. Synopsis Crystal structures of keratin 1/keratin 10 1B tetramers reveal a knob-pocket interaction important for the assembly of mature intermediate filaments. An epidermolytic palmoplantar keratoderma-related mutation is localized in the pocket region and causes aberrant filament formation. Symmetrical knob-pocket interactions in the 1B domain termini of type II keratins drive A11-tetramer formation. Mutation of 1B domain knob residues is detrimental to mature full-length intermediate filament formation in K1/K10, K8/K18, and vimentin. Keratin 1 mutation S233L, associated with epidermolytic palmoplantar keratoderma, causes aberrant hydrophobic interactions between K1/K10-1B tetramers in an octameric crystal structure. Introduction One of the most critical questions in keratin biology is how keratin heterodimers assemble into keratin intermediate filaments (KIFs). Multiple biophysical studies have defined the stages of IF assembly as: One type I keratin and one type II keratin pair to form a parallel heterodimer; heterodimers then bind to form an anti-parallel tetramer; tetramers then merge to form a protofibril/unit-length filament; and finally, protofibrils assemble into the complete KIF (Aebi et al, 1983; Parry et al, 2001; Herrmann & Aebi, 2016). A major knowledge gap exists in understanding the biochemical determinants of KIF assembly at atomic resolution. Recent X-ray crystal structures of the keratin 1/10 and keratin 5/14 helix 2B heterodimers provided key insights into heterodimer structure, such as the electrostatic and hydrophobic chemistry of the molecular surface (Lee et al, 2012; Bunick & Milstone, 2017). These structures did not, aside from a disulfide linkage related to inter-filament organization, capture information on how heterodimers assemble into KIFs. Four modes of axial alignment of keratin heterodimers within a filament have been proposed based on keratin 1/10 and 5/14 cross-linking studies (Steinert et al, 1993a; Steinert et al, 1993b; Fig 1A). Two modes contain heterodimers in an anti-parallel, staggered alignment such that either the 1B coiled-coil segments are in phase (A11 mode) or the 2B coiled-coil segments are in phase (A22 mode). One mode contains two anti-parallel heterodimers in almost exact register (A12 mode), but without any specific coiled-coil region being in phase with itself. The fourth mode is a head-to-tail alignment of the helical rod domain (i.e., helices 1A, 1B, 2A, 2B) with ~ 10 residues overlapping between the 1A helix from one heterodimer and the 2B helix from another (ACN mode). To date, there have been no crystal structures of human keratins that elucidate the molecular mechanisms of any of these axial alignments. For other types of IF proteins, however, crystal structures of vimentin (type III IF) and lamin A (type V IF) domains have provided molecular insights into the A11 and ACN modes of tetramer assembly, respectively (Strelkov et al, 2004; Aziz et al, 2012; Chernyatina et al, 2012, 2015). In the case of lamin A, it was proposed that head-to-tail association occurs because clusters of positively charged arginine residues in the head and tail domains interact with negatively charged residues in the ends of the helical rod domain (Strelkov et al, 2004). The arginine clusters, however, are not conserved among keratin heads and tails. This difference highlights why it is necessary to study further the structural mechanisms governing higher order IF assemblies, especially for keratins. Figure 1. Molecular surface properties of the wild-type K1/K10-1B dimer and tetramer A. The four proposed modes of keratin tetramer alignment in filament formation. B. Ribbon diagram of the wild-type K1/K10-1B tetramer crystal structure at 3.0 Å resolution. Helices within one heterodimer (the crystal asymmetric unit) are oriented parallel, whereas the two heterodimers in the tetramer are anti-parallel. C. Electrostatic surface potential mapped onto the K1/K10-1B heterodimer structure demonstrates a polarization of charge: The N-terminus has some basic charge (blue), while the majority of the distal 1B dimer is acidic (red). D. Close-up view of the N-terminal basic patches on the K1/K10-1B heterodimer. Three K1 (R239, R240, and R241) and three K10 (K198, K201, and K207) residues contribute positive charge. One basic patch (left) surrounds a hydrophobic pocket involved in tetramer formation (asterisk). E, F. Electrostatic surface potential mapped onto the K1/K10-1B tetramer structure demonstrates it is overwhelmingly acidic: The anti-parallel orientation of the dimers within the tetramer eliminates the small basic potential at the dimer N-terminus. Unique molecular surface contours are present in the K1/K10-1B tetramer that do not exist in the dimer: One tetramer face has a long, linear, highly acidic surface groove (top of panel E, arrows; panel F, asterisks), whereas the other face contains a central concave pocket (bottom of panel E, asterisk) flanked by angled grooves (bottom of panel E, arrows). G. Residues forming the central concave pocket on one face of the K1/K10-1B tetramer are shown and colored based on residue property (red, acidic; dark blue, basic; orange, hydrophobic; light blue, polar). The asterisk marks the twofold symmetry axis in the tetramer. H. Close-up view of one “angled groove” (arrow) in the K1/K10-1B tetramer colored to demonstrate a portion of all four tetramer helices contributes to groove formation. Some of the anchoring knob/hydrophobic pocket residues are accessible in the groove (yellow asterisk). Download figure Download PowerPoint After we determined the keratin 1/10-2B heterodimer structure (Bunick & Milstone, 2017), the next logical coiled-coil domain to target for structural studies was the 1B region—the second longest helical domain in keratins (~ 106 amino acids). While the 1A and 2B helices are the most commonly mutated domains in keratinopathies (human skin diseases caused by keratin mutation), the 1B domain also harbors pathogenic mutations. For example, a Ser233Leu missense mutation in K1-1B causes epidermolytic palmoplantar keratoderma (EPPK). Histologic and electron microscopic examination of skin from patients with EPPK due to S233LK1 mutation revealed KIFs that formed tubular assemblies with enlarged 43 nm diameters rather than normal 10-nm-diameter KIFs (Wevers et al, 1991; Terron-Kwiatkowski et al, 2006). This tubular morphology was described as “tonotubular” keratin as opposed to the “tonofilamentous” keratin observed in healthy skin. Our goal was to characterize the biochemical and structural properties of the wild-type keratin 1/10 helix 1B heterodimer and the impact of S233LK1 mutation on that structure. However, efforts to determine the structure of wild-type keratin 1/10-1B serendipitously led to the structure of the keratin 1/10-1B tetramer, a far more valuable structure given the need for atomic resolution information on higher order keratin filament assembly. The work reported here advances intermediate filament biology in several ways: It provides an atomic resolution basis for the A11 mode of axial alignment in keratin filaments; it identifies an anchoring knob/hydrophobic pocket mechanism that drives helix 1B tetramer assembly not just for keratins, but for other non-keratin intermediate filaments as well; and it establishes insight into the pathogenic mechanisms of tonotubular keratin formation associated with EPPK. First, we determined a 3.0 Å resolution crystal structure of the wild-type keratin 1/10-1B tetramer. Second, multi-angle light scattering and circular dichroism measurements demonstrated the S233LK1 mutation alters the aggregation state of keratin 1/10-1B in solution but not the secondary structure. Third, we determined a 2.39 Å resolution crystal structure of K1/10-1B containing the pathogenic S233LK1 mutation. Fourth, we identified and validated through mutagenesis an “anchoring knob/hydrophobic pocket mechanism” for tetramer assembly. This research addresses knowledge gaps in keratin filament assembly and how pathogenic mutations can lead to human skin disease by altering that assembly. Results Wild-type K1/K10-1B structure Using the divide-and-conquer approach (Strelkov et al, 2001), the X-ray crystal structure of the human K1/K10 helix 1B heterotetrameric complex was determined at 3.0 Å resolution (Fig 1B and Table 1; Eldirany et al, 2018). The tetramer is composed of two K1/K10-1B heterodimers arranged anti-parallel (one heterodimer is the crystal asymmetric unit). The K1 and K10 molecules within the heterodimer structure form a parallel coiled-coil, spanning K1 residues (226–331) and K10 residues (195–296). Key molecular interactions along the K1/K10-1B heterodimer interface are detailed (Fig EV1A and B). The K1/K10-1B tetramer did not exhibit supercoiling of the coiled-coil heterodimers. Throughout this manuscript, we will denote protein–protein interactions occurring between the two anti-parallel dimers of the tetramer by associating a prime symbol with the residue(s) from the second dimer (e.g., K1-K1′). Table 1. Data collection and refinement statistics Crystal Wild-type K1/K10-1B K1S233L/K10-1B Diffraction dataaa Data collection was performed on 07-06-2017. Space group P 31 2 1 P 64 2 2 Unit cell dimensions a, b, c (Å) 106.68, 106.68, 70.32 93.30, 93.30, 124.74 α, β, γ (°) 90, 90, 120 90, 90, 120 Resolution range (outer shell), Å 46.20–2.98 (3.05–2.98)bb Values in parentheses are for highest resolution (outer) shell. 46.65–2.39 (2.43–2.39) I/σI 11.72 (0.64) 20.2 (1.92) Resolution (Å) where I/σI ~ 1.9 3.46 2.39 CC(1/2) in outer shell, % 64.0 78.7 Completeness, % 89.5 (69.9) 99.9 (99.5) Rmerge 0.132 (1.185) 0.139 (0.969) No. of crystals used 1 1 No. of unique reflections 8,414 13,342 Redundancy 8.0 (5.0) 13.3 (10.0) Wilson B-factor, Å2 86.2 67.3 Refinement Rwork, % 0.279 (0.417) 0.271 (0.349) Rfree, % 0.298 (0.478) 0.294 (0.371) No. of non-hydrogen atoms Protein 1,731 1,751 Ligands/Ions 9 4 Waters 35 60 R.m.s. deviations Bond lengths (Å) 0.006 0.004 Angles (°) 0.876 0.587 Chirality 0.036 0.029 Planarity 0.004 0.005 Dihedral (°) 18.415 17.408 Average B-factor (overall), Å2 146.0 106.3 a Data collection was performed on 07-06-2017. b Values in parentheses are for highest resolution (outer) shell. Click here to expand this figure. Figure EV1. Structure analysis of wild-type K1/K10-1B and mutant K1S233L/K10-1B heterodimer structures The amino acid contacts at the heterodimer interface for both wild-type K1/K10-1B and mutant K1S233L/K10-1B X-ray crystal structures were analyzed and plotted onto a single residue contact map. Intramolecular (within K1 or K10 only) and inter-molecular (between K1 and K10) salt bridges are plotted red. Hydrogen bonds are plotted green. Interactions between hydrophobic residues are plotted orange (hydrophobic residues are defined as A, I, L, F, V, P, M, W), including hydrophobic interaction with the aromatic residue tyrosine. Other types of molecular contacts are plotted black. Analyses were performed using WHAT IF (defines atoms as “in contact” when the distance between their van der Waals surfaces is < 1.0 Å), ESBRI, and PDBePISA. Since S233K1 is a surface-exposed residue, its mutation to L233 does not impact the heterodimer interface. Hence, the analysis of both heterodimer interfaces was used to obtain the contact map. The wild-type K1/K10-1B and mutant K1S233L/K10-1B heterodimer structures were superimposed and have a root-mean-square deviation (RMSD) of 0.736 Å. The superposition shows slight variation in the positioning of the K10 C-terminus. Download figure Download PowerPoint The electrostatic surface potential of the K1/K10-1B heterodimer is similar to that observed in the K1/K10-2B heterodimer (Bunick & Milstone, 2017): There is polarization of charge with the distal three-fourths of the complex being acidic, whereas the proximal one-fourth is more basic (Fig 1C). The basic patch at the N-terminus of K1/K10-1B contains residues from both K1 (R239, R240, R241) and K10 (K198, K201, K207; Fig 1D); this is in contrast to the 2B heterodimer, where a linear N-terminal basic patch was solely formed by nine K1 residues (Bunick & Milstone, 2017). Acidic groove on molecular surface of 1B tetramer Due to anti-parallel alignment of K1/K10-1B heterodimers in the tetramer, the basic electrostatic surface potential at the N-terminus of the heterodimer is diminished by the strength of the adjacent acidic C-terminus in the tetramer (Fig 1E). The electrostatic surface potential of the K1/K10-1B tetramer is mainly acidic. There are unique surface contours present in the K1/K10-1B tetramer that are not present in the heterodimer structure (Fig 1E and H). One face of the tetramer contains a linear groove that extends from one end all the way to the other; this groove has the highest acidic electrostatic surface potential in the K1/K10-1B tetramer structure (Fig 1E and F). In contrast, the tetramer face 180° opposite the acidic linear groove contains a central concave pocket ~ 66.7 Å long by 17.7 Å wide (Fig 1G), flanked by two symmetric angled grooves ~ 54.9 Å long at either end of the molecule (Fig 1H). Hydrophobic interactions drive 1B tetramer formation Mapping of hydrophobic surface potential onto the K1/K10-1B heterodimer structure demonstrates that one heterodimer face contains multiple surface-exposed hydrophobic residues, whereas the face 180° opposite is largely polar with only a few exposed hydrophobic residues (Fig 2A). The hydrophobic face of the K1/K10-1B heterodimer contains the molecular determinants of tetramer assembly. They can be divided into four key segments from N- to C-terminus: a K1 hydrophobic pocket, a K10 hydrophobic stripe, K1 interaction residues, and a K1 anchoring knob (Fig 2B). Figure 2. Biochemical basis for K1/K10-1B heterotetramer formation The molecular surface of the K1/K10-1B heterodimer can be divided into a predominantly hydrophobic face (top) and a predominantly polar face (bottom). The molecular surface is colored according to hydrophobic potential: Hydrophobic residues are orange, and polar residues are blue (color intensity indicates magnitude of potential). Select residues are labeled blue (K1) or pink (K10). Four key regions along the hydrophobic face of the K1/K10-1B heterodimer drive tetramer formation: an N-terminal K1 hydrophobic pocket (gold), a K10 hydrophobic stripe (purple), K1 interaction residues (green), and a C-terminal K1 anchoring knob (yellow). All of the following panels show tetramer interactions. One end of the K1/K10-1B tetramer depicting the anchoring knob residues (yellow sticks) binding into the hydrophobic pocket (gold surface). Stick representation of the anchoring knob/hydrophobic pocket mechanism in tetramer formation. F314K1′ and L318K1′ wedge between L227K1, Y230K1, F231K1, and F234K1; the aromatic ring of F314K1′ stacks against that of F234K1. A salt bridge between K207K10 and E311K1′ is shown. Ten K10 residues (purple) in the hydrophobic stripe of one dimer (K10 helix backbone pink) interact with 12 K1′ residues (green) from the partner dimer (K1′ backbone light blue) to create an anti-parallel tetramer interface. Close-up view of the one region in the K1/K10-1B tetramer where K10 and K10′ interact; this is facilitated by L236K10 interacting with L244K10′. Mutant K1S233L/K10-1B (K1S233L, dark blue; K10, dark red) is superimposed on wild-type K1/K10-1B (K1, light blue; K10, pink). N240K10 is the center of the twofold symmetry in the tetramer; hence, L236-L244′ and the reciprocal L236′-L244 interactions are on either side of N240. Download figure Download PowerPoint At the N-terminus of the K1/K10-1B heterodimer, there is a hydrophobic pocket formed by four K1 residues (L227, Y230, F231, and F234). The concavity between the aromatic residues is the receptor site for the C-terminal anchoring knob on a neighboring K1/K10-1B heterodimer, facilitating tetramer formation (Fig 2C). The C-terminal anchoring knob is composed of two K1 residues (F314 and L318). F314K1′ binds by wedging between F231K1 and F234K1, creating a ring-stacking interaction with F234K1 (Fig 2D). L318K1′ interacts with F231K1 and Y230K1 (~ 3.3 and 4.2 Å, respectively), and knob/pocket docking brings A321K1′ near Y230K1 (~ 3.6 Å) and L318K1′ near L227K1 (~ 4.6 Å). Adjacent to the hydrophobic pocket, and aligned along the outer aspect of the α-helical ridge, are several K10 residues constituting a predominantly hydrophobic stripe (Fig 2A and B). A type I keratin “hydrophobic stripe” was identified from modeling analyses of K6/K16/K17 dimers (Bernot et al, 2005); this work showed that most, but not all (e.g., K10), type I keratins contained a consensus hydrophobic sequence at alternating b- and f- positions of the heptad repeat (L-x-x-x-(I/V)-x-x-A-x-x-x-L) contributing to tetramer stability. However, K10 has threonine in the second position of this motif, and in our K1/K10-1B tetramer structure, the function of this protein region proves complex—there exists an interacting stripe, but the interactions are not strictly hydrophobic. The K10 helical ridge on the N-terminal half of the K1/K10-1B heterodimer is defined by 11 K10 residues: K207, L211, T215, A218, N219, L221, L222, N226, L229, K237, and L236. Several of these residues are not hydrophobic (K207, T215, N219, N226, K237) but make meaningful interactions to stabilize tetramer assembly and thus are considered part of the stripe (Fig 2E). K207K10 forms a salt bridge with E311K1′, while T215K10 interacts with M296K1′ and D300K1′ (Fig 2D and E). K10 hydrophobic residues L211, A218, L221, L222, and L229 all have interactions with K1′ residues < 5 Å apart. L236, on the other hand, is involved in K10-K10′ interactions only (Fig 2F). The hydrophobic face of the K1/10-1B heterodimer contains a segment consisting of “K1 interaction residues” between the K10 hydrophobic stripe and the C-terminal anchoring knob. K1 interaction residues exist on the K1 α-helix whose helical ridge forms most of the distal hydrophobic face. In the K1/10-1B tetramer, 12 K1 residues from this segment have hydrophobic or electrostatic interactions with 10 K10 hydrophobic stripe residues from the binding heterodimer (Fig 2E). S233LK1 mutation drives aggregation of K1/K10-1B in solution Keratin 1 containing the missense mutation S233L, which is pathogenic for epidermolytic palmoplantar keratoderma, was produced and purified to investigate how the mutation affects K1/K10-1B heterodimer structure and function (Fig 3A). After His-tag removal from K10, wild-type K1/K10-1B and mutant K1S233L/K10-1B complexes were analyzed by gel filtration. Wild-type K1/K10-1B separated into two main peaks (Fig 3B, solid line), whereas K1S233L/K10-1B formed one major peak (Fig 3B, dotted line) that eluted earlier than the wild-type complex. This suggested K1S233L/K10-1B formed a higher molecular weight complex in solution than wild-type K1/K10-1B. Figure 3. Biophysical analysis of wild-type and K1S233L mutant keratin 1/10-1B in solution Bacterial expression lysates for recombinant His6-tagged K10-1B (1), wild-type K1-1B (2), and K1S233L-1B (3). Wild-type K1/K10-1B before (4) and after (5) nickel affinity purification. Wild-type K1/10-1B (6) and K1S233L/K10-1B (7) after thrombin cleavage of His6-tag on K10 and subsequent gel filtration (untagged K10 overlaps with K1 after tag removal). Gel filtration of wild-type K1/K10-1B (solid line) produced two peaks from 52 to 67 ml, whereas the K1S233L/K10-1B mutant (dotted line) produced one major peak from 50 to 55 ml that eluted earlier than wild type. V0 = void volume. Multi-angle light scattering demonstrated wild-type K1/K10-1B (solid line) exists mostly as a tetramer (observed MW 49,100; calculated tetramer MW 49,700), with a small amount of dimer (observed MW 26,160; calculated MW 24,840), in 100 mM NaCl solution. This does not change in 200 mM NaCl. K1S233L/K10-1B forms higher MW aggregates than wild type in both 100 mM NaCl (observed MW 62,640) and 200 mM NaCl (observed MW 86,870) solutions. Circular dichroism shows identical helical secondary structure for wild-type K1/K10-1B and mutant K1S233L/K10-1B. Download figure Download PowerPoint To characterize the oligomerization state of these complexes, K1/K10-1B and K1S233L/K10-1B were analyzed by multi-angle light scattering in either 100 mM NaCl or 200 mM NaCl solutions (Fig 3C). Wild-type K1/K10-1B (solid line) formed a tetramer species (peak 2, ~ 49 kDa) and a dimer species (peak 3, ~ 24–26 kDa) in both 100 and 200 mM NaCl conditions (wild-type heterodimer calculated MW is 24,840). In contrast, K1S233L/K10-1B (dotted line) formed a single species of ~ 62 kDa in 100 mM NaCl solution and ~ 86 kDa in 200 mM NaCl solution. This demonstrated that K1S233L/K10-1B formed higher molecular weight aggregates than wild-type K1/K10-1B in solution. The increased MW for the mutant complex under higher ionic strength is consistent with enhanced hydrophobic interaction. Circular dichroism measurements demonstrated that S233LK1 does not alter the secondary structure of K1/K10-1B (Fig 3D). Both wild-type K1/K10-1B (solid line) and K1S233L/K10-1B (dotted line) complexes had identical α-helical secondary structure in solution. Pseudo-tonotubular keratin in mutant K1S233L/K10-1B octamer structure To further investigate how S233LK1 mutation impacts K1/K10-1B structure, the K1S233L/K10-1B crystal structure was determined at 2.39 Å resolution (Table 1). Both S233 from the wild-type K1/K10-1B structure and L233 from the mutant K1S233L/K10-1B structure occupy solvent-exposed positions at the N-terminus of the 1B heterodimer. The S233LK1 mutation changes the surface potential at this site from polar (wild-type) to hydrophobic (mutant; Fig 4A). Near position 233, along the inter-molecular interface of the 1B heterodimer, are two critical K1 phenylalanines (F231 and F234) involved in heterodimer stabilization and in forming the hydrophobic pocket. Figure 4. Structural features of the mutant K1S233L/K10-1B octamer The N-terminus of the wild-type K1/K10-1B (left) and mutant K1S233L/K10-1B (right) heterodimer structures is depicted as a ribbon (top) and molecular surface (bottom). Both S233K1 and S233LK1 are surface-exposed, but S233LK1 generates a new hydrophobic surface patch compared to wild-type S233K1. Crystal structure of K1S233L/K10-1B octamer presented as a ribbon diagram. Close-up view of the biochemical interactions between two K1/K10 tetramers (T1, T2) caused by the L233K1 mutation (red). L233K1 mediates hydrophobic assembly of the octamer by interacting with five residues from the opposing tetramer: Y230K1′, L233K1′, F234K1′, F314K1′, and A317K1′. Section of the K1S233L/K10-1B crystal lattice demonstrating “pseudo-tonotubular” structures (corresponding to the octamer) with diameter of 45 Å. One end of the K1S233L/K10-1B tetramer structure depicting L227K1 interacting with L318K1′ to form the N-terminal boundary of the hydrophobic pocket. The hydrophobic pocket from one dimer is depicted as a transparent molecular surface and colored according to hydrophobic potential (orange, hydrophobic; white to blue, polar); only the anchoring knob residues from the partner dimer are shown (yellow sticks). Cadmium ions (green spheres) bound to the N-terminal methionine of K1-1B and to C-terminal residues E322K1 and H287K10 caused small structural changes at the termini (e.g., position of L227K1) compared to the S233LK1 mutant structure. Given its higher resolution and absence of heavy atoms, the mutant structure likely represents the more accurate and physiologic positioning of L227K1. Download figure Download PowerPoint The increased hydrophobic surface potential created by S233LK1 mutation did not alter heterodimer or tetramer formation, but rather altered how tetramers interacted with each other (Fig 4B). This explains why the K1S233L/K10-1B structure was determined as an octamer. Specifically, L233K1 from one tetramer bound to five residues from a different tetramer (the aromatic portion of Y230K1′, L233K1′, F234K1′, F314K1′, and Ala317K1′) to drive hydrophobic assembly of an octamer (Fig 4C). Due to the anti-parallel symmetry of the tetramer, the same interactions by L233K1 occur at both ends of the octamer. L233K1 closely interacts with itself, L233K1′ (~ 3.8 Å), and Ala317K1′ (~ 3.9 Å). L233K1 additionally interacts with three aromatic residues over slightly longer distances: 4.3 Å (F314K1′), 4.8 Å (Y230K1′), and 5.5 Å (F234K1′). All three of these aromatic residues are involved in the anchoring knob/hydrophobic pocket mechanism of tetramer assembly. As two tetramers bind in the K1S233L/K10-1B octamer, Y230 from one hydrophobic pocket binds with Y230 from the adjacent pocket (Fig 4C). Examination of K1S233L/K10-1B crystal lattice packing revealed a repetitive arrangement of a circular structure (the K1S233L/K10-1B octamer; Fig 4D). At first glance, it appears the octamer mimics the tonotubular keratin observed under electron microscopy from EPPK skin. The diameter of the octamer, however, is only ~ 45 Å (4.5 nm), which is about one-tenth the diameter of the observed in vitro tonotubular keratin (430 Å or 43 nm; Wevers et al, 1991). Hence, we refer to the octamer as pseudo-tonotubular keratin. Comparing wild-type K1/K10-1B and mutant K1S233L/K10-1B tetramer structures, the major interactions between the hydrophobic pocket and anchoring knob, as described above for the wild-type structure, are preserved in the mutant." @default.
• W2943595942 created "2019-05-09" @default.
• W2943595942 creator A5020785070 @default.
• W2943595942 creator A5030268989 @default.
• W2943595942 creator A5030400118 @default.
• W2943595942 creator A5032070796 @default.
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• W2943595942 date "2019-04-29" @default.
• W2943595942 modified "2023-10-12" @default.
• W2943595942 title "Human keratin 1/10‐1B tetramer structures reveal a knob‐pocket mechanism in intermediate filament assembly" @default.
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