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Crystal Structure of Human Profilaggrin S100 Domain and Identification of Target Proteins Annexin II, Stratifin, and HSP27

  • Christopher G. Bunick
    Correspondence
    Department of Dermatology, Yale University, 333 Cedar Street, LCI 501, PO Box 208059, New Haven, Connecticut 06520-8059, USA
    Affiliations
    Department of Dermatology, Yale University, New Haven, Connecticut, USA

    Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, USA

    The first two authors contributed equally to this work.
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  • Richard B. Presland
    Affiliations
    Department of Oral Health Sciences, University of Washington, Seattle, Washington, USA

    Department of Medicine, Division of Dermatology, University of Washington, Seattle, Washington, USA

    The first two authors contributed equally to this work.
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  • Owen T. Lawrence
    Affiliations
    Department of Oral Health Sciences, University of Washington, Seattle, Washington, USA

    Present Address: South African Association for Marine Biological Research, Marine Parade 4056, Durban, KwaZulu-Natal, South Africa (DJP); Department of Biological Structure, University of Washington, Seattle, Washington, USA (OTL)
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  • David J. Pearton
    Affiliations
    Department of Oral Health Sciences, University of Washington, Seattle, Washington, USA

    Present Address: South African Association for Marine Biological Research, Marine Parade 4056, Durban, KwaZulu-Natal, South Africa (DJP); Department of Biological Structure, University of Washington, Seattle, Washington, USA (OTL)
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  • Leonard M. Milstone
    Affiliations
    Department of Dermatology, Yale University, New Haven, Connecticut, USA
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  • Thomas A. Steitz
    Affiliations
    Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, USA
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      The fused-type S100 protein profilaggrin and its proteolytic products including filaggrin are important in the formation of a normal epidermal barrier; however, the specific function of the S100 calcium-binding domain in profilaggrin biology is poorly understood. To explore its molecular function, we determined a 2.2 Å-resolution crystal structure of the N-terminal fused-type S100 domain of human profilaggrin with bound calcium ions. The profilaggrin S100 domain formed a stable dimer, which contained two hydrophobic pockets that provide a molecular interface for protein interactions. Biochemical and molecular approaches demonstrated that three proteins, annexin II/p36, stratifin/14-3-3 sigma, and heat shock protein 27, bind to the N-terminal domain of human profilaggrin; one protein (stratifin) co-localized with profilaggrin in the differentiating granular cell layer of human skin. Together, these findings suggest a model where the profilaggrin N-terminus uses calcium-dependent and calcium-independent protein–protein interactions to regulate its involvement in keratinocyte terminal differentiation and incorporation into the cornified cell envelope.

      Abbreviations

      ASA
      accessible surface area
      CABD
      calcium-binding domain
      KH
      keratohyalin
      MW
      molecular weight
      PF-CABD
      N-terminal S100 fused-type calcium-binding domain of human profilaggrin
      PF-NT
      human profilaggrin N-terminus containing CABD plus B domain
      Y2H
      yeast two-hybrid

      Introduction

      Profilaggrin is an~400 kDa human protein critical for normal skin barrier development. It is principally expressed in a differentiation-dependent manner in the stratum granulosum (
      • Presland R.B.
      • Rothnagel J.A.
      • Lawrence O.T.
      Profilaggrin and the Fused S100 Family of Calcium-Binding Proteins.
      ). Loss-of-function mutations in the profilaggrin (FLG) gene associate it with the skin diseases atopic dermatitis and ichthyosis vulgaris (
      • Palmer C.N.
      • Irvine A.D.
      • Terron-Kwiatkowski A.
      • et al.
      Common loss-of-function variants of the epidermal barrier protein filaggrin are a major predisposing factor for atopic dermatitis.
      ;
      • Sandilands A.
      • O'Regan G.M.
      • Liao H.
      • et al.
      Prevalent and rare mutations in the gene encoding filaggrin cause ichthyosis vulgaris and predispose individuals to atopic dermatitis.
      ;
      • Smith F.J.
      • Irvine A.D.
      • Terron-Kwiatkowski A.
      • et al.
      Loss-of-function mutations in the gene encoding filaggrin cause ichthyosis vulgaris.
      ;
      • Akiyama M.
      FLG mutations in ichthyosis vulgaris and atopic eczema: spectrum of mutations and population genetics.
      ). Furthermore, variation in copy number of filaggrin monomers within the profilaggrin gene is associated with a dry skin phenotype in the general population (
      • Brown S.J.
      • Kroboth K.
      • Sandilands A.
      • et al.
      Intragenic copy number variation within filaggrin contributes to the risk of atopic dermatitis with a dose-dependent effect.
      ).
      In all mammals examined, profilaggrin has four distinct domains: (1) a 92 amino acid N-terminal S100 fused-type calcium-binding domain (PF-CABD); (2) highly basic B domain containing nuclear localization signal; (3) variable number (species dependent) of filaggrin units; and (4) short C-terminal domain (
      • Presland R.B.
      • Haydock P.V.
      • Fleckman P.
      • et al.
      Characterization of the human epidermal profilaggrin gene. Genomic organization and identification of an S-100-like calcium binding domain at the amino terminus.
      ,
      • Presland R.B.
      • Rothnagel J.A.
      • Lawrence O.T.
      Profilaggrin and the Fused S100 Family of Calcium-Binding Proteins.
      ). Profilaggrin is highly phosphorylated at Ser/Thr residues within filaggrin units and packaged into keratohyalin (KH) granules within the stratum granulosum (
      • Dale B.A.
      • Lonsdale-Eccles J.D.
      • Holbrook K.A.
      Stratum corneum basic protein: an interfilamentous matrix protein of epidermal keratin.
      ;
      • Lonsdale-Eccles J.D.
      • Haugen J.A.
      • Dale B.A.
      A phosphorylated keratohyalin-derived precursor of epidermal stratum corneum basic protein.
      ). During terminal differentiation of the epidermis, when granular cells transition to dead corneocytes, profilaggrin is dephosphorylated and processed by multiple proteases to generate the discrete N-terminal peptide containing the CABD and B domains (PF-NT) and individual filaggrin units (
      • Kam E.
      • Resing K.A.
      • Lim S.K.
      • et al.
      Identification of rat epidermal profilaggrin phosphatase as a member of the protein phosphatase 2 A family.
      ;
      • Resing K.A.
      • Thulin C.
      • Whiting K.
      • et al.
      Characterization of profilaggrin endoproteinase 1. A regulated cytoplasmic endoproteinase of epidermis.
      ;
      • Presland R.B.
      • Rothnagel J.A.
      • Lawrence O.T.
      Profilaggrin and the Fused S100 Family of Calcium-Binding Proteins.
      ). The PF-NT can translocate (nuclear localization signal within B domain) into the keratinocyte nucleus both in vitro and in vivo (
      • Ishida-Yamamoto A.
      • Takahashi H.
      • Presland R.B.
      • et al.
      Translocation of profilaggrin N-terminal domain into keratinocyte nuclei with fragmented DNA in normal human skin and loricrin keratoderma.
      ;
      • Pearton D.J.
      • Dale B.A.
      • Presland R.B.
      Functional analysis of the profilaggrin N-terminal peptide: identification of domains that regulate nuclear and cytoplasmic distribution.
      ). It is postulated that PF-NT, once in the nucleus, provides cellular signals directing events of terminal differentiation; it may act as a negative feedback signal controlling epidermal proliferation and homeostasis (
      • Aho S.
      • Harding C.R.
      • Lee J.M.
      • et al.
      Regulatory role for the profilaggrin N-terminal domain in epidermal homeostasis.
      ).
      Profilaggrin, along with six other epidermal proteins, has an S100 domain fused to additional protein sequences, defining a “fused S100-type” subfamily within the larger S100 family (
      • Kizawa K.
      • Takahara H.
      • Unno M.
      • et al.
      S100 and S100 fused-type protein families in epidermal maturation with special focus on S100A3 in mammalian hair cuticles.
      ). An important unsolved question is why the epidermis expresses several S100 fused-type proteins, whose genes cluster on chromosome 1q21. Although isolated S100 proteins have been studied extensively, the functions of fused-type S100 proteins in the epidermis are less clear. Thus, we investigated the structure and binding interactions of the most abundant and best understood member of the S100 fused-type family, profilaggrin. Although structures for isolated S100 proteins exist, no atomic resolution structures have been determined for an S100 fused-type calcium-binding protein, nor for any region of profilaggrin.
      We report here advances in the molecular structure and biochemical function of the N-terminus of human profilaggrin. First, we determined the 2.2 Å resolution x-ray crystal structure of the S100 fused-type CABD of profilaggrin. Second, several biochemical and yeast two-hybrid (Y2H) studies demonstrated that the N-terminal fragment of profilaggrin exists both in vitro and in vivo as a homodimer. Third, co-immunoprecipitation and mass spectrometry approaches identified three protein targets for the N-terminus of profilaggrin. This research addresses a specific knowledge gap in human epidermal barrier biology correlating protein structure with molecular function.

      Results

      Biochemical and structural basis for profilaggrin dimerization

      The influence of calcium ions on the aggregation state of PF-CABD (residues 1–92, inclusive of N-terminal Met) was analyzed using size-exclusion chromatography. In the presence of 1 mM EDTA, and the absence of calcium ions, recombinant PF-CABD separated into a single major peak (Figure 1a, solid line). In contrast, PF-CABD in 5 mM calcium chloride separated into numerous higher-than-expected molecular weight (MW) aggregates (Figure 1a, dotted line). SDS-PAGE of the collected size-exclusion fractions demonstrated that PF-CABD eluted in a narrow range of fractions in the absence of calcium (Figure 1b) but in a broad range of higher-than-expected MW fractions in the presence of 5 mM calcium (Figure 1c). These data suggest that PF-CABD undergoes calcium-dependent aggregation.
      Figure thumbnail gr1
      Figure 1Calcium-independent dimerization and calcium-dependent self-aggregation of profilaggrin N-terminal S-100 fused-type calcium-binding domain. (a) Gel filtration (GF) of PF-CABD with 1 mM EDTA (solid line) produced a single peak (180–191 ml) but with 5 mM CaCl2 (dotted line) produced multiple high MW aggregates (100–186 ml). Void volume (Vo). (b) SDS-PAGE of GF eluted protein (1 mM EDTA) within 169–197 ml fractions demonstrated PF-CABD protein; none was present in 105 ml fraction. (c) SDS-PAGE of GF eluted protein (5 mM CaCl2) in 101–169 ml fractions demonstrated PF-CABD high MW aggregates eluting earlier than expected. L=loaded sample. (d) Light scattering of PF-CABD with 1 mM EDTA (solid lines) demonstrated a single homogenous peak with MW (22,590 Da) of a PF-CABD dimer (scattering distribution labeled ‘D’) but with 5 mM CaCl2 (dotted lines) demonstrated multiple peaks of higher than expected MW (scattering distributions labeled 1–3). (e) Increased ANS fluorescence emission for PF-CABD (2 μM) in 2 mM CaCl2 (dotted line) compared with PF-CABD in 2 mM EDTA (solid line) indicated calcium-dependent structural opening of the protein to expose hydrophobic residues. MW, molecular weight; PF-CABD, N-terminal S100 fused-type calcium-binding domain of human profilaggrin.
      To further characterize these distinct PF-CABD aggregates, multiple angle light scattering was performed on PF-CABD in the absence and presence of calcium. PF-CABD in 1 mM EDTA without added calcium demonstrated a monodisperse peak of 22,590 Da (Figure 1d, solid lines); this is double the expected calculated MW of 11,194 Da for a PF-CABD monomer. In the presence of 5 mM (Figure 1d, dotted lines) or 15 mM (data not shown) calcium chloride, PF-CABD aggregated into multiple peaks corresponding to complexes ranging from 30,550 Da to 117,300 Da. Thus, in the absence of calcium, PF-CABD exists as a stable dimer; however, in the 5–15 mM calcium environment tested PF-CABD aggregates in vitro into higher MW species.
      The calcium-dependent conformational opening of PF-CABD to expose hydrophobic residues was demonstrated by fluorescence spectroscopy (Figure 1e) using the molecular probe 8-anilinonaphthalene-1-sulfonic acid (ANS), which fluoresces greater when bound to hydrophobic surface. Fluorescence results confirm that PF-CABD undergoes calcium-dependent conformational/structural change and suggest non-specific hydrophobic interactions as the mechanism for higher-order (beyond dimer) protein aggregation.
      We subsequently determined the 2.2 Å resolution x-ray crystal structure of PF-CABD bound to calcium (Table 1). The structure demonstrates that PF-CABD is a dimer (Figure 2a). Each PF-CABD monomer forms a four-helix domain similar to other EF-hand calcium-binding proteins and binds two calcium ions (Supplementary Figures S1 and S2 online). The crystal asymmetric unit contains two PF-CABD dimers bound together for four total copies of PF-CABD per asymmetric unit; eight calcium ions (two per PF-CABD) are bound (Figure 2b,Supplementary Figure S3 online). Comparison of PF-CABD inter-helical angles with other select S100 proteins demonstrates unique helical orientations, particularly for helix I/IV and helix II/III interfaces (Supplementary Table S1 online); the four PF-CABD subunits in the asymmetric unit have similar helical orientations (average root mean square deviation 0.82 Å).
      Table 1Data collection and refinement statistics
      CrystalPF-CABD
      Diffraction Data
      Space groupP212121
      Unit cell dimensions,
      a, b, c (Å)40.49, 85.28, 96.90
      α, β, γ (°)90, 90, 90
      Resolution range (outer shell), Å50–2.20 (2.24–2.20)
      Values in parentheses are for highest-resolution shell.
      I/σI12.7 (1.69)
      CC(1/2) in outer shell, %59.2
      Completeness, %92.9 (93.3)
      Rmerge0.13 (0.854)
      Rpim0.067 (0.439)
      Rmeas0.147 (0.966)
      No. of crystals used1
      No. of unique reflections16,492
      Redundancy (outer shell)4.2 (4.1)
      Wilson B-factor, Å226.9
      Refinement
       Rwork, %19.8 (25.2)
       Rfree, %24.2 (32.2)
       Rfree test set size, %5.13
      No. of non-hydrogen atoms
       Protein2,978
       Ligands/ions (2PE, Ca)64
       Waters120
      r.m.s. Deviations
       Bond lengths, (Å)0.003
       Angles, (°)0.719
       Chirality0.027
       Planarity0.004
       Dihedral, (°)16.61
      Average B-factor, (overall) Å246.0
       Protein/Ca/2PE/waters47.21/36.70/44.85/42.0
      Abbreviations: PF-CABD, N-terminal S100 fused-type calcium-binding domain of human profilaggrin; r.m.s., root mean square.
      1 Values in parentheses are for highest-resolution shell.
      Figure thumbnail gr2
      Figure 2Crystal structure of the N-terminal S100 fused-type calcium-binding domain of human profilaggrin. (a) PF-CABD is biologically a dimer (protein 1, magenta; protein 2, blue with prime symbol). PF-CABD monomer forms a four-helix bundle. (b) The crystal AU contained two PF-CABD dimers (dimer 1, magenta/blue; dimer 2, orange/gray). The tetramer core is composed of four helix IV helices. (c) PF-CABD dimer rotated forward 120° about x axis compared with a to display the antiparallel helix IV plane connected to a schematic of remaining profilaggrin sequence. (d) Molecular surface of hydrophobic pocket in PF-CABD dimer illustrating two hydrophobic pockets (orange color gradient based on the degree of hydrophobicity); polar residues=blue; orientation 40° backward rotation about x axis compared with panel a. (e) Electrostatic surface potential of PF-CABD dimer, demonstrating acidic calcium-binding loops (red) and uncharged pocket (white). Basic residues=blue. (f) Only five residues are conserved in the hydrophobic pocket (labeled, magenta) across S100 fused-type protein family. Non-conserved residues colored blue. Calcium ions=green. AU, asymmetric unit; B, B domain; F, filaggrin units; H, helix; L, interhelical linker; PF-CABD, N-terminal S100 fused-type calcium-binding domain of human profilaggrin.
      The dimerization interface is formed primarily by α-helices I and IV from interdigitating monomers. Helices I and IV from one monomer form a V-shaped groove that is occupied by helix I from the dimerization partner. Monomer interdigitation creates two α-helical planes: “helix I” plane (two antiparallel N-terminal helices of homodimer) and “helix IV” plane (two antiparallel C-terminal helices of homodimer). This antiparallel dual-plane helical architecture structurally positions the remainder of profilaggrin on opposite sides of the homodimer in a sterically feasible state (Figure 2c). Residues stabilizing the interhelical interfaces are mainly hydrophobic or aromatic (Supplementary Table S1 online).
      Stabilizing interactions exist for the N-terminus (Leu3 and Leu4) and C-terminus (Tyr85; Supplementary Table S1 online). Tyr85 is stabilized by a four aromatic ring cluster; Tyr85 of one monomer stacks against several phenylalanine residues derived from the opposing dimer molecule: Phe14, Phe70, and Phe73 (Supplementary Figure S4 online). The total combined area of the molecular surface buried between dimerizing subunits is ~3,000 Å2.
      Y2H experiments examined the ability of the human profilaggrin N-terminus (PF-NT) (CABD (residues 1–92) plus B domain (residues 93–293)) to form homodimers in vivo. PF-NT homodimerization was observed only when both bait and prey constructs contained full-length PF-NT (Supplementary Figures S5 and S6 online); a similar positive interaction was seen using full PF-NT and a slightly shorter profilaggrin peptide (259 residues; AB-259). No Y2H interaction was seen with shorter constructs containing only part of the B domain. PF-CABD was unable to form homodimers in yeast, under conditions where S100A2 could form a functional protein interaction. Western analysis with PF-NT antibodies showed that all B domain constructs were abundantly expressed in yeast cells; however, PF-CABD constructs were less well expressed but detectable on immunoblots (data not shown). The in vivo Y2H experiments complement the in vitro biochemical data and together reveal a dimerization capacity for human PF-NT.

      Profilaggrin N-terminal S100 domain contains a unique hydrophobic pocket

      The molecular surface of the PF-CABD dimer demonstrates two distinct, symmetric hydrophobic pockets arranged ~115° opposite each other (Figures 2d and e). Each pocket (one per PF-CABD subunit) is formed by accessible surface area (ASA) contributions from 24 residues, 14 of which are hydrophobic and account for 47.4% of the pocket ASA (Supplementary Table S2 online). In addition, there are five polar (31.8% ASA), two basic (14.4% ASA), and three acidic (6.4% ASA) residues contributing to the molecular surface properties of the pocket. The two pockets are connected by a small hydrophobic surface patch (~226 Å2) formed by adjacent Phe 78 residues. Previous investigations on target selectivity in S100 proteins showed, despite sharing a common EF-hand structural motif and moderate sequence similarity, that S100 proteins have unique surface properties at their target protein binding sites, enabling functional diversity (
      • Bhattacharya S.
      • Bunick C.G.
      • Chazin W.J.
      Target selectivity in EF-hand calcium binding proteins.
      ). One dimer subunit contained a polyethylene glycol 400 molecule bound in the hydrophobic pocket (Supplementary Figure S7 online).
      To analyze primary sequence and molecular surface conservation across all seven members of the S100 fused-type protein family, a multiple sequence alignment was generated (Supplementary Figure S8 online) and conserved and non-conserved residues mapped onto the PF-CABD structure (Figure 2f). Excluding the N-terminal Met, S100 fused-type proteins conserve 26 residues, defining “conserved” as at least 6 out of 7 family members contain the same amino acid at a given position. Of those 26, only 5 are within the hydrophobic pocket; thus, of 24 residues comprising the hydrophobic pocket (Supplementary Table S2 online), only 21% are conserved. This illustrates how S100 fused-type proteins utilize sequence diversity within the hydrophobic pocket to generate unique binding interfaces for potential protein targets in the skin.

      Identification of human profilaggrin N-terminus-associated proteins

      To identify proteins that associate with the 32 kDa human PF-NT, we performed immunoprecipitation of human foreskin cell lysates with profilaggrin N-terminal B1 antibody and fractionated the total precipitate using SDS-PAGE. Western analysis of immunoprecipitate with profilaggrin B1 antibody confirmed the presence of this peptide in the mixture. Eight bands visible by SDS-PAGE after immunoprecipitation with profilaggrin B1 antibody were selected for trypsin digestion and mass spectrometry. Three proteins (in addition to profilaggrin N-terminus itself) were identified by mass spectrometry: annexin II (p36 subunit), stratifin (14-3-3 sigma), and heat shock protein 27 (HSP27). Each protein was represented by three or more peptides encompassing 10.6–21% of the identified polypeptide (Supplementary Table S3 online). PF-NT was represented by 19 peptides spanning 128/293 residues (43.7%).

      Stratifin and annexin II directly interact with human profilaggrin N-terminus

      Y2H studies were conducted to further dissect interactions of PF-NT with stratifin and annexin II in vivo (Table 2; Figure 3 and Supplementary Figure S9 online). Human stratifin associated with both PF-NT and prey proteins containing the CABD plus varying amounts of the B domain (residues 120–293); however, stratifin did not interact with human PF-CABD alone. Stratifin additionally interacted with mouse PF-AB (Table 2; Supplementary Figure S9 online). Stratifin also forms homodimers in vivo (
      • Yaffe M.B.
      • Rittinger K.
      • Volinia S.
      • et al.
      The structural basis for 14-3-3: phosphopeptide binding specificity.
      ), but in yeast this interaction is relatively weak compared with the association with human or mouse PF-NT. Association of human profilaggrin B domain with stratifin in a calcium-independent manner is consistent with the presence of several potential 14-3-3 binding sites in profilaggrin (Supplementary Table S4 online).
      Table 2Summary of yeast two-hybrid data: interaction of profilaggrin N-terminus with human annexin II/p36 and stratifin/14-3-3σ
      Bait protein (pOBD construct)Prey protein (pOAD construct)Strength of interaction
      +++, robust growth on 10mM 3-amino 1,2,4-triazole (3-AT), ++, weaker growth (and less colonies) on 10mM 3-AT; +, weak growth on 10mM 3-AT.
      Annexin II, 1-339Human AB+++
      Annexin II, 1-339Human AB (S100 mutant)
      This protein contains mutations in key amino acids involved in calcium binding (see text for details).
      ±
      Very weak but detectable growth on histidine-deficient media containing 10mM 3-AT.
      Annexin II, 1-339Human A++
      Annexin II, 1-339Mouse AB++
      Annexin II, 1-339Mouse A++
      Annexin II, 1-44Human AB++
      Annexin II, 1-44Mouse A++
      Annexin II, 15-339Human AB
      Annexin II, 15-339Mouse A
      StratifinHuman AB
      Stratifin exhibited a positive interaction with all human profilaggrin constructs containing varying lengths of the B domain––i.e., PF-AB 120, PF-AB 140, PF-AB 160, PF-AB 218, PF-AB 259, and PF-AB 293. Growth was similar (++) for all bait–prey combinations on histidine-deficient media containing 10mM 3-AT.
      ++
      StratifinHuman AB (S100 mutant)
      This protein contains mutations in key amino acids involved in calcium binding (see text for details).
      ++
      StratifinHuman A
      StratifinHuman B+
      StratifinMouse AB++
      StratifinMouse A+
      StratifinMouse B+
      StratifinStratifin±
      Very weak but detectable growth on histidine-deficient media containing 10mM 3-AT.
      1 +++, robust growth on 10 mM 3-amino 1,2,4-triazole (3-AT), ++, weaker growth (and less colonies) on 10 mM 3-AT; +, weak growth on 10 mM 3-AT.
      2 This protein contains mutations in key amino acids involved in calcium binding (see text for details).
      3 Very weak but detectable growth on histidine-deficient media containing 10 mM 3-AT.
      4 Stratifin exhibited a positive interaction with all human profilaggrin constructs containing varying lengths of the B domain––i.e., PF-AB 120, PF-AB 140, PF-AB 160, PF-AB 218, PF-AB 259, and PF-AB 293. Growth was similar (++) for all bait–prey combinations on histidine-deficient media containing 10 mM 3-AT.
      Figure thumbnail gr3
      Figure 3The profilaggrin S100 domain interacts with the N-terminus of annexin II. Yeast two-hybrid (Y2H) analysis (a and b) demonstrated that the A (S100) domain of profilaggrin interacts with the N-terminus of annexin II. (a) Bait (green) and prey (pink) plasmid combinations were plated on histidine, leucine, and tryptophan-deficient media containing 10 mM 3-amino-1,2,4-triazole. Both full length ANXA2 and a truncated N-terminal protein (ANXA2, 1–44) interacted with human and mouse profilaggrin N-terminus, but an ANXA2 protein lacking the first 14 amino acids (ANXA2, 15-339) showed no Y2H signal. (b) Control (leucine and tryptophan-deficient media) plate showing confluent growth of bait/prey combinations. (c) Association of profilaggrin N-terminus and annexin II in vitro is calcium dependent. Epidermal proteins were immunoprecipitated with either profilaggrin B domain (B1) antibody, a p21/WAF1 antibody, or a pre-immune rabbit control. Immunoprecipitated proteins were separated on SDS/polyacrylamide gels and immunoblotted with annexin II antibody. The lanes show immunoprecipitation with the following: B1 antibody, with no additions; B1/Ca, B1 antibody with the addition of 5 mM CaCl2; B1/E, B1 antibody with the addition of 5 mM EDTA; and p21/WAF1 antibody or pre-immune serum, with no additions. E represents a control epidermal extract to show annexin II.
      Annexin II/p36 interacts with human and mouse PF-CABD domain in a calcium-dependent manner, as well as the corresponding PF-AB regions (Table 2; Figure 3; Supplementary Discussion online). Removal of the N-terminal 14 amino acids from annexin II, which is the domain that binds S100A10 (
      • Bharadwaj A.
      • Bydoun M.
      • Holloway R.
      • et al.
      Annexin A2 heterotetramer: structure and function.
      ), prevented its interaction with human PF-NT (Figure 3). These studies demonstrate that PF-NT and S100A10 bind the same N-terminal domain of annexin II. Immunoprecipitation experiments using human epidermal lysates confirmed that annexin II associates with PF-NT; however, detectable annexin II could only be pulled down with profilaggrin N-terminal antibody in the presence of added calcium (Figure 3c).
      To investigate the role of calcium coordination on annexin II binding and homodimerization, six mutations were made to key calcium coordination residues in PF-NT (two in N-terminal pseudo/non-canonical EF-hand (D21A, E31V) and four in C-terminal canonical EF-hand (D61A, I62T, D63A, E72V)). Mutant PF-NT showed diminished binding affinity with annexin II in yeast (Table 2); by contrast, alteration of calcium binding did not diminish the strength of interaction with stratifin (Supplementary Figure S9 online). Homodimerization of mutant human PF-NT protein in yeast was not affected (data not shown), consistent with our biochemical data showing stable homodimer formation in the absence of calcium.

      Stratifin and human profilaggrin N-terminus co-localize in the epidermal granular layer

      To determine whether stratifin and profilaggrin N-terminus co-localize in human epithelial cells, we performed double-label immunofluorescence microscopy on paraformaldehyde-fixed human skin (Figures 4a and b). The results confirm that PF-NT and stratifin are co-expressed in the stratum granulosum of human skin and co-localize in vivo, primarily at the cell periphery (Figure 4b).
      Figure thumbnail gr4
      Figure 4Profilaggrin N-terminus and stratifin co-localize in human epidermal granular cells. Double label immunofluorescence was performed on fixed adult human skin using antibodies directed against the profilaggrin N-terminus (green) and stratifin (red). Panel a shows that stratifin is expressed throughout the epidermis, whereas profilaggrin is restricted to the granular layer and anuclear stratum corneum. (b) Shown is a vertical section of adult human skin immunolabeled with stratifin (red) and profilaggrin N-terminus (green) antibodies, with nuclei counterstained with DAPI. Stratifin is localized through the cytoplasm in spinous cells, but in the upper granular layer it is concentrated at the cell periphery where it co-localizes with profilaggrin N-terminus (orange labeling, arrows). Profilaggrin is also present in keratohyalin granules in the characteristic granular pattern but shows little or no association with stratifin when present in the granular (profilaggrin) form. (c) Proposed biological functions of a calcium-dependent and calcium-independent protein interaction network for profilaggrin in human epidermis (based on the current study and the previous study by
      • Yoneda K.
      • Nakagawa T.
      • Lawrence O.T.
      • et al.
      Interaction of the profilaggrin N-terminal domain with loricrin in human cultured keratinocytes and epidermis.
      ). DAPI, 4′,6-diamidino-2-phenylindole.

      Discussion

      Few atomic resolution structures support the biology currently known about terminal epidermal differentiation or the stratum corneum. X-ray crystal structures of the major proteins known to exist in the stratum corneum (e.g., involucrin, keratins 1/10, loricrin, profilaggrin) to our knowledge have not been previously reported. This study correlates profilaggrin structure with its function and establishes an important foundation for future structural studies in epidermal biology. Moreover, it shows that at least one member of the fused-type S100 family follows the paradigm of S100 proteins––i.e., calcium binding, peptide homodimerization, and physical association with other intracellular proteins. This is important because the function of fused-type S100 domains in other epidermal proteins is unknown.
      Advances presented here establish several principles for epidermal barrier biology: (1) profilaggrin’s N-terminus undergoes calcium-independent homodimerization and calcium-dependent non-specific hydrophobic aggregation; (2) the S100 fused-type CABD contains a unique hydrophobic target binding site; (3) profilaggrin participates in protein interactions with stratifin, annexin II, and HSP27; and (4) the S100 fused-type CABD and B domain both contribute to target binding selectivity. Furthermore, we propose a model whereby calcium promotes aggregation of profilaggrin into KH storage granules, and in which the identified binding partners associate with profilaggrin or the cleaved, soluble N-terminal peptide, regulating both its nuclear role in keratinocyte terminal differentiation and incorporation into the functionally important cornified cell envelope (Figure 4c).
      Profilaggrin is stored within KH granules in a calcium-rich environment; molecular driving forces for KH granule assembly are not fully known. It has been proposed that both extensive phosphorylation of filaggrin units and calcium binding by the N-terminus are required for KH granule formation (
      • Dale B.
      • Resing K.
      • Presland R.
      Keratohyalin granules.
      ). The PF-CABD structure offers several mechanisms of how this domain may contribute to KH granule assembly: (1) homodimerization facilitates aggregation of profilaggrin as there are 50% less free molecules; (2) calcium-bound PF-CABD adopts an open conformation with exposed hydrophobic patches; (3) hydrophobic patches, through self-aggregation or target binding, offer a protein–protein interaction mechanism for assembly. HSP27, for example, co-localized with profilaggrin to KH granules, and loss of HSP27 is associated with hyperkeratinization and misprocessing of profilaggrin (
      • O'Shaughnessy R.F.
      • Welti J.C.
      • Cooke J.C.
      • et al.
      AKT-dependent HspB1 (Hsp27) activity in epidermal differentiation.
      ).
      Similar to most S100 proteins (
      • Isobe T.
      • Ishioka N.
      • Okuyama T.
      Structural relation of two S-100 proteins in bovine brain; subunit composition of S-100a protein.
      ;
      • Hermann A.
      • Donato R.
      • Weiger T.M.
      • et al.
      S100 calcium binding proteins and ion channels.
      ), profilaggrin CABD formed homodimers. PF-NT (PF-CABD+B domain), the physiologically relevant proteolytic product, also formed homodimers. In the Y2H system, however, PF-CABD alone did not form homodimers (Supplementary Figure S5 online). The likely explanation for this discrepancy is that the shorter human PF-CABD protein, but not the longer PF-CABD+B, is prevented from forming homodimers in yeast by the attached yeast GAL4 transcription factor domains. Despite one inconsistent experiment, all other biochemical, structural, and Y2H studies supported dimerization of PF-NT mediated by the S100 domain. Additional studies are needed to determine whether full-length profilaggrin dimerizes; for PF-CABD, dimerization potentially enables binding of two similar or different macromolecules simultaneously. This may impart stability to the stratum corneum; for example, some PF-NT is retained in the stratum corneum and may be cross-linked as part of the cornified envelope (
      • Presland R.B.
      • Kimball J.R.
      • Kautsky M.B.
      • et al.
      Evidence for specific proteolytic cleavage of the N-terminal domain of human profilaggrin during epidermal differentiation.
      ).
      Identification of annexin II, stratifin, and HSP27 as target proteins for PF-NT demonstrates that human profilaggrin may be an epidermal signaling molecule or involved in protein trafficking, in addition to its role in cytokeratin reorganization and tissue moisturization (Figure 4c). We previously found that PF-NT binds loricrin, a component of the cornified cell envelope (
      • Yoneda K.
      • Nakagawa T.
      • Lawrence O.T.
      • et al.
      Interaction of the profilaggrin N-terminal domain with loricrin in human cultured keratinocytes and epidermis.
      ). Similar to loricrin, stratifin (epithelial-specific member of the 14-3-3 protein family also known as 14-3-3 sigma) interacted with PF-NT and not with the CABD domain alone. 14-3-3 proteins including stratifin bind phosphorylated proteins, often to R-X-X-pS sequences (
      • Yaffe M.B.
      • Rittinger K.
      • Volinia S.
      • et al.
      The structural basis for 14-3-3: phosphopeptide binding specificity.
      ). Human and mouse PF-NT contain several RXXS sequences (five in human, six in mouse; Supplementary Table S4 online) located in the B domain, which may function as stratifin binding sites. Whereas both the S100 and B domains cooperatively bound stratifin in a calcium-independent manner, annexin II bound only to the S100 domain in a calcium-dependent manner. Y2H experiments showed that the N-terminal 14 amino acids of annexin II mediated the interaction with PF-CABD. Analysis of the PF-CABD structure bound to the N-terminal 11 residues of annexin II using in silico molecular docking suggests that six annexin II (Thr2, Val3, Ile6, Leu7, Lys9, Leu10) and seven profilaggrin (Gln42, Phe40, Met52, Phe56, Met76, Leu80, and Tyr84) residues mediate the interaction (Supplementary Figure S10 online). There is a debate whether Y2H experiments can discriminate between calcium-regulated and calcium-independent interactions (
      • Deloulme J.C.
      • Gentil B.J.
      • Baudier J.
      Monitoring of S100 homodimerization and heterodimeric interactions by the yeast two-hybrid system.
      ); however, profilaggrin is a fused-type S100 protein distinct from isolated S100 domains, with evidence that downstream protein domains (e.g., B domain) influence target binding affinity, likely through domain cooperativity. To confirm identified protein interactions, multiple techniques were utilized besides Y2H.
      In summary, we determined the crystal structure of the N-terminal S100 CABD of human profilaggrin, analyzed its suspected target-binding site in molecular detail, and identified protein binding partners for each domain of the cleaved N-terminus. This work enhances our understanding of profilaggrin as a multifunctional epidermal protein; it is a precursor to stratum corneum structure and function, engages in a protein interaction network in human epidermis, and may be active in epidermal protein–protein signaling and protein trafficking.

      Materials and Methods

      Profilaggrin, annexin II (p36), and stratifin constructs

      Design and production of constructs are detailed in Supplementary Materials and Methods and Supplementary Table S5 online.

      Antibodies

      Primary antibodies were directed against annexin II (Invitrogen, Carlsbad, CA), stratifin (Upstate Biotechnology, Lake Placid, NY), p21 (Cell Signaling Technology, Danvers, MA), and the A (PF-CABD) and B domains of the human profilaggrin N-terminus (
      • Presland R.B.
      • Kimball J.R.
      • Kautsky M.B.
      • et al.
      Evidence for specific proteolytic cleavage of the N-terminal domain of human profilaggrin during epidermal differentiation.
      ).

      Protein production and purification

      N-terminal calcium binding domain of human profilaggrin (PF-CABD) was produced and purified (detailed in Supplementary Table S6 online).

      Multi-angle light scattering

      Apo-PF-CABD (5.2 mg ml-1) in 50 mM Tris-HCl buffer (pH 7.8) containing 0.5M NaCl and 1 mM EDTA was applied at 0.5 ml per minute to Superdex 75 gel filtration column in-line with DAWN HELEOS II light scattering instrument (Wyatt Technology, Santa Barbara, CA; laser wavelength 658.0 nm). Data collection/analysis used Astra software (Wyatt Technology) version 5.3.4.20. After preparation by overnight dialysis against 50 mM Tris-HCl buffer (pH 7.8) containing 0.5M NaCl and either 5 mM or 15 mM CaCl2, calcium-bound samples of PF-CABD were analyzed similar to apo state.

      Fluorescence spectroscopy

      Fluorescence measurements were made using a SLM Aminco spectrofluorometer (SLM Instruments, Urbana, IL) following established protocol (
      • Bunick C.G.
      • Nelson M.R.
      • Mangahas S.
      • et al.
      Designing sequence to control protein function in an EF-hand protein.
      ).

      Crystallization and X-ray data collection

      PF-CABD (4.6 mg ml-1) in 50 mM Tris-HCl buffer (pH 7.8) containing 0.5M NaCl and 1 mM EDTA was crystallized by sitting drop vapor diffusion using a reservoir solution of 40% polyethylene glycol 4,000, 50 mM Tris-HCl buffer (pH 8.5), and 0.1M CaCl2. Drops were prepared by mixing 1 μl PF-CABD with 1 μl reservoir solution. Crystals grew over 24–48 hours at 25 °C. PF-CABD crystals were soaked 1–3 minutes in cryoprotectant solution containing 10% polyethylene glycol 400 in mother liquor prior to flash cooling of the crystal by direct immersion into a crystal puck storage system maintained under liquid nitrogen. A native data set on a single crystal maintained at ~100 K was collected using the X-29 beamline (λ=1.075 Å) at National Synchrotron Light Source in Brookhaven National Laboratory. PF-CABD crystallized in the orthorhombic space group P212121 (cell dimensions: a=40.49 Å, b=85.28 Å, c=96.90 Å, α=β=γ=90°) with a unit cell solvent content of 34%. Diffraction data were processed using HKL2000 (HKL Research, Charlottesville, VA).

      Structure determination, refinement, and analysis

      PF-CABD structure was determined by molecular replacement with MOLREP (
      • Vagin A.
      • Teplyakov A.
      Molecular replacement with MOLREP.
      ) using calcium-bound S100A12 (PDB 1E8A) as the search model. A simulated annealing composite omit map generated in PHENIX (
      • Adams P.D.
      • Afonine P.V.
      • Bunkóczi G.
      • et al.
      PHENIX: a comprehensive Python-based system for macromolecular structure solution.
      ) was necessary to overcome the initial model bias. The PF-CABD structure underwent iterative rounds of model building (Coot (
      • Emsley P.
      • Cowtan K.
      Coot: model-building tools for molecular graphics.
      )) and refinement (PHENIX). Final model of asymmetric unit contained four PF-CABD molecules (two homodimers), 8 calcium ions, 120 water molecules, and two polyethylene glycol 400 molecules. Final Ramachandran statistics: residues in favorable regions, 95.1%; in allowed regions, 4.0% and; in outlier regions, 0.9%. Structural analyses were performed with Coot, UCSF Chimera (Resource for Biocomputing, Visualization, and Informatics, University of California, San Francisco), INTERHLX (K. Yap, University of Toronto), and PDBePISA (The European Bioinformatics Institute, European Molecular Biology Laboratory, UK). Electrostatics calculated using PDB2PQR (
      • Dolinsky T.J.
      • Nielsen J.E.
      • McCammon J.A.
      • et al.
      PDB2PQR: an automated pipeline for the setup of Poisson-Boltzmann electrostatics calculations.
      ) and Adaptive Poisson-Boltzmann Software (
      • Baker N.A.
      • Sept D.
      • Joseph S.
      • et al.
      Electrostatics of nanosystems: application to microtubules and the ribosome.
      ). Figures prepared using UCSF Chimera or the PyMOL Molecular Graphics System (Version 1.5.0.4, Schrödinger, New York, NY).

      Yeast two-hybrid assays, expression analysis, and profilaggrin protein interactions

      Details for yeast experiments, immunoprecipitation and mass spectrometry studies, and immunofluorescence microscopy are in Supplementary Materials and Methods online.

      Statement on use of human materials

      Normal human neonatal and adult skin samples, utilized for immunofluorescence microscopy and immunoprecipitation studies, were obtained through the Division of Dermatology with approval from the University of Washington Institutional Review Board.

      Acknowledgments

      We thank Joe Watson and Bill Eliason for assistance with purification and light scattering experiments, respectively, Ivan Lomakin for ANS fluorescence assistance, Daniel Eiler and Jimin Wang for crystallographic discussions, Rebecca Hjorten for performing immunohistochemistry experiments, and Jessica Huard and Bradley Sainsbury for Y2H assistance. We thank Professor Walter J Chazin for critical manuscript review. Work was supported by the Dermatology Foundation through a Dermatologist Investigator Research Fellowship and a Career Development Award (to CGB), the National Institutes of Health/NIAMS Dermatology Training Grant to Yale (PI: Richard Edelson) T32 AR007016 (to CGB), and by grant R01 AR49183 from the NIH (to RBP). DJP was partially supported by grant P01 AM21557 from the NIH. Atomic coordinates and structure factors have been deposited in the Protein Data Bank under accession code 4PCW.

      Supplementary Material

      Supplementary material is linked to the online version of the paper at http://www.nature.com/jid

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