Structural properties of target binding by profilaggrin A and B domains and other S100 fused-type calcium-binding proteins


      • S100 fused-type proteins have distinct surface chemistries at target binding site
      • Inter-EF-hand linker residues help S100 fused-type proteins anchor substrate
      • Profilaggrin B domain works with A domain to bind and stabilize protein targets
      • Profilaggrin AB complex binds coiled-coil region of keratin intermediate filaments
      • Annexin 2 domains I and II are positioned to interact with profilaggrin B domain



      Profilaggrin belongs to the S100 fused-type protein family expressed in keratinocytes and is important for skin barrier integrity. Its N-terminus contains an S100 (“A”) domain and a unique “B” domain with a nuclear localization sequence.


      To determine whether profilaggrin B domain cooperates with the S100 domain to bind macromolecules. To characterize the biochemical and structural properties of the profilaggrin N-terminal “AB” domain and compare it to other S100 fused-type proteins.


      We used biochemical (protease protection, light scattering, fluorescence spectroscopy, pull-down assays) and computational techniques (sequence analysis, molecular modeling with crystallographic structures) to examine human profilaggrin and S100 fused-type proteins.


      Comparing profilaggrin S100 crystal structure with models of the other S100 fused-type proteins demonstrated each has a unique chemical composition of solvent accessible surface around the hydrophobic binding pocket. S100 fused-type proteins exhibit higher pocket hydrophobicity than soluble S100 proteins. The inter-EF-hand linker in S100 fused-type proteins contains conserved hydrophobic residues involved in binding substrates. Profilaggrin B domain cooperates with the S100 domain to bind annexin II and keratin intermediate filaments in a calcium-dependent manner using exposed cationic surface. Using molecular modeling we demonstrate profilaggrin B domain likely interacts with annexin II domains I and II. Steric clash analysis shows annexin II N-terminal peptide is favored to bind profilaggrin among S100 fused-type proteins.


      The N-terminal S100 and B domains of profilaggrin cooperate to bind substrate molecules in granular layer keratinocytes to provide epidermal barrier functions.


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        • Bhattacharya S.
        • Bunick C.G.
        • Chazin W.J.
        Target selectivity in EF-hand calcium binding proteins.
        Biochim. Biophys. Acta. 2004; 1742: 69-79
        • Kizawa K.
        • et al.
        S100 and S100 fused-type protein families in epidermal maturation with special focus on S100A3 in mammalian hair cuticles.
        Biochimie. 2011; 93: 2038-2047
        • Kypriotou M.
        • Huber M.
        • Hohl D.
        The human epidermal differentiation complex: cornified envelope precursors, S100 proteins and the’ fused genes’ family.
        Exp. Dermatol. 2012; 21: 643-649
        • Choi J.
        • et al.
        Hornerin is involved in breast Cancer progression.
        J. Breast Cancer. 2016; 19: 142-147
        • Steinert P.M.
        • et al.
        Characterization of a class of cationic proteins that specifically interact with intermediate filaments.
        Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 4097-4101
        • Bunick C.G.
        • et al.
        Crystal structure of human profilaggrin S100 domain and identification of target proteins annexin II, Stratifin and hsp27.
        J. Invest. Dermatol. 2015;
        • 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.
        J. Invest. Dermatol. 2002; 119: 661-669
        • Ishida-Yamamoto A.
        • et al.
        Translocation of profilaggrin N-terminal domain into keratinocyte nuclei with fragmented DNA in normal human skin and loricrin keratoderma.
        Lab. Invest. 1998; 78: 1245-1253
        • Aho S.
        • et al.
        Regulatory role for the profilaggrin N-terminal domain in epidermal homeostasis.
        J. Invest. Dermatol. 2012; 132: 2376-2385
        • Yoneda K.
        • et al.
        Interaction of the profilaggrin N-terminal domain with loricrin in human cultured keratinocytes and epidermis.
        J. Invest. Dermatol. 2012; 132: 1206-1214
        • Brown S.J.
        • et al.
        Intragenic copy number variation within filaggrin contributes to the risk of atopic dermatitis with a dose-dependent effect.
        J. Invest. Dermatol. 2012; 132: 98-104
        • Akiyama M.
        FLG mutations in ichthyosis vulgaris and atopic eczema: spectrum of mutations and population genetics.
        Br. J. Dermatol. 2010; 162: 472-477
        • Sandilands A.
        • et al.
        Comprehensive analysis of the gene encoding filaggrin uncovers prevalent and rare mutations in ichthyosis vulgaris and atopic eczema.
        Nat. Genet. 2007; 39: 650-654
        • Palmer C.N.
        • et al.
        Common loss-of-function variants of the epidermal barrier protein filaggrin are a major predisposing factor for atopic dermatitis.
        Nat. Genet. 2006; 38: 441-446
        • Sandilands A.
        • et al.
        Prevalent and rare mutations in the gene encoding filaggrin cause ichthyosis vulgaris and predispose individuals to atopic dermatitis.
        J. Invest. Dermatol. 2006; 126: 1770-1775
        • Smith F.J.
        • et al.
        Loss-of-function mutations in the gene encoding filaggrin cause ichthyosis vulgaris.
        Nat. Genet. 2006; 38: 337-342
        • Eldirany S.A.
        • et al.
        Human keratin 1/10-1B tetramer structures reveal a knob-pocket mechanism in intermediate filament assembly.
        EMBO J. 2019; 38
        • Bunick C.G.
        • et al.
        Designing sequence to control protein function in an EF-hand protein.
        J. Am. Chem. Soc. 2004; 126: 5990-5998
        • Waterhouse A.
        • et al.
        SWISS-MODEL: homology modelling of protein structures and complexes.
        Nucleic Acids Res. 2018; 46: W296-W303
        • Lynley A.M.
        • Dale B.A.
        The characterization of human epidermal filaggrin. A histidine-rich, keratin filament-aggregating protein.
        Biochim. Biophys. Acta. 1983; 744: 28-35
        • Scott I.R.
        • Harding C.R.
        • Barrett J.G.
        Histidine-rich protein of the keratohyalin granules. Source of the free amino acids, urocanic acid and pyrrolidone carboxylic acid in the stratum corneum.
        Biochim. Biophys. Acta. 1982; 719: 110-117
        • Zhang M.
        • Tanaka T.
        • Ikura M.
        Calcium-induced conformational transition revealed by the solution structure of apo calmodulin.
        Nat. Struct. Biol. 1995; 2: 758-767
        • Gasymov O.K.
        • Glasgow B.J.
        ANS fluorescence: potential to augment the identification of the external binding sites of proteins.
        Biochim. Biophys. Acta. 2007; 1774: 403-411
        • Matsui T.
        • Amagai M.
        Dissecting the formation, structure and barrier function of the stratum corneum.
        Int. Immunol. 2015; 27: 269-280
        • Candi E.
        • Schmidt R.
        • Melino G.
        The cornified envelope: a model of cell death in the skin.
        Nat. Rev. Mol. Cell Biol. 2005; 6: 328-340
        • Santamaria-Kisiel L.
        • Rintala-Dempsey A.C.
        • Shaw G.S.
        Calcium-dependent and -independent interactions of the S100 protein family.
        Biochem. J. 2006; 396: 201-214
        • Hermann A.
        • et al.
        S100 calcium binding proteins and ion channels.
        Front. Pharmacol. 2012; 3: 67
        • Quiroz F.G.
        • et al.
        Liquid-liquid phase separation drives skin barrier formation.
        Science. 2020; 367
        • Hughes M.P.
        • et al.
        Atomic structures of low-complexity protein segments reveal kinked β sheets that assemble networks.
        Science. 2018; 359: 698-701
        • Banani S.F.
        • et al.
        Biomolecular condensates: organizers of cellular biochemistry.
        Nat. Rev. Mol. Cell Biol. 2017; 18: 285-298
        • Shin Y.
        • Brangwynne C.P.
        Liquid phase condensation in cell physiology and disease.
        Science. 2017; 357
        • Presland R.B.
        • et al.
        Evidence for specific proteolytic cleavage of the N-terminal domain of human profilaggrin during epidermal differentiation.
        J. Invest. Dermatol. 1997; 108: 170-178
        • Pearton D.J.
        • et al.
        Proprotein convertase expression and localization in epidermis: evidence for multiple roles and substrates.
        Exp. Dermatol. 2001; 10: 193-203
        • O’Shaughnessy R.F.
        • et al.
        AKT-dependent HspB1 (Hsp27) activity in epidermal differentiation.
        J. Biol. Chem. 2007; 282: 17297-17305
        • Kayser J.
        • et al.
        The small heat shock protein Hsp27 affects assembly dynamics and structure of keratin intermediate filament networks.
        Biophys. J. 2013; 105: 1778-1785
        • Vishwanatha J.K.
        • Jindal H.K.
        • Davis R.G.
        The role of primer recognition proteins in DNA replication: association with nuclear matrix in HeLa cells.
        J. Cell. Sci. 1992; 101: 25-34
        • Eberhard D.A.
        • et al.
        Control of the nuclear-cytoplasmic partitioning of annexin II by a nuclear export signal and by p11 binding.
        J. Cell. Sci. 2001; 114: 3155-3166
        • Liu J.
        • Vishwanatha J.K.
        Regulation of nucleo-cytoplasmic shuttling of human annexin A2: a proposed mechanism.
        Mol. Cell. Biochem. 2007; 303: 211-220
        • Gutowska-Owsiak D.
        • et al.
        Orchestrated control of filaggrin-actin scaffolds underpins cornification.
        Cell Death Dis. 2018; 9: 412
        • Ma A.S.
        • Ozers L.J.
        Annexins I and II show differences in subcellular localization and differentiation-related changes in human epidermal keratinocytes.
        Arch. Dermatol. Res. 1996; 288: 596-603
        • Hayes M.J.
        • et al.
        Regulation of actin dynamics by annexin 2.
        EMBO J. 2006; 25: 1816-1826
        • Mack J.W.
        • Steven A.C.
        • Steinert P.M.
        The mechanism of interaction of filaggrin with intermediate filaments. The ionic zipper hypothesis.
        J. Mol. Biol. 1993; 232: 50-66
        • Lee C.H.
        • et al.
        Structural basis for heteromeric assembly and perinuclear organization of keratin filaments.
        Nat. Struct. Mol. Biol. 2012; 19: 707-715
        • Lomakin I.B.
        • et al.
        Crystal structure of keratin 1/10(C401A) 2B heterodimer demonstrates a proclivity for the C-terminus of helix 2B to form higher order molecular contacts.
        Yale J. Biol. Med. 2020; 93
        • Bunick C.G.
        • Milstone L.M.
        The X-Ray crystal structure of the keratin 1-Keratin 10 Helix 2b heterodimer reveals molecular surface properties and biochemical insights into human skin disease.
        J. Invest. Dermatol. 2017; 137: 142-150
        • Sandilands A.
        • et al.
        Filaggrin in the frontline: role in skin barrier function and disease.
        J. Cell. Sci. 2009; 122: 1285-1294
        • Lonsdale-Eccles J.D.
        • Teller D.C.
        • Dale B.A.
        Characterization of a phosphorylated form of the intermediate filament-aggregating protein filaggrin.
        Biochemistry. 1982; 21: 5940-5948
        • Dale B.A.
        • et al.
        Transient expression of epidermal filaggrin in cultured cells causes collapse of intermediate filament networks with alteration of cell shape and nuclear integrity.
        J. Invest. Dermatol. 1997; 108: 179-187
        • Brown S.J.
        • McLean W.H.
        One remarkable molecule: filaggrin.
        J. Invest. Dermatol. 2012; 132: 751-762
        • Dolinsky T.J.
        • et al.
        PDB2PQR: an automated pipeline for the setup of Poisson-Boltzmann electrostatics calculations.
        Nucleic Acids Res. 2004; 32: W665-W667