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

Abstract

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.

Figure 1. Calcium-independent dimerization and calcium-dependent self-aggregation of profilaggrin N-terminal S-100 fused-type calcium-binding domain

Gel filtration (GF) of PF-CABD with 1mM EDTA (solid line) produced a single peak [180-191mL], but with 5mM CaCl₂ (dotted line) produced multiple high MW aggregates [100-186mL]. Void volume (V_o). (b) SDS-PAGE of GF eluted protein (1mM EDTA) within 169-197mL fractions demonstrated PF-CABD protein; none was present in 105mL fraction. (c) SDS-PAGE of GF eluted protein (5mM CaCl₂) in 101-169mL fractions demonstrated PF-CABD high MW L = loaded sample. (d) Light scattering of PF-CABD with 1mM EDTA (solid lines) demonstrated a single homogenous peak with MW (22,590 Da) of a PF-CABD dimer (scattering distribution labeled “D”), but with 5mM 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 2mM CaCl₂ (dotted line) compared to PF-CABD in 2mM EDTA (solid line) indicated calcium-dependent structural opening of the protein to expose hydrophobic residues.

Figure 2. Crystal 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 to panel 2a to display the antiparallel helix IV plane connected to a schematic of remaining profilaggrin sequence. B = B domain; F = filaggrin units. (d) Molecular surface of hydrophobic pocket in PF-CABD dimer illustrating two hydrophobic pockets (orange color gradient based on degree of hydrophobicity); polar residues = blue; orientation 40° backward rotation about x-axis compared to Figure 2a. (e) Electrostatic surface potential of PF-CABD dimer, demonstrating acidic calcium-binding loops (red) and uncharged pocket (white). Basic residues = blue. (f) Only 5 residues are conserved in the hydrophobic pocket (labeled, magenta) across S100 fused-type protein family. Non-conserved residues colored blue. Calcium ions = green. L = interhelical linker; H = helix.

Figure 3. The profilaggrin S100 domain interacts with the N-terminus of annexin II

Yeast two-hybrid analysis (panels a and b) demonstrated 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 HLT-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 (LT-deficient) 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 immunblotted with annexin II antibody. The lanes show immunoprecipitation with: 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; p21/WAF1 antibody or pre-immune serum, with no additions. E represents a control epidermal extract to show annexin II.

Figure 4. Profilaggrin 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, while 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 PNT (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 PNT (orange labeling, arrows). Profilaggrin is also present in KHGs 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 et al., 2012).