Conformational and thermodynamic properties of peptide binding to the human S100P protein

Abstract

S100P is a member of the S100 subfamily of calcium-binding proteins that are believed to be associated with various diseases, and in particular deregulation of S100P expression has been documented for prostate and breast cancer. Previously, we characterized the effects of metal binding on the conformational properties of S100P and proposed that S100P could function as a Ca 2+ conformational switch. In this study we used fluorescence and CD spectroscopies and isothermal titration calorimetry to characterize the target-recognition properties of S100P using a model peptide, melittin. Based on these experimental data we show that S100P and melittin can interact in a Ca 2+ -dependent and -independent manner. Ca 2+ -independent binding occurs with low affinity (K d ≈ 0.2 mM), has a stoichiometry of four melittin molecules per S100P dimer and is presumably driven by favorable electrostatic interactions between the acidic protein and the basic peptide. In contrast, Ca 2+ -dependent binding of melittin to S100P occurs with high affinity (K d ≈ 5 M) has a stoichiometry of two molecules of melittin per S100P dimer, appears to have positive cooperativity, and is driven by hydrophobic interactions. Furthermore, Ca 2+ -dependent S100P-melittin complex formation is accompanied by significant conformational changes: Melittin, otherwise unstructured in solution, adopts a helical conformation upon interaction with Ca 2+ -S100P. These results support a model for the Ca 2+ -dependent conformational switch in S100P for functional target recognition.Keywords: Isothermal titration calorimetry; circular dichroism spectroscopy; fluorescence spectroscopy; structural thermodynamics; conformational transitionThe human protein S100P belongs to the S100 subfamily of calcium-binding proteins that share a common Ca 2+ -binding structural motif, the EF-hand (Kretsinger 1976;Chazin 1995). Twenty members of the subfamily have been identified to date (Donato 1999;Gribenko et al. 2001). These proteins have two EF-hand motifs in their primary structure, various metal-binding properties, and are expressed in a tissue-and cell-type-specific fashion (Hilt and Kligman 1991;Zimmer et al. 1995;Schafer and Heizmann 1996). Increased or altered expression of S100 proteins has been documented for many human diseases and tumors (Donato 1999). S100P was shown to be differentially expressed in androgen-dependent and androgen-independent prostate cancer cell lines (Averboukh et al. 1996). Recently it has also been shown that S100P overexpression is associated with immortalization of human breast epithelial cells in vitro and early stages of breast cancer development in vivo (Guerreiro Da Silva et al. 2000).In our previous report we characterized the oligomerization and divalent cation-binding properties of S100P and proposed a Ca 2+ /Mg 2+ switch model for S100P function

Elimination of the C-cap in ubiquitin—structure, dynamics and thermodynamic consequences

“…The buffer used in all titration experiments and in measurements of fluorescence emission spectra was 25mM sodium acetate, pH 5.5. Concentration of the stock solution of ANS (8-anilino-1-naphtalene sulfonic acid) in buffer was determined spectrophotometrically using an extinction coefficient of 5000M − 1 cm − 1 at 355 nm [9]. Stock solutions of protein and ANS were mixed to the final concentrations 10μM and 25 μM, respectively.…”

S‐100 proteins in the human peripheral nervous system

“…The so-called S100A1 or S100␣ (93 amino-acid residues, 10.4 kDa), and S100B or S100␤ (91 amino-acid residues, 10.5 kDa) are the main forms present in nervous tissues (Haimoto et al, 1987;Sugimura et al, 1989;Zimmer and Van Eldik, 1987), and they are combined as homo or heterodimers to form S100a0 (␣␣), S100a (␣␤) and S100b (␤␤), but also higher-order aggregates (for a review, see Heizmann, 2002;Heizmann et al, 2002;Schafer and Heizmann, 1996). At present 14 subfamilies have been identified in the S100␣-group (Pietas et al, 2002), and new families are emerging, i.e., S100P (Gribenko et al, 2002), S100Z (Gribenko et al, 2001) S100 proteins participate in the regulation of intracellular Ca ϩϩ homeostasis (Baimbridge et al, 1992;Barger and Van Eldik, 1992;Heizmann, 2002), although in neurons they are trigger or activation proteins and not buffers of Ca 2ϩ . In addition, S100␤ has neurotrophic activity enhancing neurite outgrowth and survival of neurons (Bhattacharyya et al, 1992;Huttunen et al, 2000;Marshak, 1990;Val Eldik et al, 1991;WinninghamMajor et al, 1989).…”

Target Peptide Recognition by S100P Protein and Role of Central Linker Region and Dimer Interface

“…The mutations were detected by sequencing. Recombinant and WT proteins were expressed and purified according to Gribenko et al [4]. All reagents were purchased from Sigma.…”

“…Melittin is a small peptide of 26 amino acid residues. It exists as an unstructured monomer at neutral pH [4]. Calcium dependent complex formation occurs with a stochiometry of two molecules of mellitin per S100P dimer.…”