Design of high-affinity S100-target hybrid proteins

Abstract

S100B and S100A10 are dimeric, EF-hand proteins. S100B undergoes a calcium-dependent conformational change allowing it to interact with a short contiguous sequence from the actin-capping protein CapZ (TRTK12). S100A10 does not bind calcium but is able to recruit the N-terminus of annexin A2 important for membrane fusion events, and to form larger multiprotein complexes such as that with the cation channel proteins TRPV5/6. In this work, we have designed, expressed, purified, and characterized two S100-target peptide hybrid proteins comprised of S100A10 and S100B linked in tandem to annexin A2 (residues 1-15) and CapZ (TRTK12), respectively. Different protease cleavage sites (tobacco etch virus, PreScission) were incorporated into the linkers of the hybrid proteins. In situ proteolytic cleavage monitored by (1)H-(15)N HSQC spectra showed the linker did not perturb the structures of the S100A10-annexin A2 or S100B-TRTK12 complexes. Furthermore, the analysis of the chemical shift assignments ((1)H, (15)N, and (13)C) showed that residues T102-S108 of annexin A2 formed a well-defined alpha-helix in the S100A10 hybrid while the TRTK12 region was unstructured at the N-terminus with a single turn of alpha-helix from D108-K111 in the S100B hybrid protein. The two S100 hybrid proteins provide a simple yet extremely efficient method for obtaining high yields of intact S100 target peptides. Since cleavage of the S100 hybrid protein is not necessary for structural characterization, this approach may be useful as a scaffold for larger S100 complexes.

Figure 1

Models of the S100A10-annexin A2 (A10A2) and Ca²⁺-S100B-TRTK12 (BT12) hybrid constructs. Ribbon representations of S100A10 in complex with (A) annexin A2 (1BT6) and (B) Ca²⁺-S100B bound to TRTK12 (1MWN) are shown in similar orientations to demonstrate how the annexin A2 and TRTK12 peptides (magenta) are joined to the S100 proteins through a linker (cyan). In each model, one of the protomers is presented in light gray (helices labeled as I–IV) and the other protomer shaded in black (helices labeled as I′–IV′). Four bound calcium ions are shown in yellow spheres for Ca²⁺-S100B. The corresponding schematic diagrams of the constructs used for A10A2 and BT12 hybrid proteins are also illustrated with S100 protein located at the N-terminus (gray), annexin A2 and TRTK12 peptides positioned at the C-terminus (magenta), and the linker (cyan) connecting the S100 proteins to the target peptides. The amino acid composition of the linker region and the corresponding protease cleavage sites are indicated. All numbering is consecutive based on the S100 protein sequences.

Figure 2

Coomassie-stained SDS–PAGE gel (16.5%) depicting the purification of S100A10-annexin A2 and S100B-TRTK12 constructs. Lane 1 contains the protein molecular weight markers, with molecular weights labeled on the left of the gel. Lanes 2 and 5 contain S100B and S100A10, while BT12 and A10A2 hybrid constructs are presented in lanes 3 and 6, respectively. Lanes 4 and 7 show S100B and S100A10 following cleavage of BT12 and A10A2 by PreScission and TEV proteases, respectively. Each of these proteins runs at a higher molecular weight than the parent due to the presence of the linker that remains appended to the C-terminus of the S100 protein. The TRTK12 and annexin A2 peptides are not observed.

Figure 3

¹H-¹⁵N HSQC spectra of 0.5 mM (monomer) uniformly (A) ¹⁵N, ¹³C-labeled A10A2 hybrid protein, (B) ¹⁵N, ¹³C-labeled S100A10 in complex with unlabeled annexin A2 peptide, and (C) the ¹⁵N, ¹³C-labeled A10A2 hybrid protein following TEV cleavage. All spectra were collected in 20 mM MOPS, 1 mM EDTA, 1 mM DTT, 50 mM arginine, 50 mM glutamic acid, 100 mM NaCl, pH 7.0 and 35°C. Assigned backbone amide cross peaks are indicated with their one letter amino acid code and number. In (A) and (C) the residues from ¹⁵N, ¹³C-labeled annexin A2 are presented in pink and those from the linker region in cyan. Pairs of resonances for side chain amide cross peaks are connected by horizontal lines. Peaks visible at lower contour levels are indicated by boxes.

Figure 4

Secondary structure determinations for annexin A2 and TRTK12 peptides in the A10A2 and BT12 hybrid constructs using the chemical shift index for Cα, C′, and Hα atoms. The secondary structure identified for each atom is shown as α-helix (•), β-strand (▪) or coil conformation (○).