S100-annexin complexes: some insights from structural studies

Several annexins have been shown to bind proteins that belong to the S100 calcium-binding protein family. The two best-characterized complexes are annexin II with p11 and annexin I with S100C, the former of which has been implicated in membrane fusion processes. We have solved the crystal structures of the complexes of p11 with annexin II N-terminus and of S100C with annexin I N-terminus. Using these structural results, as well as electron microscopy observations of liposome junctions formed in the presence of such complexes (Lambert et al., 1997 J Mol Biol 272, 42-55), we propose a computer generated model for the entire annexin II/p11 complex.     

S100-annexin complexes--structural insights

Annexins and S100 proteins represent two large, but distinct, calcium-binding protein families. Annexins are made up of a highly alpha-helical core domain that binds calcium ions, allowing them to interact with phospholipid membranes. Furthermore, some annexins, such as annexins A1 and A2, contain an N-terminal region that is expelled from the core domain on calcium binding. These events allow for the interaction of the annexin N-terminus with target proteins, such as S100. In addition, when an S100 protein binds calcium ions, it undergoes a structural reorientation of its helices, exposing a hydrophobic patch capable of interacting with its targets, including the N-terminal sequences of annexins. Structural studies of the complexes between members of these two families have revealed valuable details regarding the mechanisms of the interactions, including the binding surfaces and conformation of the annexin N-terminus. However, other S100-annexin interactions, such as those between S100A11 and annexin A6, or between dicalcin and annexins A1, A2 and A5, appear to be more complicated, involving the annexin core region, perhaps in concert with the N-terminus. The diversity of these interactions indicates that multiple forms of recognition exist between S100 proteins and annexins. S100-annexin interactions have been suggested to play a role in membrane fusion events by the bridging together of two annexin proteins, bound to phospholipid membranes, by an S100 protein. The structures and differential interactions of S100-annexin complexes may indicate that this process has several possible modes of protein-protein recognition.     

The S100A10-annexin A2 complex provides a novel asymmetric platform for membrane repair

Membrane repair is mediated by multiprotein complexes, such as that formed between the dimeric EF-hand protein S100A10, the calcium- and phospholipid-binding protein annexin A2, the enlargeosome protein AHNAK, and members of the transmembrane ferlin family. Although interactions between these proteins have been shown, little is known about their structural arrangement and mechanisms of formation. In this work, we used a non-covalent complex between S100A10 and the N terminus of annexin A2 (residues 1-15) and a designed hybrid protein (A10A2), where S100A10 is linked in tandem to the N-terminal region of annexin A2, to explore the binding region, stoichiometry, and affinity with a synthetic peptide from the C terminus of AHNAK. Using multiple biophysical methods, we identified a novel asymmetric arrangement between a single AHNAK peptide and the A10A2 dimer. The AHNAK peptide was shown to require the annexin A2 N terminus, indicating that the AHNAK binding site comprises regions on both S100A10 and annexin proteins. NMR spectroscopy was used to show that the AHNAK binding surface comprised residues from helix IV in S100A10 and the C-terminal portion from the annexin A2 peptide. This novel surface maps to the exposed side of helices IV and IV' of the S100 dimeric structure, a region not identified in any previous S100 target protein structures. The results provide the first structural details of the ternary S100A10 protein complex required for membrane repair.     

S100A1 and S100B interactions with annexins

Members of the annexin protein family interact with members of the S100 protein family thereby forming heterotetramers in which an S100 homodimer crossbridges two copies of the pertinent annexin. Previous work has shown that S100A1 and S100B bind annexin VI in a Ca(2+)-dependent manner and that annexin VI, but not annexin V, blocks the inhibitory effect of S100A1 and S100B on intermediate filament assembly. We show here that both halves of annexin VI (i.e., the N-terminal half or annexin VI-a and the C-terminal half or annexin VI-b) bind individual S100s on unique sites and that annexin VI-b, but not annexin VI-a, blocks the ability of S100A1 and S100B to inhibit intermediate filament assembly. We also show that the C-terminal extension of S100A1 (and, by analogy, S100B), that was previously demonstrated to be critical for S100A1 and S100B binding to several target proteins including intermediate filament subunits, is not part of the S100 surface implicated in the recognition of annexin VI, annexin VI-a, or annexin VI-b. Evaluation of functional properties with a liposome stability and a calcium influx assay reveals the ability of both S100 proteins to permeabilize the membrane bilayer in a similar fashion like annexins. When tested in combinations with different annexin proteins both S100 proteins mostly lead to a decrease in the calcium influx activity although not all annexin/S100 combinations behave in the same manner. Latter observation supports the hypothesis that the S100-annexin interactions differ mechanistically depending on the particular protein partners.     

Insights into S100 target specificity examined by a new interaction between S100A11 and annexin A2

S100 proteins are a group of EF-hand calcium-signaling proteins, many of which interact with members of the calcium- and phospholipid-binding annexin family of proteins. This calcium-sensitive interaction enables two neighboring membrane surfaces, complexed to different annexin proteins, to be brought into close proximity for membrane reorganization, using the S100 protein as a bridging molecule. S100A11 and S100A10 are two members of the S100 family found to interact with the N-termini of annexins A1 and A2, respectively. Despite the high degree of structural similarity between these two complexes and the sequences of the peptides, earlier studies have shown that there is little or no cross-reactivity between these two S100s and the annexin peptides. In the current work the specificity and the affinity of the interaction of the N-terminal sequences of annexins A1 and A2 with Ca2+-S100A11 were investigated. Through the use of alanine-scanning peptide array experiments and NMR spectroscopy, an approximate 5-fold tighter interaction was identified between Ca2+-S100A11 and annexin A2 (approximately 3 microM) compared to annexin A1 (approximately 15 microM). Chemical shift mapping revealed that the binding site for annexin A2 on S100A11 was similar to that observed for the annexin A1 but with distinct differences involving the C-terminus of the annexin A2 peptide. In addition, kinetic measurements based on NMR titration data showed that annexin A2 binding to Ca2+-S100A11 occurs at a comparable rate (approximately 120 s(-1)) to that observed for membrane fusion processes such as endo- and exocytosis.     

Identification of regions responsible for the open conformation of S100A10 using chimaeric S100A11-S100A10 proteins

S100A11 is a dimeric EF-hand calcium-binding protein. Calcium binding to S100A11 results in a large conformational change that uncovers a broad hydrophobic surface used to interact with phospholipid-binding proteins (annexins A1 and A2) and facilitate membrane vesiculation events. In contrast with other S100 proteins, S100A10 is unable to bind calcium due to deletion and substitution of calcium-ligating residues. Despite this, calcium-free S100A10 assumes an 'open' conformation that is very similar to S100A11 in its calcium-bound state. To understand how S100A10 is able to adopt an open conformation in the absence of calcium, seven chimaeric proteins were constructed where regions from calcium-binding sites I and II, and helices II-IV in S100A11 were replaced with the corresponding regions of S100A10. The chimaeric proteins having substitutions in calcium-binding site II displayed increased hydrophobic surface exposure as assessed by bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'disulfonic acid, dipotassium salt) fluorescence and phenyl-Sepharose binding in the absence of calcium. This response is similar to that observed for Ca2+-S100A11 and calcium-free S100A10. Further, this substitution resulted in calcium-insensitive binding to annexin A2 for one chimaeric protein. The results indicate that residues within site II are important in stabilizing the open conformation of S100A10 and presentation of its target binding site. In contrast, S100A11 chimaeric proteins with helical substitutions displayed poorer hydrophobic surface exposure and, consequently, unobservable annexin A2 binding. The present study represents a first attempt to systematically understand the molecular basis for the calcium-insensitive open conformation of S100A10.     

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

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 Ca2+ 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 Ca2+-dependent and -independent manner. Ca2+-independent binding occurs with low affinity (Kd approximately 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, Ca2+-dependent binding of melittin to S100P occurs with high affinity (Kd approximately 5 microM) has a stoichiometry of two molecules of melittin per S100P dimer, appears to have positive cooperativity, and is driven by hydrophobic interactions. Furthermore, Ca2+-dependent S100P-melittin complex formation is accompanied by significant conformational changes: Melittin, otherwise unstructured in solution, adopts a helical conformation upon interaction with Ca2+-S100P. These results support a model for the Ca2+-dependent conformational switch in S100P for functional target recognition.     

Resonance assignments of Ca2+-bound human S100A11

The S100 family belongs to the EF-hand calcium-binding proteins regulating a wide range of important cellular processes via protein–protein interactions. Most S100 proteins adopt a conformation of non-covalent homodimer for their functions. Calcium binding to the EF-hand motifs of S100 proteins is essential for triggering the structural changes, promoting exposure of hydrophobic regions necessary for target protein interactions. S100A11 is a protein found in diverse tissues and possesses multiple functions upon binding to different target proteins. RAGE is a multiligand receptor binding to S100A11 and the interactions at molecular level have not been reported. However, the three-dimensional structure of human S100A11 containing 105 amino acids is still not available for further interaction studies. To determine the solution structure, for the first time we report the ¹H, ¹⁵N and ¹³C resonance assignments and protein secondary structure prediction of human S100A11 dimer in complex with calcium using a variety of triple resonance NMR experiments and the chemical shift index (CSI) method, respectively.     

Functional expression of the epithelial Ca(2+) channels (TRPV5 and TRPV6) requires association of the S100A10-annexin 2 complex

TRPV5 and TRPV6 constitute the Ca(2+) influx pathway in a variety of epithelial cells. Here, we identified S100A10 as the first auxiliary protein of these epithelial Ca(2+) channels using yeast two-hybrid and GST pull-down assays. This S100 protein forms a heterotetrameric complex with annexin 2 and associates specifically with the conserved sequence VATTV located in the C-terminal tail of TRPV5 and TRPV6. Of these five amino acids, the first threonine plays a crucial role since the corresponding mutants (TRPV5 T599A and TRPV6 T600A) exhibited a diminished capacity to bind S100A10, were redistributed to a subplasma membrane area and did not display channel activity. Using GST pull-down and co-immunoprecipitation assays we demonstrated that annexin 2 is part of the TRPV5-S100A10 complex. Furthermore, the S100A10-annexin 2 pair colocalizes with the Ca(2+) channels in TRPV5-expressing renal tubules and TRPV6-expressing duodenal cells. Importantly, downregulation of annexin 2 using annexin 2-specific small interfering RNA inhibited TRPV5 and TRPV6-mediated currents in transfected HEK293 cells. In conclusion, the S100A10-annexin 2 complex plays a crucial role in routing of TRPV5 and TRPV6 to plasma membrane.     

Crystal structure of metastasis-associated protein S100A4 in the active calcium-bound form

S100A4 (metastasin) is a member of the S100 family of calcium-binding proteins that is directly involved in tumorigenesis. Until recently, the only structural information available was the solution NMR structure of the inactive calcium-free form of the protein. Here we report the crystal structure of human S100A4 in the active calcium-bound state at 2.03 Å resolution that was solved by molecular replacement in the space group P6₅ with two molecules in the asymmetric unit from perfectly merohedrally twinned crystals. The Ca^2 +-bound S100A4 structure reveals a large conformational change in the three-dimensional structure of the dimeric S100A4 protein upon calcium binding. This calcium-dependent conformational change opens up a hydrophobic binding pocket that is capable of binding to target proteins such as annexin A2, the tumor-suppressor protein p53 and myosin IIA. The structure of the active form of S100A4 provides insight into its interactions with its binding partners and a better understanding of its role in metastasis.     

Ions binding to S100 proteins. I. Calcium- and zinc-binding properties of bovine brain S100 alpha alpha, S100a (alpha beta), and S100b (beta beta) protein: Zn2+ regulates Ca2+ binding on S100b protein

Flow dialysis measurements of calcium binding to bovine brain S100 alpha alpha, S100a (alpha beta), and S100b (beta beta) proteins in 20 mM Tris-HCl buffer at pH 7.5 and 8.3 revealed that S100 proteins bind specifically 4 Ca2+ eq/mol of protein dimer. The specific calcium-binding sites had, therefore, been assigned to typical amino acid sequences on the alpha and beta subunit. The protein affinity for calcium is much lower in the presence of magnesium and potassium. Potassium strongly antagonizes calcium binding on two calcium-binding sites responsible for most of the Ca2+-induced conformational changes on S100 proteins (probably site II alpha and site II beta). Zinc-binding studies in the absence of divalent cations revealed eight zinc-binding sites/mol of S100b protein dimer that we assumed to correspond to 4 zinc-binding sites/beta subunit. Zinc binding to S100b studied with UV spectroscopy methods showed that the occupation of the four higher affinity sites and the four lower affinity sites on the protein dimer were responsible for different conformational changes in S100b structure. Zinc binding on the higher affinity sites regulates calcium binding to S100b by increasing the protein affinity for calcium and decreasing the antagonistic effect of potassium on calcium binding. Zinc-binding studies on S100a and S100 alpha alpha protein showed that the Trp-containing S100 proteins bind zinc more weakly than S100b protein. Calcium-binding studies on zinc-bound S100a proved that calcium- and zinc-binding sites were distinct although there was no increase in zinc-bound S100a affinity for calcium, as in S100b protein. Finally we provide evidence that discrepancies between previously published results on the optical properties of S100b protein probably result from oxidation of the sulfhydryl groups in the protein.     

Structural and thermodynamic characterization of the recognition of the S100-binding peptides TRTK12 and p53 by calmodulin

Calmodulin (CaM) is a multifunctional messenger protein that activates a wide variety of signaling pathways in eukaryotic cells in a calcium-dependent manner. CaM has been proposed to be functionally distinct from the S100 proteins, a related family of eukaryotic calcium-binding proteins. Previously, it was demonstrated that peptides derived from the actin-capping protein, TRTK12, and the tumor-suppressor protein, p53, interact with multiple members of the S100 proteins. To test the specificity of these peptides, they were screened using isothermal titration calorimetry against 16 members of the human S100 protein family, as well as CaM, which served as a negative control. Interestingly, both the TRTK12 and p53 peptides were found to interact with CaM. These interactions were further confirmed by both fluorescence and nuclear magnetic resonance spectroscopies. These peptides have distinct sequences from the known CaM target sequences. The TRTK12 peptide was found to independently interact with both CaM domains and bind with a stoichiometry of 2:1 and dissociations constants K_d,C-term = 2 ± 1 µM and K_d,N-term = 14 ± 1 µM. In contrast, the p53 peptide was found to interact only with the C-terminal domain of CaM, K_d,C-term =2 ± 1 µM, 25°C. Using NMR spectroscopy, the locations of the peptide binding sites were mapped onto the structure of CaM. The binding sites for both peptides were found to overlap with the binding interface for previously identified targets on both domains of CaM. This study demonstrates the plasticity of CaM in target binding and may suggest a possible overlap in target specificity between CaM and the S100 proteins.     

Identification of the binding site on S100B protein for the actin capping protein CapZ.

The calcium-binding protein S100B binds to several potential target proteins, but there is no detailed information showing the location of the binding site for any target protein on S100B. We have made backbone assignments of the calcium-bound form of S100B and used chemical-shift changes in spectra of 15N-labeled protein to locate the site that binds a peptide corresponding to residues 265-276 from CapZ alpha, the actin capping protein. The largest chemical-shift changes are observed for resonances arising from residues around the C terminus of the C-terminal helix of S100B and residues Val-8 to Asp-12 of the N-terminal helix. These residues are close to but not identical to residues that have been identified by mutational analysis to be important in other S100 protein-protein interactions. They make up a patch across the S100B dimer interface and include some residues that are quite buried in the structure of calcium-free S100B. We believe we may have identified a binding site that could be common to many S100 protein-protein interactions.     

Identification of a dimeric intermediate in the unfolding pathway for the calcium-binding protein S100B

The S100 proteins comprise 25 calcium-signalling members of the EF-hand protein family. Unlike typical EF-hand signalling proteins such as calmodulin and troponin-C, the S100 proteins are dimeric, forming both homo- and heterodimers in vivo. One member of this family, S100B, is a homodimeric protein shown to control the assembly of several cytoskeletal proteins and regulate phosphorylation events in a calcium-sensitive manner. Calcium binding to S100B causes a conformational change involving movement of helix III in the second calcium-binding site (EF2) that exposes a hydrophobic surface enabling interactions with other proteins such as tubulin and Ndr kinase. In several S100 proteins, calcium binding also stabilizes dimerization compared to the calcium-free states. In this work, we have examined the guanidine hydrochloride (GuHCl)-induced unfolding of dimeric calcium-free S100B. A series of tryptophan substitutions near the dimer interface and the EF2 calcium-binding site were studied by fluorescence spectroscopy and showed biphasic unfolding curves. The presence of a plateau near 1.5 M GuHCl showed the presence of an intermediate that had a greater exposed hydrophobic surface area compared to the native dimer based on increased 4,4-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid fluorescence. Furthermore, ¹H-¹⁵N heteronuclear single quantum coherence analyses as a function of GuHCl showed significant chemical shift changes in regions near the EF1 calcium-binding loop and between the linker and C-terminus of helix IV. Together these observations show that calcium-free S100B unfolds via a dimeric intermediate.     

The three-dimensional solution structure of Ca(2+)-bound S100A1 as determined by NMR spectroscopy

S100A1 is an EF-hand-containing Ca(2+)-binding protein that undergoes a conformational change upon binding calcium as is necessary to interact with protein targets and initiate a biological response. To better understand how calcium influences the structure and function of S100A1, the three-dimensional structure of calcium-bound S100A1 was determined by multidimensional NMR spectroscopy and compared to the previously determined structure of apo. In total, 3354 nuclear Overhauser effect-derived distance constraints, 240 dihedral constraints, 160 hydrogen bond constraints, and 362 residual dipolar coupling restraints derived from a series of two-dimensional, three-dimensional, and four-dimensional NMR experiments were used in its structure determination (>21 constraints per residue). As with other dimeric S100 proteins, S100A1 is a symmetric homodimer with helices 1, 1', 4, and 4' associating into an X-type four-helix bundle at the dimer interface. Within each subunit there are four alpha-helices and a short antiparallel beta-sheet typical of two helix-loop-helix EF-hand calcium-binding domains. The addition of calcium did not change the interhelical angle of helices 1 and 2 in the pseudo EF-hand significantly; however, there was a large reorientation of helix 3 in the typical EF-hand. The large conformational change exposes a hydrophobic cleft, defined by residues in the hinge region, the C terminus, and regions of helix 3, which are important for the interaction between S100A1 and a peptide (TRTK-12) derived from the actin-capping protein CapZ.     

Interaction of S100A13 with C2 domain of receptor for advanced glycation end products (RAGE)

S100A13 is involved in several key biological functions like angiogenesis, tumor formation and cell apoptosis. It is a homodimeric protein that belongs to the S100 protein family. S100A13 is co-expressed with acidic fibroblast growth factor (FGF1) and interleukin-1α which are key angiogenesis inducers. The S100 proteins have been shown to be involved in several cellular functions such as calcium homeostasis, cell growth and differentiation dynamic of cytoskeleton. Its biological functions are mainly mediated through the receptor for advanced glycation end products (RAGE) signaling. RAGE is involved in inflammatory processes and is associated with diabetic complications, tumor outgrowth, and neurodegenerative disorders. RAGE induces cellular signaling upon binding of different ligands, such as S100 proteins, glycated proteins, and HMGB1. RAGE signaling is complex, and it depends on the cell type and concentration of the ligand. Molecular level interactions of RAGE and S100 proteins are useful to understand the RAGE signaling diversity. In this report we focus on the molecular level interactions of S100A13 and RAGE C2 domain. The binding between RAGE C2 and S100A13 is moderately strong (K_d ~ 1.3 μM). We have solved the solution structure of the S100A13–RAGE C2 complex and pronounce the interface regions in S100A13–RAGE C2 complex which are helpful for drug development of RAGE induced diseases.     

15N relaxation studies of Apo-Mts1: a dynamic S100 protein

Mts1 is a member of the S100 family of EF-hand calcium-binding proteins. Like most S100 proteins, Mts1 exists as a dimer in solution and contains one canonical and one pseudo-EF-hand motif per monomer, each of which consists of two alpha helices connected by a loop capable of coordinating a calcium ion. The backbone dynamics of murine apo-Mts1 homodimer have been examined by nuclear magnetic resonance spectroscopy. Longitudinal and transverse relaxation data and steady-state (1)H- (15)N nuclear Overhauser effects were analyzed using model-free formalism. The extracted global correlation time is 9.94 ns. Results indicate that the protein backbone is most rigid at the dimer interface, made up of helices 1 and 4 from each monomer with mean S (2) ( S avg (2)) values approximately 0.9, flanked by helices 2 and 3 with lower S avg (2) values of 0.84 and 0.77, respectively. Each calcium-binding site along with the hinge joining the two EF-hands and the N- and C-termini are considerably more flexible than the dimer interface on a range of time scales and more flexible than the corresponding regions of other S100 proteins studied to date. As the hinge and the C-terminal tail are believed to interact with target proteins, these dynamic characteristics may have implications for Mts1 activity.     

S100B-Mediated Inhibition of the Phosphorylation of GFAP Is Prevented by TRTK-12

S100B belongs to a family of calcium-binding proteins involved in cell cycle and cytoskeleton regulation. We observed an inhibitory effect of S100B on glial fibrillary acidic protein (GFAP) phosphorylation, when stimulated by cAMP or Ca2+/calmodulin, in a cytoskeletal fraction from primary astrocyte cultures. We found that S100B has no direct effect on CaM KII activity, the major kinase in this cytoskeletal fraction able to phosphorylate GFAP. The inhibition of GFAP phosphorylation is most likely due to the binding of S100B to the phosphorylation sites on this protein and blocking the access of these sites to the protein kinases. This inhibition was dependent on Ca2+. However, Zn2+ could substitute for Ca2+. The inhibitory effect of S100B was prevented by TRTK-12, a peptide that blocks S100B interaction with several target proteins including glial fibrillary acidic protein. These data suggest a role for S100B in the assembly of intermediate filaments in astrocytes.     

Two functional S100A4 monomers are necessary for regulating nonmuscle myosin-IIA and HCT116 cell invasion

S100A4, a member of the Ca(2+)-activated S100 protein family, regulates the motility and invasiveness of cancer cells. Moreover, high S100A4 expression levels correlate with poor patient survival in several cancers. Although biochemical, biophysical, and structural data indicate that S100A4 is a noncovalent dimer, it is unknown if two functional S100A4 monomers are required for the productive recognition of protein targets and the promotion of cell invasion. To address this question, we created covalently linked S100A4 dimers using a glycine rich flexible linker. The single-chain S100A4 (sc-S100A4) proteins exhibited wild-type affinities for calcium and nonmuscle myosin-IIA, retained the ability to regulate nonmuscle myosin-IIA assembly, and promoted tumor cell invasion when expressed in S100A4-deficient colon carcinoma cells. Mutation of the two calcium-binding EF-hands in one monomer, while leaving the other monomer intact, caused a 30-60-fold reduction in binding affinity for nonmuscle myosin-IIA concomitant with a weakened ability to regulate the monomer-polymer equilibrium of nonmuscle myosin-IIA. Moreover, sc-S100A4 proteins with one monomer deficient in calcium responsiveness did not support S100A4-mediated colon carcinoma cell invasion. Cross-linking and titration data indicate that the S100A4 dimer binds a single myosin-IIA target peptide. These data are consistent with a model in which a single peptide forms interactions in the vicinity of the canonical target binding cleft of each monomer in such a manner that both target binding sites are required for the efficient interaction with myosin-IIA.     

Design of high‐affinity S100‐target hybrid proteins

S100B and S100A10 are dimeric, EF‐hand proteins. S100B undergoes a calcium‐dependant 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 ¹H‐¹⁵N 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 (¹H, ¹⁵N, and ¹³C) showed that residues T102‐S108 of annexin A2 formed a well‐defined α‐helix in the S100A10 hybrid while the TRTK12 region was unstructured at the N‐terminus with a single turn of α‐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.