A Novel S100 Target Conformation Is Revealed by the Solution Structure of the Ca2+-S100B-TRTK-12 Complex*

Kimberly A. McClintock and Gary S. ShawDagger

From the Department of Biochemistry and McLaughlin Macromolecular Structure Facility, the University of Western Ontario, London, Ontario N6A 5C1, Canada

Received for publication, October 17, 2002, and in revised form, December 9, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The Alzheimer-linked neural protein S100B is a signaling molecule shown to control the assembly of intermediate filament proteins in a calcium-sensitive manner. Upon binding calcium, a conformational change occurs in S100B exposing a hydrophobic surface for target protein interactions. The synthetic peptide TRTK-12 (TRTKIDWNKILS), derived from random bacteriophage library screening, bears sequence similarity to several intermediate filament proteins and has the highest calcium-dependent affinity of any target molecule for S100B to date (Kd <1 µM). In this work, the three-dimensional structure of the Ca2+-S100B-TRTK-12 complex has been determined by NMR spectroscopy. The structure reveals an extended, contiguous hydrophobic surface is formed on Ca2+-S100B for target interaction. The TRTK-12 peptide adopts a coiled structure that fits into a portion of this surface, anchored at Trp7, and interacts with multiple hydrophobic contacts in helices III and IV of Ca2+-S100B. This interaction is strikingly different from the alpha -helical structures found for other S100 target peptides. By using the TRTK-12 interaction as a guide, in combination with other available S100 target structures, a recognition site on helix I is identified that may act in concert with the TRTK-12-binding site from helices III and IV. This would provide a larger, more complex site to interact with full-length target proteins and would account for the promiscuity observed for S100B target protein interactions.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The S100 proteins are low molecular weight (10-12 kDa) members of the EF-hand family of calcium-binding proteins. Many of these proteins, including several of the S100s, the muscle contractile protein troponin C, and the ubiquitous protein calmodulin act as signaling molecules by converting an influx of cellular calcium into a biological response. Calcium binding to these EF-hand proteins triggers a conformational change and allows the protein to interact with an appropriate target molecule. The S100 proteins are unique among this family because, unlike troponin C or calmodulin, they exist in solution as homo- or heterodimers. Each S100 monomer contains two helix-loop-helix calcium-binding motifs as follows: a basic N-terminal pseudo EF-hand comprising 14 residues (site I), and a canonical and acidic C-terminal EF-hand of 12 residues (site II). A central linker region joining the two EF-hands along with the extreme N and C termini of these proteins exhibit the most sequence divergence among family members and are therefore believed to provide specificity for target protein interactions. This feature, along with the dimeric state of the S100 proteins, likely indicates these calcium-signaling proteins have the distinctive ability to interact with more than one target molecule at a time.

S100B,1 a homodimer of 91-residue S100beta monomers, is found primarily in glial cells and has been implicated in neurological diseases including Alzheimer's disease and Down's syndrome (1-3). More than 20 calcium-sensitive in vitro binding partners have been identified for S100B (4) including several cellular architecture proteins such as tubulin (5) and GFAP (6), where S100B can inhibit polymerization of these oligomeric molecules. Furthermore, S100B inhibits the phosphorylation of multiple kinase substrates including the Alzheimer protein tau (7, 8) and neuromodulin (GAP-43) (9) through a calcium-sensitive interaction with the protein substrates. Consistent with the calcium-induced conformational change mechanism, a comparison of three-dimensional structures of apo- and calcium-bound S100B reveals that binding of calcium leads to the exposure of a hydrophobic surface(s) for protein-protein interactions (10).

The 12-residue peptide TRTK-12, derived from random bacteriophage library experiments, has been used in previous studies as a model for S100B-target protein interactions. Experiments utilizing this peptide have indicated that peptides containing the consensus sequence (R/K)(L/I)(XWXXIL) bind specifically to S100B in a calcium-sensitive manner (11). Furthermore, this consensus sequence is conservatively found in the cytoskeletal proteins tubulin, desmin, vimentin, and GFAP, shown previously to interact with Ca2+-S100B (12). The TRTK-12 peptide competes with S100B target proteins including CapZ-alpha and GFAP for Ca2+-S100B binding (6, 11) and has the highest affinity (Kd ~260 nM) (13) of any known S100B target. Fluorescence studies (13) have shown that Trp7 of TRTK-12 is a key residue for the calcium-sensitive interaction with S100B becoming buried at the protein-peptide interface. In addition, deletion of residues 85-91 from S100B leads to a >2000-fold decrease in affinity for TRTK-12 indicating the C-terminal helix is an important site of interaction.

To date, only three structures are available for S100-target peptide complexes. In each case the target peptide adopts a 2.5 turn alpha -helical structure that interacts via two distinct modes with the S100 protein. The structures of human S100A10 and S100A11 in complex with peptides from the binding regions of annexin II and I, respectively, are nearly identical (14, 15) with the annexin peptides bridging the two S100 monomers through contacts in the linker region and C terminus of one monomer and the N terminus of the other monomer. Surprisingly, this similarity of interaction occurs despite little sequence similarity between the annexin peptides. In contrast, the interaction of rat S100B with a 23-residue peptide from the tumor suppressor protein p53 shows each S100beta monomer binds to a single peptide through interactions with helix III and a portion of helix IV (16). This orients the alpha -helical p53 peptide about 90° from that found for the annexin peptides with respect to their S100 partners. These structural variations of the S100A10, S100A11, and S100B complexes indicate that recognition differences in the protein and the target must exist for S100 proteins. In an effort to clarify these interactions, we present the three-dimensional solution structure of Ca2+-S100B in complex with the TRTK-12 peptide. The amino acid sequence of TRTK-12 does not correspond to a natural sequence for any known S100B target. However, its unusually high affinity for S100B may indicate that it contains structural determinants that remain to be uncovered for recognition of an S100 binding partner. The binding interaction of TRTK-12 is unexpected with the peptide adopting an extended and reversed orientation compared with the p53 interaction. Furthermore, there is no interaction with helix I of S100B as found in the S100A11/S100A10 annexin structures thus providing a novel third mode of recognition for an S100-target protein complex.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Sample Preparation-- Human S100B protein was expressed in Escherichia coli (strain N99) and purified as described previously (17). Uniformly 15N-labeled or 15N/13C-labeled S100B was prepared using M9 minimal media containing 1 g/liter 99% 15NH4Cl or 1 g/liter 99% 15NH4Cl and 2 g/liter [13C]glucose. Uniformly 2H/15N-labeled S100B was prepared in M9 minimal media containing 1 g/liter 99% 15NH4Cl and 99% D2O. Unlabeled TRTK-12 peptide (Ac-TRTKIDWNKILS-NH2) and [13C]isoleucine/[13C]acetyl-labeled peptide (*Ac-TRTK*IDWNK*ILS-NH2) were synthesized by the Queen's Peptide Synthesis Lab (Queen's University, Kingston, Canada). Purity was confirmed using reversed phase-high pressure liquid chromatography and mass spectrometry. Typically, NMR samples contained 1 mM S100beta monomer, 1.2 mM TRTK-12 peptide, 35 mM KCl, 4 mM CaCl2, and 5 mM dithiothreitol in 90% H2O, 10% D2O (v/v), pH 7.05. For a single sample, a mixed 13C/12C S100B dimer was prepared by incubating 20 mg each of 13C-labeled S100B and unlabeled S100B (total S100B concentration) in D2O at 37 °C for 150 h. Evolution of the mixed dimer over time was monitored by mass spectrometry. The final concentration of 13C/12C S100B dimer in the NMR sample was ~1.5 mM, and the sample was prepared in 100% D2O. Experimental conditions were otherwise identical to those reported above.

NMR Spectroscopy-- NMR experiments were performed at 35 °C on Varian 500-, 600-, and 800-MHz spectrometers with pulsed field gradient probes. Backbone resonances for Ca2+-S100B in the complex were sequentially assigned using HNCACB (18), CBCA(CO)NH (19), and HNCO (20) experiments. Side chain assignments were made using C(CO)NH (21), H(CCO)NH (21), 15N-edited TOCSY, and HCCH-TOCSY (22) experiments. Some aromatic ring protons were assigned using (HB)CB(CGCD)HD and (HB)CB(CGCDCE)HE experiments (23). TRTK-12 1H assignments were determined from homonuclear two-dimensional NOESY and two-dimensional TOCSY experiments with wet and watergate water suppression, respectively, using uniformly 15N,2H-labeled S100B and unlabeled TRTK-12. Side chain 13C resonances were obtained from a natural abundance 13C-HSQC. 13C and 1H assignments for the two isoleucine residues (Ile5 and Ile10) were confirmed using specifically labeled TRTK-12. Spectra were processed and analyzed on a Silicon Graphics work station using NMRPipe, NMRDraw (24), and Pipp and Stapp (25) programs.

Structure Determination-- Approximate interproton distances in S100B were determined using 15N-filtered NOESY (26)(tau mix = 150 ms) and simultaneous 13C/15N-separated three-dimensional NOESY-HSQC (27)(tau mix = 100 ms) experiments. NOEs derived from the 15N-filtered NOESY were calibrated based on known dHNalpha and sequential dalpha HN distances. The 13C/15N-separated three-dimensional NOESY-HSQC was calibrated to geminal protons or protons on adjacent carbon atoms. In cases where direct calibration was not possible, the maximum distance of 6.0 Å was used. phi  and psi  angles for Ca2+-S100B and the bound TRTK-12 peptide were incorporated where greater than 9 of 10 predictions fell within the same region of the Ramachandran plot based on the TALOS program (28). Minimum standard deviations of ±20° and ±30° were used for phi  and psi , respectively. phi  angle restraints were confirmed with 3JNH-Halpha coupling constants derived from an HNHA experiment (29). Hydrogen bond distance restraints of rNH-O = 1.4-2.7 Å and rN-O = 2.4-3.7 Å were implemented based on the identification of helical regions in initial structures. NOEs between the two S100beta monomers were unambiguously assigned using a mixed 13C/12C S100B dimer and a 13C F1-edited F3-filtered NOESY-HMQC experiment (30) which detects only NOEs between a proton attached to 13C and a proton attached to12C. These NOEs were calibrated in the same fashion as cross-peaks from the 13C/15N-separated three-dimensional NOESY-HSQC.

Secondary structure of the TRTK-12 peptide was determined using a two-dimensional wetNOESY experiment and 15N,2H S100B protein. Dihedral restraints for the peptide were included using the same stipulations as for Ca2+-S100B. Intermolecular NOEs between S100B and TRTK-12 were unambiguously identified using the same 13C F1-edited F3-filtered NOESY-HMQC experiment (30) described above with 13C/15N-labeled S100B protein and unlabeled TRTK-12 as well as a simultaneously 13C/15N-separated three-dimensional NOESY-HSQC using 15N-labeled S100B and specifically labeled TRTK-12.

Initial structures were calculated using the hybrid distance geometry and simulated annealing protocol in the Crystallography and NMR System program, version 1.1. The two monomeric subunits of S100B were constrained to be identical using the non-crystallographic symmetry definition and a force constant of 10 kcal/mol. The same constraint was applied for the two molecules of TRTK-12.

Circular Dichroism Spectroscopy-- Experiments were performed at 25 °C on a Jasco J-810 spectropolarimeter. For the TRTK-12, Ca2+-S100B, and Ca2+-S100B-TRTK-12 samples, spectra from three scans (200-250 nM) in a 1-mm path length cuvette were averaged, and the buffer background was subtracted. Protein samples were prepared in 50 mM KCl, 10 mM MOPS, pH 7.2, 1 mM dithiothreitol, 1 mM CaCl2 to final protein concentrations of 100 µM for S100beta and 100 µM for TRTK-12 and 100 µM S100beta ; 100 µM TRTK-12 for the complex.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Binding of the TRTK-12 peptide has been monitored previously by NMR and fluorescence spectroscopy and found to have a Kd = 260 nM (13). In this work, formation of the complex was measured using a 15N-labeled S100B sample and monitoring the change in resonances as a function of added TRTK-12 peptide. Complex formation was complete at a ratio of 2:1 TRTK-12:S100B indicating that two TRTK-12 peptides bind to each S100B dimer protein. The resulting 1H-15N HSQC spectrum of this complex showed a single set of resonances in the NMR spectra for most residues (Fig. 1) indicating the arrangement of the Ca2+-S100B-TRTK-12 complex dimer is symmetric. Exceptions were noted for residues Ser1, Leu3, Lys5, Ser41, and His42 which have duplicate peaks resulting from the presence of formyl and desformyl N-terminal methionine S100B species as observed previously for apo- and Ca2+-S100B (31). Several residues in the N-terminal calcium-binding loop have weak (Ser18, Glu21, His25, Lys26, Leu27, and Lys28) or absent (Gly22 and Asp23) amide correlations in the 1H-15N-HSQC likely due to exchange with the H2O solvent. Gly66 shows a substantial downfield 1H shift characteristic of calcium binding to the C-terminal calcium loop. A comparison of this spectrum with that for Ca2+-S100B alone indicated that several residues including Ile47, Val53, Ala78, Ala83, and Cys84 had shifted upon TRTK-12 binding.


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Fig. 1.   1H-15N HSQC spectrum (500 MHz) of Ca2+-S100B bound to TRTK-12. Backbone amide resonances are indicated. All backbone amides were identified with the exception of Gly22 and Asp23. Correlations of side chain amide groups are indicated by horizontal lines. Duplicate peaks are observed for residues at the extreme N terminus (S1, L3, and K5) and in the linker region (S41 and H42) due to the presence of both formyl and desformyl N-terminal methionine S100B forms.

To identify the specific interactions between TRTK-12 and human Ca2+-S100B, the solution structure of the complex (24 kDa) was determined using 2072 experimental distance restraints in combination with 536 dihedral and 136 hydrogen bond restraints. This generated a family of 17 well defined structures (Fig. 2) based on the superposition of the 8 helices in the dimer (root mean square deviation 0.63 ± 0.07) (Table I). The TRTK-12 peptide was well defined between residues 4 and 12 forming a coil structure that folds back on itself. As observed in the rat S100B-p53 structure (16), each S100B monomer binds one TRTK-12 molecule.


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Fig. 2.   Structure of the Ca2+-S100B-TRTK-12 complex. The N and C termini are labeled for one S100beta monomer and TRTK-12, and helices are indicated for S100beta . A, stereo view of the backbone superposition of the final ensemble of 17 NMR derived structures of the complex. S100beta monomers are shown in magenta and blue, and the TRTK-12 peptide molecules are shown in light blue. B, ribbon structure of the complex. Each monomer consists of four alpha -helices (helix I, blue; II, magenta; III, green; and IV, yellow) and two anti-parallel beta -strands (orange, blue). TRTK-12 is shown in dark blue.

                              
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Table I
NMR restraints and structure statistics

Structure of Ca2+-S100B in the Complex-- Overall, the structure of human Ca2+-S100B in the TRTK-12 complex retains many structural features of the calcium-bound form alone (32). The dimer interface is characterized by antiparallel interactions between helices I and I' (interhelical angle (Omega ) = 164 ± 4°) and helices IV and IV' (Omega  = 172 ± 4°) forming an X-type four helix bundle (Fig. 2). Each S100beta monomer consists of an N-terminal pseudo EF-hand motif (site I) comprising helix I (Leu3-Tyr17), calcium binding loop I (Ser18-Glu31), and helix II (Ser30-Glu39) and a C-terminal canonical EF-hand (site II) comprising helix III (Gln50-Leu60), calcium binding loop II (Asp61-Glu72), and helix IV (Phe70-Cys84). Calcium binding loops I and II are associated through a short antiparallel beta -sheet (Lys26-Lys28 and Glu67-Asp69). The arrangement of helices I and II in site I (Omega  = 129 ± 3°) is similar to that of Ca2+-S100B (Omega  = 138 ± 4°) and calbindin D9k (Omega  = 130 ± 2°), indicating that TRTK-12 binding to Ca2+-S100B results in little conformational change to this region. In contrast, significant changes are identified in site II. The helix III-IV interhelical angle (Omega  = 104 ± 4°) is dramatically opened by more than 90° than found in apo-S100B (Omega  = -166 ± 1°). Furthermore, the arrangement of helices III-IV shows distinct differences from human Ca2+-S100B (Omega  = 148 ± 4°) being more similar to the III-IV arrangement found in the S100A11-annexin I complex (Omega  = 103°) and more "open" compared with either the S100B-p53 (Omega  = 110 ± 1°) or S100A10-annexin II (Omega  = 117°) complexes. These observations suggest that the relative orientations of helices III and IV may be a governing factor for target specificity.

Structure of TRTK-12 in the Complex-- Our previous studies (33) have shown that the TRTK-12 peptide is unstructured in the absence of Ca2+-S100B. Consistent with this, the circular dichroism spectrum of TRTK-12 (Fig. 3) showed an ellipticity minimum at <200 nm indicative of a random coiled structure. Upon binding, TRTK-12 assumes a coiled conformation that folds back upon itself (Fig. 2). The structure determination of TRTK-12 was aided by using a peptide having uniform 13C-labeling at Ile5 and Ile10 and the N-terminal acetyl CH3 positions thus providing 13C markers throughout the peptide length. The coiled structure of TRTK-12 was supported by definitive NOEs between the side chains of residues Trp7-Leu11, and peptide spectra were characterized by an absence of alpha -helical NOEs and alpha -proton (and alpha C for Ile5 and Ile10) chemical shifts not consistent with alpha -helical structure. These findings are supported by CD spectra of Ca2+-S100B (Fig. 3) that show ellipticity minima at 208 and 222 nm typical of an alpha -helical protein. Upon addition of 1 eq of TRTK-12 per S100beta monomer (Fig. 3). little difference in this spectrum occurs indicating the TRTK-12 peptide does not assume an alpha -helical conformation. Furthermore, the theta 208/theta 222 ratio for the complex appears nearly identical to that of Ca2+-S100B, indicating little change in helix-helix interactions occurs.


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Fig. 3.   Far-UV circular dichroism spectra of Ca2+-S100B in the presence of the TRTK-12 peptide. The spectra show TRTK-12 (), Ca2+-S100B (black-triangle), and Ca2+-S100B in the presence of TRTK-12 (black-diamond ). The complex was formed using a 1:2 ratio of Ca2+-S100B (50 µM) and TRTK-12 (100 µM) consistent with the binding of one TRTK-12 peptide for each S100beta monomer.

Ca2+-S100B-TRTK-12 Interactions-- The interface between Ca2+-S100B and TRTK-12 is defined by several residues in helix III (Val52, Lys55, Val56, Thr59, and Leu60) and helix IV (Phe76, Ala80, Ala83, and Cys84). In particular, 13C-labeled Ile5 and Ile10 revealed definitive interactions to residues Thr82 in helix IV and Val52 and Val56 in helix III, respectively (Fig. 4). Furthermore, residues Lys9, Ile10, and Leu11 in the C terminus of TRTK-12 have multiple contacts in helix III, and Thr1, Thr3, and Lys4 are in close proximity to Ala83 in helix IV. These interactions position the N terminus of the TRTK-12 peptide near helix IV and the C terminus near helix III resulting in a peptide orientation opposite to that observed for the S100B-p53 complex. Although 13C labeling of the N-terminal acetyl group in TRTK-12 was used, no observable peptide-protein cross-peaks resulting from this label were observed in 13C-edited NOE spectra, indicating the extreme N terminus of TRTK-12 is exposed to solvent and does not interact with residues in helix I as the annexin peptides do in the S100A10 and S100A11 structures. A large hydrophobic cavity exists on Ca2+-S100B where TRTK-12 is bound (Fig. 5). This observation is consistent with previous results showing that calcium binding to human S100B results in exposure of a hydrophobic surface (10). Trp7 of the peptide is the anchoring residue nestled in a hydrophobic pocket including residues Val56, Thr59, Phe76, and Val80 of S100B. This environment results in the observed blue shift and an increase in fluorescence intensity for Trp7 upon protein binding (33). Comparison of the exposed surface area between Ca2+-S100B alone and in complex with TRTK-12 shows the side chains of Val56, Thr59, and Ala83 decrease their surface exposure by 86, 96, and 99%, respectively, upon TRTK-12 binding. Similarly, residues Trp7 and Ile10 of TRTK-12 have more than 84 and 90%, respectively, of their surface area buried in the complex. Recent fluorescence studies of the S100B-TRTK-12 interaction have indicated that deletion of the C-terminal 7 residues in S100B results in a drastic decrease in TRTK-12 binding affinity (13). This was attributed to a loss in alpha -helical structure near Ala83 in the truncated S100B protein. Presumably this would considerably alter the arrangement of residues Val80, Ala83, and Cys84 resulting in a significantly reduced affinity.


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Fig. 4.   Ca2+-S100B-TRTK-12 interactions. A, strip plots extracted from tau mix = 100-ms 13C/15N-edited NOESY-HSQC spectrum of Ca2+-S100B bound to [13C]Ile-labeled TRTK-12 peptide (Ile5 and Ile10, 1st 3 panels) and tau mix = 100-ms 13C F1-edited, F3-filtered NOESY-HMQC experiment of 13C-labeled Ca2+-S100B (Ala83 and Lys55, last 2 panels) bound to unlabeled TRTK-12. Each strip represents the 13C plane and 1H chemical shift of the indicated residue. Asterisks indicate NOEs involving TRTK-12 residues. B, structural details of the binding interface of the Ca2+-S100B-TRTK-12 complex. The TRTK-12 peptide (blue) shows residues Thr1, Lys4, Ile5, Lys9, and Ile10 (*), where NOE contacts were observed to residues Val52, Lys55, Val56, Thr82, and Ala83 in Ca2+-S100B (magenta) as shown in A.


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Fig. 5.   Electrostatic potential surface showing the TRTK-12-binding site on Ca2+-S100B. Negative potential is indicated in blue, and positive potential is shown in red for Ca2+-S100B. A, location of the two TRTK-12 molecules (green) on opposite sides of Ca2+-S100B. The TRTK-12 peptide fits into the hydrophobic cleft generated by helices III and IV of each monomer. Note that on either side of the TRTK-12-binding site a significant uncharged region exists. B, environment of the anchoring Trp7 residue from TRTK-12 (green) showing interactions with S100B residues (blue) in helix III (Val56 and Thr59) and IV (Phe76 and Val80).


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Examination of the structure of Ca2+-S100B-TRTK-12 and comparison to the existing structures of S100A10-annexin II, S100A11-annexin I, and S100B-p53 allows a distinction to be made between target recognition by these S100 proteins. Furthermore, the TRTK-12 sequence was identified from random bacteriophage peptide selection and therefore may contain unique binding features in comparison to the natural sequences for the annexin and p53 peptides. In the current structure, the TRTK-12 peptide lies in a hydrophobic cleft formed between helices III and IV maximizing the interaction of the hydrophobic residues Ile5, Trp7, Ile10, and Leu11 with residues in both helices of S100B. Nearly all the interaction is hydrophobic in nature reaffirming earlier work that the S100B-TRTK-12 interaction is tighter at increased ionic strength (13). The interaction is distinct from that of the rat S100B-p53 peptide (16), likely due to the sequence differences of the interacting peptides. The target sequence for p53 (SRHKKLMFKT) bears little similarity to TRTK-12 containing a highly positively charged C terminus that orients near the highly acidic N terminus of helix III (EEIKEEQE). In contrast, the charged residues in TRTK-12 are more dispersed throughout its sequence and most are occupied through potential hydrogen-bonding interactions. Nevertheless, some similarities between the target peptide molecules exist. The anchoring residues for the p53 peptide are Leu383 and Phe385, spaced only one residue apart, as are Ile5 and Trp7 in TRTK-12. The distinguishing feature of these two residues in each peptide is their positions with respect to S100B. In the current work Trp7 is positioned in an analogous region to Leu383 in p53, whereas the side chain of Ile5 is located nearby that of Phe385 in p53, albeit somewhat shifted due to a different orientation. The reverse nature of these two interactions together with the differential charge balance in the peptides likely results in the reversal of orientation of TRTK-12 to p53 upon binding to S100B.

The regions of interaction of TRTK-12 and p53 differ significantly from that of both annexin I and II with S100A11 and S100A10, respectively. Both of the annexin peptides have interactions with several residues in the N-terminal helix I of these S100 proteins (Fig. 6A). An examination of the sequences and interactions reveals three key residues in the annexin peptides (Val3, Glu5, and Leu7) that have identical interactions with the N-terminal residues Glu5, Met8, Glu9, and Met12 in S100A10 (Glu9, Ile12, Glu13, and Ile16 in S100A11). This pattern in the target peptide, where two hydrophobes are separated by three residues, including a central acidic residue, is not found in either the TRTK-12 or p53 peptides. Furthermore, the side chain carboxylate of Glu9 in S100A10 (Glu13 in S100A11) has conserved hydrogen bonds to the Thr2 and Val3 amides in annexin II (Met2 and Val3, in S100A11). In S100B the position of Glu9 (Glu13) is held by a hydrophobe (Val8) removing its side chain hydrogen bonding ability. Interestingly, this position is among the least conserved within the S100 protein family. This sequence divergence in both the S100B and S100A10/S100A11 proteins and in the peptide motif is likely responsible for the different target peptide recognition sites between the proteins. Consistent with this, previous experiments have shown that TRTK-12 shows no observable interaction with S100A11 (33) when monitored by Trp7 fluorescence.


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Fig. 6.   Interactions of S100 proteins with annexin I, annexin II, and TRTK-12. A, aligned sequences of helices I (top) for S100B, S100A11, and S100A10 along with observed interactions to their respective target peptides (). The annexin I and II peptides were aligned based on the structurally conserved interactions to the N-terminal residues of S100A11 and S10010, respectively (green), and involving the identical residues (Val, Glu, and Leu) in the annexin peptides (dark shading). The conserved interaction involving Glu9 in S100A10 (Glu13 in S100A11) is unlikely in S100B where a valine residue occupies this position (blue). B, the aligned sequences of helix III and the C-terminal portion of the linker are shown together along with the common interactions to Trp7 (pink) in the TRTK-12 peptide and Trp11 in the annexin I peptide (). Interactions found between S100B and TRTK-12, but not in annexin I, are also shown (- - -). C, proposed full-length target site for S100B. The accessible surface for Ca2+-S100B was calculated based on the complex with TRTK-12 in the absence of the peptide. Residues where the side chain interactions with the TRTK-12 peptide (green) and those where a proposed site exists based on the S100A11-annexin I structure (yellow) are shown based on the observations in A. Residues that have common contacts to the bridging Trp residue, as shown in B, are shown in red. A comparison of the structures reveals that the hydrophobic surface used by S100B for recognition of TRTK-12 (green) is largely exposed in the S100A11-annexin I structure and vice versa.

The interaction of Trp7 in TRTK-12 likely contributes to the tightest binding for any S100B target to date. It is also interesting that replacement of Phe385 in the p53 peptide with a Trp residue increases its affinity for rat S100B by about 5-fold (34). In addition, important similarities exist between the environments of Trp7 (TRTK-12) and Trp11 (annexin I) even though the orientation of the two target peptides is different with respect to the protein helices in each complex. A comparison of the anchoring Trp7 of TRTK-12 reveals side chain interactions (<6.5 Å) with Ile47, Val52, and Val56 in a majority of the NMR structures. The analogous interactions, involving Gln52, Val57, and Met61 exist for S100A11 (Fig. 6B). Despite this similarity, the differences of the peptide orientations for TRTK-12 and annexin I result in a significant hydrophobic surface for each protein that remains exposed, even in the presence of the bound peptide (Fig. 5A and Fig. 6C). This may occur simply because the target peptides presently used are too short or that the interaction can be modulated through a second region of interaction. As a result, it has been suggested recently (35) that S100 proteins may bind full-length targets using more than one site. The similarity of the locations for Trp7 (TRTK-12) and Trp11 (annexin I) would provide a unique bridging residue to encompass a larger, contiguous binding region that includes an alpha -helical annexin type interaction, utilizing the VEL residues found in annexin I and II, and an extended TRTK-12 type interaction based on hydrophobic interactions anchored by Trp7 (Fig. 6C). It is interesting that the recent structure of the calmodulin-calmodulin kinase kinase peptide complex displays exactly these features, where the N-terminal portion of the peptide forms an alpha -helical structure and the C-terminal region is more extended (36), folding back on the helix in addition to having important protein contacts. As in calmodulin, this larger interaction site would also provide a rationale for the promiscuity of S100B target interactions, including cellular architecture proteins such as tubulin, vimentin, desmin, GFAP, and caldesmon, which contain the TRTK-12 consensus motif (12). It remains to be seen whether the natural sequences for the intermediate filament proteins interact in a similar manner as TRTK-12 or via the alpha -helical mode found for either annexin or p53 peptides.

Coordinates-- Chemical shift assignments for S100B and TRTK-12 in the complex have been deposited in the BioMagResBank (accession number 5377).

    ACKNOWLEDGEMENTS

We thank Kathryn Barber for technical support; Frank Delaglio and Dan Garrett (National Institutes of Health) for NMRPipe and Pipp; Lewis Kay (University of Toronto) for all pulse sequences; and The National High Field Nuclear Magnetic Resonance Facility (NANUC, Edmonton, Alberta, Canada) for acquisition of 13C F1-edited F3-filtered NOESY-HMQC experiments. Funding for 500 and 600 MHz NMR spectrometers was made possible by grants from the Canada Foundation for Innovation, the Medical Research Council of Canada, and the Academic Development Fund of the University of Western Ontario and generous gifts from R. Samuel McLaughlin Foundation and London Life Insurance Company of Canada.

    FOOTNOTES

* This work was supported by operating and maintenance grants from the Canadian Institute for Health Research (to G. S. S.) and graduate studentships from the Natural Sciences and Engineering Research Council (to K. A. M.). This work was presented at the XXth International Conference on Magnetic Resonance in Biological Systems, August 25-30, 2002, Toronto, Ontario, Canada.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and the structure factors (code 1MQ1) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

Dagger To whom correspondence should be addressed. Fax: 519-661-3175; E-mail: gshaw1@uwo.ca.

Published, JBC Papers in Press, December 11, 2002, DOI 10.1074/jbc.M210622200

    ABBREVIATIONS

The abbreviations used are: S100B, dimeric S100beta ; GFAP, glial fibrillary acidic protein; TRTK-12, acetyl-TRTKIDWNKILS-NH2; NOE, nuclear Overhauser effect; MOPS, 4-morpholinepropanesulfonic acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Marshak, D. R., Pesce, S. A., Stanley, L. C., and Griffin, W. S. T. (1991) Neurobiol. Aging 13, 1-7
2. Van Eldik, L. J., and Griffin, W. S. T. (1994) Biochim. Biophys. Acta 1223, 398-403[Medline] [Order article via Infotrieve]
3. Griffin, W. S. T., Stanley, L. C., Ling, C., White, L., MacLeod, V., Perrot, L. J., White, C. L., and Araoz, C. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 7611-7615[Abstract]
4. Donato, R. (1999) Biochim. Biophys. Acta 1450, 191-231[Medline] [Order article via Infotrieve]
5. Donato, R. (1988) J. Biol. Chem. 263, 106-110[Abstract/Free Full Text]
6. Bianchi, R., Garbuglia, M., Verzini, M., Giambanco, I., Ivanenkov, V. V., Dimlich, R. V. W., Jamieson, G. A., Jr., and Donato, R. (1996) Biochim. Biophys. Acta 1313, 258-267[CrossRef][Medline] [Order article via Infotrieve]
7. Baudier, J., Mochly-Rosen, D., Newton, A., Lee, S.-H., Koshland, D. E., and Cole, R. D. (1987) Biochemistry 26, 2886-2893[Medline] [Order article via Infotrieve]
8. Baudier, J., and Cole, R. D. (1988) J. Biol. Chem. 263, 5876-5883[Abstract/Free Full Text]
9. Lin, L.-H., Van Eldik, L. J., Osheroff, N., and Norden, J. J. (1994) Mol. Brain Res. 25, 297-304[Medline] [Order article via Infotrieve]
10. Smith, S. P., and Shaw, G. S. (1998) Structure 6, 211-222[Medline] [Order article via Infotrieve]
11. Ivanenkov, V. V., Jamieson, G. A., Jr., Gruenstein, E., and Dimlich, R. V. W. (1995) J. Biol. Chem. 270, 14651-14658[Abstract/Free Full Text]
12. McClintock, K. A., and Shaw, G. S. (2000) Protein Sci. 9, 2043-2046[Abstract]
13. McClintock, K. A., Van Eldik, L. J., and Shaw, G. S. (2002) Biochemistry 41, 5421-5428[CrossRef][Medline] [Order article via Infotrieve]
14. Rety, S., Sopkova, J., Renouard, M., Osterloh, D., Gerke, V., Tabaries, S., Russo- Marie, F., and Lewit-Bentley, A. (1999) Nat. Struct. Biol. 6, 89-95[CrossRef][Medline] [Order article via Infotrieve]
15. Rety, S., Osterloh, D., Arie, J.-P., Tabaries, S., Seeman, J., Russo-Marie, F., Gerke, V., and Lewit-Bentley, A. (2000) Structure 8, 175-184[CrossRef][Medline] [Order article via Infotrieve]
16. Rustandi, R. R., Baldisseri, D. M., and Weber, D. J. (2000) Nat. Struct. Biol. 7, 570-574[CrossRef][Medline] [Order article via Infotrieve]
17. Smith, S. P., Barber, K. R., Dunn, S. D., and Shaw, G. S. (1996) Biochemistry 35, 8805-8814[CrossRef][Medline] [Order article via Infotrieve]
18. Wittekind, M., and Mueller, L. (1993) J. Magn. Reson. Ser. B. 101, 171-180
19. Grzesiek, S., and Bax, A. (1992) J. Am. Chem. Soc. 114, 6291-6293
20. Kay, L. E., Xu, G. Y., and Yamazaki, T. (1994) J. Magn. Reson. Ser. A 109, 129-133[CrossRef]
21. Grzesiek, S., Anglister, J., and Bax, A. (1993) J. Mag. Reson. 101, 114-119[CrossRef]
22. Kay, L. E., Xu, G., Singer, A. U., Muhandiram, D. R., and Forman-Kay, J. D. (1993) J. Magn. Reson. Ser. B 101, 333-337[CrossRef]
23. Yamazaki, T., Forman-Kay, J. D., and Kay, L. E. (1993) J. Am. Chem. Soc. 115, 11054-11055
24. Delaglio, F., Grzesiek, S., Vuister, G. W., Zhu, G., Pfeifer, J., and Bax, A. (1995) J. Biomol. NMR 263, 277-293
25. Garrett, D. S., Powers, R., Gronenborn, A. M., and Clore, G., M. (1991) J. Magn. Reson. 95, 214-220
26. Zhang, O., Kay, L. E., Olivier, J. P., and Foreman-Kay, J. D. (1994) J. Biomol. NMR 4, 845-858[Medline] [Order article via Infotrieve]
27. Pascal, S. M., Muhandiram, D. R., Yamazaki, T., Forman-Kay, J. D., and Kay, L. E. (1994) J. Mag. Reson. Ser. B 103, 197-201
28. Cornilescu, G., Delaglio, F., and Bax, A. (1999) J. Biomol. NMR 13, 289-302[CrossRef][Medline] [Order article via Infotrieve]
29. Vuister, G. W., and Bax, A. (1993) J. Am. Chem. Soc. 115, 7772-7777
30. Lee, W., Revington, M., Arrowsmith, C., and Kay, L. E. (1994) FEBS Lett. 350, 87-90[CrossRef][Medline] [Order article via Infotrieve]
31. Smith, S. P., Barber, K. R., and Shaw, G. S. (1997) Protein Sci. 6, 1110-1113[Abstract/Free Full Text]
32. Smith, S. P., and Shaw, G. S. (1997) J. Biomol. NMR 10, 77-88[Medline] [Order article via Infotrieve]
33. Barber, K. R., McClintock, K. A., Jamieson, G. A., Jr., Dimlich, R. V., and Shaw, G. S. (1999) J. Biol. Chem. 274, 1502-1528[Abstract/Free Full Text]
34. Rustandi, R. R., Drohat, A. C., Baldisseri, D. M., Wilder, P. T., and Weber, D. J. (1998) Biochemistry 37, 1951-1960[CrossRef][Medline] [Order article via Infotrieve]
35. Otterbein, L. R., Kordowska, J., Witte-Hoffmann, C., Wang, C.-L. A., and Dominguez, R. (2002) Structure 10, 557-567[CrossRef][Medline] [Order article via Infotrieve]
36. Kurokawa, H., Osawa, M., Kurihara, H., Katayama, N., Tokumitsu, H., Swindells, M. B., Kainosho, M., and Ikura, M. (2001) J. Mol. Biol. 312, 59-68[CrossRef][Medline] [Order article via Infotrieve]
37. Laskowski, R. A., MacArthur, M. W., Moss, D. S., and Thornton, J. M. (1993) J. Appl. Crystallogr 26, 283-290[CrossRef]


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