Expression, purification, and characterization of a structurally disordered and functional C-terminal autoinhibitory domain (AID) of the 70 kDa 40S ribosomal protein S6 kinase-1 (S6K1)

S6K1 is a member of the AGC subfamily of serine-threonine protein kinases, whereby catalytic activation requires dual phosphorylation of critical residues in the conserved T-loop (T229) and hydrophobic motif (HM; T389) peptide regions of its catalytic kinase domain (residues 1-398). In addition to its kinase domain, S6K1 contains a C-terminal autoinhibitory domain (AID; residues 399-502), which prevents T-loop and HM phosphorylation; and autoinhibition is relieved on multi-site Ser-Thr phosphorylation of the AID (S411, S418, T421, and S424). Interestingly, 66 of the 104 C-terminal AID amino acid residues were computer predicted to exist in structurally disordered peptide regions, begetting interest as to how such dynamics could be coupled to autoregulation. To begin addressing this issue, we developed and optimized protocols for efficient AID expression and purification. Consistent with computer predictions, aberrant mobilities in both SDS-PAGE and size-exclusion chromatography, as well as low chemical shift dispersion in (1)H-(15)N HSQC NMR spectra, indicated purified recombinant AID to be largely unfolded. Yet, trans-addition of purified AID effectively inhibited PDK1-catalyzed T-loop phosphorylation of a catalytic kinase domain construct of S6K1. Using an identical purification protocol, similar protein yields of a tetraphospho-mimic mutant AID(D(2)ED) construct were obtained; and this construct displayed only weak inhibition of PDK1-catalyzed T229 phosphorylation. Purification of the structurally 'disordered' and functional C-terminal AID and AID(D(2)ED) constructs will facilitate studies aimed to understand the role of conformational plasticity and protein phosphorylation in modulating autoregulatory domain-domain interactions.     

Structural characterization of the ribosome maturation protein, RimM

The RimM protein has been implicated in the maturation of the 30S ribosomal subunit. It binds to ribosomal protein S19, located in the head domain of the 30S subunit. Multiple sequence alignments predicted that RimM possesses two domains in its N- and C-terminal regions. In the present study, we have produced Thermus thermophilus RimM in both the full-length form (162 residues) and its N-terminal fragment, spanning residues 1 to 85, as soluble proteins in Escherichia coli and have performed structural analyses by nuclear magnetic resonance spectroscopy. Residues 1 to 80 of the RimM protein fold into a single structural domain adopting a six-stranded beta-barrel fold. On the other hand, the C-terminal region of RimM (residues 81 to 162) is partly folded in solution. Analyses of 1H-15N heteronuclear single quantum correlation spectra revealed that a wide range of residues in the C-terminal region, as well as the residues in the vicinity of a hydrophobic patch in the N-terminal domain, were dramatically affected upon complex formation with ribosomal protein S19.     

Defining the minimal interacting regions of the tight junction protein MAGI-1 and HPV16 E6 oncoprotein for solution structure studies

The oncoprotein E6 produced by tumorigenic high-risk genital human papillomaviruses targets a number of cellular proteins containing PDZ domains for proteasome-mediated degradation. In particular, E6 targets the tight junction protein MAGI-1 by binding to its PDZ1 domain. Using light scattering and NMR, we explored different fragments of both the HPV16 E6 and the MAGI-1 PDZ1 domain to define the best-behaving complex for solution structure studies. We showed that the 70-residue HPV16 E6 C-terminal domain (E6C) can be efficiently substituted by a peptide spanning the 11 C-terminal residues of E6. The construct of MAGI-1 PDZ1 best suited for solution structure analysis presents a 14-residue N-terminal extension and a 26-residue C-terminal extension as compared to the construct used for the recently solved X-ray structure of a MAGI-1 PDZ1/HPV18 E6 complex. These data suggest a stabilizing role for the interdomain linker regions which separate the PDZ1 domain from its neighboring domains.     

The periplasmic domain of Escherichia coli outer membrane protein A can undergo a localized temperature dependent structural transition

Gram-negative bacteria such as Escherichia coli are surrounded by two membranes with a thin peptidoglycan (PG)-layer located in between them in the periplasmic space. The outer membrane protein A (OmpA) is a 325-residue protein and it is the major protein component of the outer membrane of E. coli. Previous structure determinations have focused on the N-terminal fragment (residues 1–171) of OmpA, which forms an eight stranded transmembrane β-barrel in the outer membrane. Consequently it was suggested that OmpA is composed of two independently folded domains in which the N-terminal β-barrel traverses the outer membrane and the C-terminal domain (residues 180–325) adopts a folded structure in the periplasmic space. However, some reports have proposed that full-length OmpA can instead refold in a temperature dependent manner into a single domain forming a larger transmembrane pore. Here, we have determined the NMR solution structure of the C-terminal periplasmic domain of E. coli OmpA (OmpA^180–325). Our structure reveals that the C-terminal domain folds independently into a stable globular structure that is homologous to the previously reported PG-associated domain of Neisseria meningitides RmpM. Our results lend credence to the two domain structure model and a PG-binding function for OmpA, and we could indeed localize the PG-binding site on the protein through NMR chemical shift perturbation experiments. On the other hand, we found no evidence for binding of OmpA^180–325 with the TonB protein. In addition, we have also expressed and purified full-length OmpA (OmpA^1–325) to study the structure of the full-length protein in micelles and nanodiscs by NMR spectroscopy. In both membrane mimetic environments, the recombinant OmpA maintains its two domain structure that is connected through a flexible linker. A series of temperature-dependent HSQC experiments and relaxation dispersion NMR experiments detected structural destabilization in the bulge region of the periplasmic domain of OmpA above physiological temperatures, which may induce dimerization and play a role in triggering the previously reported larger pore formation.     

Structural and functional analysis of ProQ: an osmoregulatory protein of Escherichia coli

Transporter ProP of Escherichia coli senses extracellular osmolality and responds by mediating cytoplasmic accumulation of organic solutes such as proline. Lesions at the proQ locus reduce ProP activity in vivo. ProQ was previously purified and characterized. Homology modeling predicted that ProQ possesses an alpha-helical N-terminal domain (residues 1-130) and a beta-sheet C-terminal domain (residues 181-232) connected by an unstructured linker. In this work, we tested the structural model for ProQ, explored the solubility and folding of full length ProQ and its domains in diverse buffers, and tested the impacts of the putative ProQ domains on ProP activity in vivo. Limited tryptic proteolysis of ProQ revealed protease resistant fragments corresponding to the predicted N-terminal and C-terminal domains. Polypeptides corresponding to the predicted N- and C-terminal domains could be overexpressed and purified to near homogeneity using nickel affinity, size exclusion and reversed phase chromatographies. Circular dichroism spectroscopy of the purified proteins revealed that the N-terminal domain was predominantly alpha-helical, whereas the C-terminal domain was predominantly beta-sheet, as predicted. The domains were soluble and folded in neutral buffers containing 0.6 M NaCl. The N-terminal domain was soluble and folded in 0.1 M MES (2-[N-morpholino]-ethane sulfonic acid) at pH 5.6. Despite high solubilities, the proteins were not well folded in Na citrate (0.1 M, pH 2.3). The ProQ domains and the linker were expressed at physiological levels, singly and in combination, in bacteria lacking the chromosomal proQ locus. Among these proteins, the N-terminal domain could partially complement the proQ deletion. The full length protein and a variant lacking only the linker restored full activity of the ProP protein.     

E1 protein of human papillomavirus is a DNA helicase/ATPase.

Replication of human papillomavirus (HPV) DNA requires the viral proteins E1 and E2. Amino acid similarities to SV40 large-T antigen had suggested that E1 is a DNA helicase/ATPase involved in initiating viral DNA replication, and this has recently been shown for bovine papillomavirus type 1 (BPV-1) E1 protein. However, in vitro analysis of HPV E1 has been hampered by the inability to produce purified protein using heterologous expression systems. We have succeeded in demonstrating ATPase and DNA helicase activities in purified HPV E1, expressed in E. coli as a maltose-binding protein fusion (MBP-E1), for the first time. As further confirmation that the ATPase and DNA helicase activities are due to E1 and not contaminating E. coli enzymes, we have shown that a fusion protein containing an amino acid change (E1 Pro-479 to Ser), predicted to inactivate ATP-binding, has impaired activities. We have carried out a structure prediction analysis which suggests that E1 may form two domains: a relatively open N-terminal domain (residues 1-125), and a highly structured C-terminal domain (170-649), with an intermediate region (125-170) predicted to form an inter-domain linker. This is consistent with the proteolytic susceptibility of MBP-E1 at a site 15-20 kD from the N-terminus of E1, and the accumulation of a 58 kD C-terminal fragment of E1. We speculate that the N-terminal domain is involved in DNA-binding, while the C-terminal 58 kD may constitute a distinct enzymatic domain. HPV E1 is of interest as a therapeutic target and the availability of pure enzyme will be invaluable in the search for antiviral compounds.     

A HECT domain E3 enzyme assembles novel polyubiquitin chains

Although polyubiquitin chains linked through Lys(29) of ubiquitin have been implicated in the targeting of certain substrates to proteasomes, the signaling properties of these chains are poorly understood. We previously described a ubiquitin-protein isopeptide ligase (E3) from erythroid cells that assembles polyubiquitin chains through either Lys(29) or Lys(48) of ubiquitin (Mastrandrea, L. D., You, J., Niles, E. G., and Pickart, C. M. (1999) J. Biol. Chem. 274, 27299-27306). Here we describe the purification of this E3 based on its affinity for a linear fusion of ubiquitin to the ubiquitin-conjugating enzyme UbcH5A. Among five major polypeptides in the affinity column eluate, the activity of interest was assigned to the product of a previously cloned human cDNA known as KIAA10 (Nomura, N., Miyajima, N., Sazuka, T., Tanaka, A., Kawarabayasi, Y., Sato, S., Nagase, T., Seki, N., Ishikawa, K., and Tabata, S. (1994) DNA Res. 1, 27-35). The KIAA10 protein is a member of the HECT (homologous to E6-AP carboxyl terminus) domain family of E3s. These E3s share a conserved C-terminal (HECT) domain that functions in the catalysis of ubiquitination, while their divergent N-terminal domains function in cognate substrate binding (Huibregtse, J. M., Scheffner, M., Beaudenon, S., and Howley, P. M. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 2563-2567). Recombinant KIAA10 catalyzed the assembly of both Lys(29)- and Lys(48)-linked polyubiquitin chains. Surprisingly, the C-terminal 428 residues of KIAA10 were both necessary and sufficient for this activity, suggesting that the ability to assemble polyubiquitin chains may be a general property of HECT domains. The N-terminal domain of KIAA10 interacted in vitro with purified 26 S proteasomes and with the isolated S2/Rpn1 subunit of the proteasome's 19 S regulatory complex, suggesting that the N-terminal domains of HECT E3s may function in proteasome binding as well as substrate binding.     

A C-terminal domain of GAP is sufficient to stimulate ras p21 GTPase activity.

The cDNA for bovine ras p21 GTPase activating protein (GAP) has been cloned and the 1044 amino acid polypeptide encoded by the clone has been shown to bind the GTP complexes of both normal and oncogenic Harvey (Ha) ras p21. To identify the regions of GAP critical for the catalytic stimulation of ras p21 GTPase activity, a series of truncated forms of GAP protein were expressed in Escherichia coli. The C-terminal 343 amino acids of GAP (residues 702-1044) were observed to bind Ha ras p21-GTP and stimulate Ha ras p21 GTPase activity with the same efficiency (kcat/KM congruent to 1 x 10(6) M-1 s-1 at 24 degrees C) as GAP purified from bovine brain or full-length GAP expressed in E. coli. Deletion of the final 61 amino acid residues of GAP (residues 986-1044) rendered the protein insoluble upon expression in E. coli. These results define a distinct catalytic domain at the C terminus of GAP. In addition, GAP contains amino acid similarity with the B and C box domains conserved among phospholipase C-II, the crk oncogene product, and the non-receptor tyrosine kinase oncogene products. This homologous region is located in the N-terminal half of GAP outside of the catalytic domain that stimulates ras p21 GTPase activity and may constitute a distinct structural or functional domain within the GAP protein.     

Hydrogen/deuterium exchange and electron-transfer dissociation mass spectrometry determine the interface and dynamics of apolipoprotein E oligomerization

Apolipoprotein E, a 34 kDa protein, plays a key role in triglyceride and cholesterol metabolism. Of the three common isoforms (ApoE2, -3, and -4), only ApoE4 is a risk factor for Alzheimer's disease. All three isoforms of wild-type ApoE self-associate to form oligomers, a process that may have functional consequences. Although the C-terminal domain, residues 216-299, of ApoE is believed to mediate self-association, the specific residues involved in this process are not known. Here we report the use of hydrogen/deuterium exchange (H/DX) coupled with enzymatic digestion to identify those regions in the sequence of full-length apoE involved in oligomerization. For this determination, we compared the results of H/DX of the wild-type proteins and those of monomeric forms obtained by modifying four residues in the C-terminal domain. The three wild-type and mutant isoforms show similar structures based on their similar H/DX kinetics and extents of exchange. Regions of the C-terminus (residues 230-270) of the ApoE isoforms show significant differences of deuterium uptake between oligomeric and monomeric forms, confirming that oligomerization occurs at these regions. To achieve single amino acid resolution, we examined the extents of H/DX by using electron transfer dissociation (ETD) fragmentation of peptides representing selected regions of both the monomeric and the oligomeric forms of ApoE4. From these experiments, we could identify the specific residues involved in ApoE oligomerization. In addition, our results verify that ApoE4 is composed of a compact structure at its N-terminal domain. Regions of C-terminal domain, however, appear to lack defined structure.     

HPV oncoprotein E6 is a structure-dependent DNA-binding protein that recognizes four-way junctions

E6 is an oncoprotein implicated in cervical cancers, produced by "high-risk" human papillomaviruses. E6 is thought to promote tumorigenesis by stimulating cellular degradation of the tumour suppressor p53, but it might display other activities. Sequence similarity was recently detected between E6 and endonuclease VII, a protein of phage T4 that recognizes and cleaves four-way DNA junctions. Here, we purified recombinant E6 proteins and demonstrated that high-risk E6 s bind selectively to four-way junctions in a structure-dependent manner. Several residues in the C-terminal zinc-binding domain, the region of E6 similar to endonuclease VII, are necessary for the junction-binding activity. E6 binds to the junction as a monomer. Comparative electrophoresis shows that E6-bound junctions migrate in an extended square conformation. Magnesium inhibits the electrophoretic migration of the complexes but does not seem to influence their formation at equilibrium. This work is the first demonstration of specific binding of purified active E6 to a well-characterized DNA ligand, and suggests new modes of action of E6 in oncogenesis.     

Engineering and characterization of the isolated C-terminal domain of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase

5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the formation of EPSP and inorganic phosphate from shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP) in the biosynthesis of aromatic amino acids. To delineate the domain-specific function, we successfully isolated the discontinuous C-terminal domain (residues 1-21, linkers, 240-427) of EPSP synthase (427 residues) by site-directed mutagenesis. The engineered C-terminal domains containing no linker (CTD), or with gly-gly (CTD(GG)) and gly-ser-ser-gly (CTD(GSSG)) linkers were purified and characterized as having distinct native-like secondary and tertiary structures. However, isothermal titration calorimetry (ITC), 15N-HSQC, and 31P-NMR revealed that neither its substrate nor inhibitor binds the isolated domain. The isolated domain maintained structural integrity, but did not function as the half of the full-length protein.     

Dimerization of the exocyst protein Sec6p and its interaction with the t-SNARE Sec9p

Vesicles in eukaryotic cells transport cargo between functionally distinct membrane-bound organelles and the plasma membrane for growth and secretion. Trafficking and fusion of vesicles to specific target sites are highly regulated processes that are not well understood at the molecular level. At the plasma membrane, tethering and fusion of secretory vesicles require the exocyst complex. As a step toward elucidation of the molecular architecture and biochemical function(s) of the exocyst complex, we expressed and purified the exocyst subunit Sec6p and demonstrated that it is a predominantly helical protein. Biophysical characterization of purified Sec6p by gel filtration and analytical ultracentrifugation experiments revealed that Sec6p is a dimer. Limited proteolysis defined an independently folded C-terminal domain (residues 300-805) that equilibrated between a dimer and monomer in solution. Removal of residues 300-410 from this construct yielded a well-folded, monomeric domain. These results demonstrate that residues 300-410 are necessary for dimerization, and the presence of the N-terminal region (1-299) increases dimer stability. Moreover, we found that the dimer of Sec6p binds to the plasma membrane t-SNARE Sec9p and inhibits the interaction between Sec9p and its partner t-SNARE Sso1p. This direct interaction between the exocyst complex and the t-SNARE implicates the exocyst in SNARE complex regulation.     

Mutational analysis of the thermostable arginine repressor from Bacillus stearothermophilus: dissecting residues involved in DNA binding properties.

Recently the crystal structure of the DNA-unbound form of the full-length hexameric Bacillus stearothermophilus arginine repressor (ArgR) has been resolved, providing a possible explanation for the mechanism of arginine-mediated repressor-operator DNA recognition. In this study we tested some of these functional predictions by performing site-directed mutagenesis of distinct amino acid residues located in two regions, the N-terminal DNA-binding domain and the C-terminal oligomerization domain of ArgR. A total of 15 mutants were probed for their capacity to repress the expression of the reporter argC - lacZ gene fusion in Escherichia coli cells. Substitutions of highly conserved amino acid residues in the alpha2 and alpha3 helices, located in the winged helix-turn-helix DNA-binding motif, reduced repression. Loss of DNA-binding capacity was confirmed in vitro for the Ser42Pro mutant which showed the most pronounced effect in vivo. In E. coli, the wild-type B. stearothermophilus ArgR molecule behaves as a super-repressor, since recombinant E. coli host cells bearing B. stearothermophilusargR on a multicopy vector did not grow in selective minimal medium devoid of arginine and grew, albeit weakly, when l -arginine was supplied. All mutants affected in the DNA-binding domain lost this super-repressor behaviour. Replacements of conserved leucine residues at positions 87 and/or 94 in the C-terminal domain by other hydrophobic amino acid residues proved neutral or caused either derepression or stronger super-repression. Substitution of Leu87 by phenylalanine was found to increase the DNA-binding affinity and the protein solubility in the context of a double Leu87Phe/Leu94Val mutant. Structural modifications occasioned by the various amino acid substitutions were confirmed by circular dichroism analysis and structure modelling.     

Polynucleotide phosphorylase interacts with ribonuclease E through a betabetaalphabetabetaalpha domain

In the present work we have used a double-hybrid assay in bacteria to identify a putative domain in E. coli PNPase required for in vivo interaction with RNase E. We used a 202 aa fragment of RNase E previously reported as the PNPase binding domain in this enzyme and a collection of 13 different fragments of 105 aa, spanning the entire sequence of 734 aa PNPase (GenBank Accession number NP_417633). Our results indicate that two clones of PNPase including residues 158-262 and residues 473-577 contain interaction sites for RNase E within a betabetaalphabetabetaalpha domain configuration. Three-dimensional modeling of the E. coli PNPase based on the S. antibioticus protein structure indicates that the putative binding domain is located on the monomer surface, facing outward from the trimeric tertiary structure. Since a copy of the betabetaalphabetabetaalpha domain is also found in RNase PH, we investigated and found an interaction with RNase E in a pull-down assay. We suggest this interaction takes place through the similar betabetaalphabetabetaalpha domain present in the tertiary structure of this enzyme. Based on these results, we propose that RNase PH and RNase E could form functional assemblies in E. coli.     

Effect of mutations in the C-terminal domain of Mu B on DNA binding and interactions with Mu A transposase

Bacteriophage Mu transposition requires two phage-encoded proteins, the transposase, Mu A, and an accessory protein, Mu B. Mu B is an ATP-dependent DNA-binding protein that is required for target capture and target immunity and is an allosteric activator of transpososome function. The recent NMR structure of the C-terminal domain of Mu B (Mu B223-312) revealed that there is a patch of positively charged residues on the solvent-exposed surface. This patch may be responsible for the nonspecific DNA binding activity displayed by the purified Mu B223-312 peptide. We show that mutations of three lysine residues within this patch completely abolish nonspecific DNA binding of the C-terminal peptide (Mu B223- 312). To determine how this DNA binding activity affects transposition we mutated these lysine residues in the full-length protein. The full-length protein carrying all three mutations was deficient in both strand transfer and allosteric activation of transpososome function but retained ATPase activity. Peptide binding studies also revealed that this patch of basic residues within the C-terminal domain of Mu B is within a region of the protein that interacts directly with Mu A. Thus, we conclude that this protein segment contributes to both DNA binding and protein-protein contacts with the Mu transposase.     

Fractionation of polyclonal antibodies to fragments of a neuroreceptor using three increasingly chaotropic solvents

We have developed specific antibodies against fragments of anaplastic lymphoma kinase (ALK) in order to develop tools for characterizing the expression and biological function of this orphan receptor. The first fragment consisted of residues 280 to 480 of the murine extracellular domain, was expressed in Escherichia coli (E. coli), purified in the presence of urea from the pellet of mechanically lysed cells and injected into rabbits as an unfolded protein in urea. The second fragment consisted of residues 1519 to 1619 of the murine sequence, corresponding to the C-terminal side of the kinase domain. It was expressed in E. coli as a soluble glutathione-S-transferase fusion protein, purified from the supernatant of broken cells and injected into rabbits as a folded protein. Both antisera were purified using antigen affinity chromatography, with the polyclonal antibodies eluted stepwise using three different buffers, 0.1 M glycine, pH 2.9, followed by 7 M urea, pH 4, followed by 6 M guanidine–HCl (GdnHCl), pH 4. Antisera prepared against either antigen contained antibodies that eluted in each of the three pools, indicating that solvents more chaotropic than acid were required to elute antibody populations that were tightly bound to the antigen column. All three antibody pools were reactive towards their respective antigens upon Western blot analysis. Purified polyclonal antibodies (pAbs) to both fragments also recognized the full-length protein expressed in Chinese hamster ovary cells. In every case, the pAbs eluting in GdnHCl were the most sensitive for detecting full-length ALK.     

A monomeric, biologically active, full-length human apolipoprotein E

Apolipoprotein E (apoE) is an exchangeable apolipoprotein that plays an important role in lipid/lipoprotein metabolism and cardiovascular diseases. Recent evidence indicates that apoE is also critical in several other important biological processes, including Alzheimer's disease, cognitive function, immunoregulation, cell signaling, and infectious diseases. Although the X-ray crystal structure of the apoE N-terminal domain was solved in 1991, the structural study of full-length apoE is hindered by apoE's oligomerization property. Using protein-engineering techniques, we generated a monomeric, biologically active, full-length apoE. Cross-linking experiments indicate that this mutant is nearly 95-100% monomeric even at 20 mg/mL. CD spectroscopy and guanidine hydrochloride denaturation demonstrate that the structure and stability of the monomeric mutant are identical to wild-type apoE. Monomeric and wild-type apoE display similar lipid-binding activities in dimyristoylphosphatidylcholine clearance assays and formation of reconstituted high-density lipoproteins. Furthermore, the monomeric and wild-type apoE proteins display an identical LDL receptor binding activity. Availability of this monomeric, biologically active, full-length apoE allows us to collect high quality NMR data for structural determination. Our initial NMR data of lipid-free apoE demonstrates that the N-terminal domain in the full-length apoE adopts a nearly identical structure as the isolated N-terminal domain, whereas the C-terminal domain appears to become more structured than the isolated C-terminal domain fragment, suggesting a weak domain-domain interaction. This interaction is confirmed by NMR examination of a segmental labeled apoE, in which the N-terminal domain is deuterated and the C-terminal domain is double-labeled. NMR titration experiments further suggest that the hinge region (residues 192-215) that connects apoE's N- and C-terminal domains may play an important role in mediating this domain-domain interaction.     

S100A6 and S100A11 are specific targets of the calcium- and zinc-binding S100B protein in vivo

In solution, S100B protein is a noncovalent homodimer composed of two subunits associated in an antiparallel manner. Upon calcium binding, the conformation of S100B changes dramatically, leading to the exposure of hydrophobic residues at the surface of S100B. The residues in the C-terminal domain of S100B encompassing Phe(87) and Phe(88) have been implicated in interaction with target proteins. In this study, we used two-hybrid technology to identify specific S100B target proteins. Using S100B as bait, we identify S100A6 and S100A11 as specific targets for S100B. S100A1, the closest homologue of S100B, is capable of interaction with S100B but does not interact with S100A6 or S100A11. S100B, S100A6, and S100A11 isoforms are co-regulated and co-localized in astrocytoma U373 cells. Furthermore, co-immunoprecipitation experiments demonstrated that Ca(2+)/Zn(2+) stabilizes S100B-S100A6 and S100B-S100A11 heterocomplexes. Deletion of the C-terminal domain or mutation of Phe(87) and Phe(88) residues has no effect on S100B homodimerization and heterodimerization with S100A1 but drastically decreases interaction between S100B and S100A6 or S100A11. Our data suggest that the interaction between S100B and S100A6 or S100A11 should not be viewed as a typical S100 heterodimerization but rather as a model of interaction between S100B and target proteins.