Calbindin D28k and S100B Have a Similar Interaction Site with the

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Calbindin D28k and S100B have a similar interaction site with the lithiuminhibitable enzyme inositol monophosphatase-1: a new drug target site Galila Agam, and Orna Almog J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm5019324 • Publication Date (Web): 09 Feb 2015 Downloaded from http://pubs.acs.org on February 14, 2015

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Journal of Medicinal Chemistry

Calbindin D28k and S100B have a similar interaction site with the lithium-inhibitable enzyme inositol monophosphatase-1: a new drug target site Galila Agam1,2 and Orna Almog1*

1

Department of Clinical Biochemistry and Pharmacology, Faculty of Health Sciences 2

Ben-Gurion University of the Negev, and Mental Health Center, Beer-Sheva, Israel

KEYWORDS. lithium; inositol monophosphatase-1; calbindin D28k; S100B; protein-protein interaction; drug target;

ABSTRACT: Li salts (Li) are used to treat bipolar disorder patients. Li inhibits inositol-monophosphatase (IMPase)-1. Calbindin D28k (calbindin) and S100B enhance IMPase-1 activity. We compared our in-silico model of the IMPase1/calbindin complex with the crystal structure of S100B. Although calbindin and S100B have a low sequence homology they seem to activate IMPase-1 in a similar mode. It is reasonable that molecules interfering with the interaction of IMPase-1 with either of its activators will have Li-like effects. INTRODUCTION Inositol monophosphatase (IMPase)-1, a lithium (Li) inhibitable key enzyme in the phosphatidylinositol signaling system, is one of the hypothesized targets of the beneficial effect of Li treatment in bipolar disorder. Berg1 gard et al. found that calbindin D28k (calbindin), a calcium binding protein abundant in neurons of the central nervous system, interacts with IMPase-1 resulting in acti2 vation of the enzyme. Similarly, Vig et al. showed that S100B, a calcium binding protein and a growth and differentiation factor, is also capable of IMPase-1 activation. Using transgenic mice of spinocerebellar ataxia type 1 (SCA1) they found co-localization of S100B with IMPase1 in cytoplasmic vacuoles of cerebellar Purkinje cells (PCs). They suggested that glial S100B internalized in PCs may be affecting physiologic processes vital for these cells sur2 vival . In the nervous system S100B is expressed in astrocytes, certain neuronal populations and Schwann cells and is constitutively excreted from astrocytes. In the extracellular space it exhibits neurotrophic or neurotoxic effects as a function of its brain extracellular space concentration. Namely, at subnanomolar to nanomolar concentrations it protects neurons against oxidative stress and promotes neurite outgrowth, while at micromolar levels, as a result of a rise in release or leakage from damaged astrocytes, it hyperactivates astrocytes and microglia. This suggests a possible role of S100B in neuroin-

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flammation and neurodegeneration . Interestingly, elevated S100B in serum was found to be associated with 6,7 8 mood disorders and schizophrenia , diseases that com9,10 prise neurodegenerative characteristics . The neurotrophic effects of S100B include stimulation of neuronal survival, cell proliferation and migration as well as inhibi8 tion of apoptosis . A neurotoxic effect of S100B is inhibition of differentiation9. In mature cells the protein regulates functions such as transcription, protein degradation, 2+ Ca homeostasis, energy metabolism and enzymes' activity by interacting with target proteins within cells, e.g. tubulin, membrane-bound guanylate cyclase, dopamine 2 D2 receptor and IMPase-1 . Translocation of S100B in vesicles plays an important role in the assembly of signaling complexes that activate specific signaling pathways. This implicates effects on brain development and activation of astrocytes during brain damage and gliomagenesis. 11

We have recently reported in-silico model results of the IMPase-1/calbindin complex indicating that the 55–66 amino-acid segment of IMPase-1 anchors calbindin via Lys59 and Lys61 with a glutamate in between (the Lys–Glu–Lys motif). The model also indicated that Lys59 (a part of the Lys-Glu–Lys motif) interacts with Asp24 and Asp26 of calbindin and Lys61 interacts with Asn158 and Asp155 of calbindin. Since calbindin and S100B share a similar function (i.e. activation of IMPase-1) and a similar three di-

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mensional structure we now hypothesized that calbindin and S100B have a similar interaction site with IMPase-1. RESULTS AND DISCUSSION We now compared our in-silico model of the IMPase-1/calbindin complex with the crystal structure of

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S100B (pdb entry 3D10). Although calbindin and S100B, both calcium binding proteins, have a low sequence homology (38% using BLAST), we found that S100B has a calbindin-like domain which includes two proximal loops containing two aspartate residues (Fig. 1). In the calbindin molecule one loop carries the aspartate residues Asp24

A

B

C

D

Figure 1. A comparison between the three dimensional structure of S100B and calbindin D28k A. The crystal structure of S100B (pdb 3D10) shown in pink superimposed on the NMR structure of calbindin (pdb 2g9b) shown in blue. As expected from the low sequence homology (38% according to BLAST) between the two proteins a different tertiary structure is demonstrated. B. The crystal structure of S100B presenting the two loops suggested as the binding site with IMPase-1. The green spheres are calcium ions. Residues Asp65 and Asp63 on one loop and residues Glu21 and Asp23 on the other loop are shown as pink and yellow sticks. C. A segment of the NMR structure of calbindin presenting the two loops suggested as the binding site with IMPase-1. The green spheres are calcium ions. Residues Asp24 and Asp26 on one loop and residues Asn158 and Asp155 on the other loop are shown as blue sticks. D. The same S100B structure as in B presenting its interaction with TRTK-12 peptide shown as green sticks and indicating a different binding site than that suggested for IMPase-1.


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and Asp26 and the other loop – Asn158 and Asp155. In S100B one loop includes Asp63 and Asp65 and the other – Glu21 and Asp23. In calbindin the distance between Asp26 and the Ca2+ ion is 2.67 Å and the distance between Asp155 and the Ca2+ ion is 2.79 Å. In S100B the distances between Asp63 and Asp65 with the Ca2+ ion are 2.27 Å and 2.39 Å, respectively. The distance between Asp23 and the other Ca2+ ion of S100B is 2.67 Å. Intriguingly, the distance between the two loops in calbindin and in S100B is similar, namely, 11.57 Å and 10.49 Å, respectively. This raises the possibility that the similar domains of calbindin and S100B interact and activate IMPase-1 by binding the same Lys–Glu–Lys motif of the enzyme. Since Li's inhibition of IMPase-1 at therapeutical12 ly-relevant concentrations is one of the hypothesized molecular mechanisms of the drug's mood stabilizing effect, and since both calbindin and S100B enhance IMPase-1 activity, it is reasonable to expect that molecules interfering with the interaction of the enzyme with either of its stimulators will have Li-like beneficial effects. The TRTK-12 peptide (T-R-T-K-I-D-W-N-K-I-LS), comprising the C-terminus of the actin-binding protein CapZ, was shown to interact with S100B and to compete with the binding of S100B with other known targets. However, the binding site of TRTK-12 to S100B is different than the site suggested by us to be involved in the interaction of S100B with IMPase-1. In the case of IMPase-1/calbindin, we have previously demonstrated that six amino acid peptides including the Lys-Glu-Lys motif or part of it inhibit calbindin-enhanced IMPase-1 activi11 ty .

CONCLUSIONS Based on our hypothesized IMPase-1/S100B mode of interaction and the similarity to the IMPase-1/calbindin mode of interaction it is conceivable that the same six amino acids peptides would also inhibit the S100Benhanced IMPase-1 activity. If approved experimentally, these peptides or small molecules resembling their effect could become Li replacement or add-on drugs.

EXPERIMENTAL SECTION Our previously described in-silico structural model of the IMPase-1/calbindin complex was calculated using the program Molfit11. The crystal structure of S100B (pdb entry 3D10) was superimposed on the calculated structure of the calbinidin/IMPase-1 complex using the program PyMol version 1.00 (2006).

AUTHOR INFORMATION Corresponding Author

*

Orna Almog, Department of Clinical Biochemistry and Pharmacology, Faculty of Health Sciences, Ben-Gurion Uni-

versity of the Negev, Beer-Sheva, Israel. [email protected]; tel: 972-8-64799330.

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. O.A. designed the study and carried out the in-situ analyzes.

Funding Sources This work was supported in part by the ministry of health grant 5924-00000-3 and by a Young Research Grant from the Israeli Institute for Psychobiology to OA.

Notes The authors declare no competing financial interest.

Abbreviations: IMPase-1 - inositol-monophosphatase-1; SCA1 - spinocerebellar ataxia type 1; PCs - Purkinje cells.

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meeting report. Eur. Neuropsychopharmacol. 2008, 18, 787-793. 10. Lieberman, J. A. Is schizophrenia a neurodegenerative disorder? A clinical and neurobiological perspective. Biol. Psychiatry 1999, 46, 729-739. 11. Levi, I.; Eskira, Y.; Eisenstein, M.; Gilon, C.; Hoffman, A.; Talgan, Y.; Fanous, J.; Bersudsky, Y.; Belmaker, R. H.; Agam, G.; Almog, O. Inhibition of inositol monophosphatase (IMPase) at the calbindin-D28k binding site: molecular and behavioral aspects. Eur. Neuropsychopharmacol. 2013, 23, 1806-1815.

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12. Ohnishi, T.; Ohba, H.; Seo, K. C.; Im, J.; Sato, Y.; Iwayama, Y.; Furuichi, T.; Chung, S. K.; Yoshikawa, T. Spatial expression patterns and biochemical properties distinguish a second myo-inositol monophosphatase IMPA2 from IMPA1. J. Biol. Chem. 2007, 282, 637-646.


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Calbindin D28k and S100B Have a Similar Interaction Site with the

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