Session 3: Calcium toolkit Lectures L3.2 L3.1 Role of zinc-calcium interplay in structure and function of human S100A12 Structural basis for intracellular Olga V. Moroz1, Elena V. Blagova1, calcium sensing and regulation Tony J. Wilkinson1, Keith S. Wilson1, Igor B. Bronstein2 Le Zheng1, Peter B. Stathopulos1, Rainer Schindl2, 1Structural Biology Laboratory, Chemistry Department, University of Guang-Yao Li1, Christoph Romanin2, Mitsu Ikura1 York, United Kingdom; 2School of Biomedical and Health Sciences, King’s College London, United Kingdom 1Division of Signaling Biology, Ontario Cancer Institute and Department e-mail: Olga Moroz <[email protected]> of Medical Biophysics, University of Toronto, Toronto, ON, Canada M5G 1L7; 2 Institute of Biophysics, University of Linz, Linz, Austria Human S100A12 is a member of the S100 family of cal- e-mail: Mitsu Ikura <[email protected]> cium-modulated EF-hand proteins. It has a role in inflam- mation and host parasite responses, and is linked to major Calcium ion (Ca2+) is a dynamically regulated cell signal diseases such as diabetes, cystic fibrosis, rheumatoid arthri- which controls a wide variety of cellular processes such as tis and atherosclerosis. Like several other S100 proteins, neuronal signaling, contraction, lymphocyte activation, cell S100A12 binds zinc in addition to calcium. Zinc binding differentiation, and fertilization. My laboratory is interested to S100A12 significantly enhances the calcium affinity [1]. in the elucidation of the structural basis on Ca2+ sensing Previous studies on the S100A12 interactions with one and regulatory mechanisms and in the most recent years of its receptors RAGE (the Receptor for the Advanced we have been focusing on the mechanism of the store op- Glycation End products) show that calcium and zinc de- erated Ca2+ entry (SOCE). Early studies showed that the pendent oligomerization is essential for target recognition stromal interaction molecule-1 (STIM1) is a key activator by S100A12 [2]. Our X-ray structures of S100A12 in sev- of SOCE in response to diminished luminal Ca2+ levels. eral states - apo, calcium, calcium+copper, and zinc in the Subsequently, we have determined the solution structure absence of calcium- suggested an explanation for zinc-in- of the Ca2+-sensing region of STIM1 consisting of the EF- duced increase in the affinity to calcium [3]. Basing on the hand and sterile a motif (SAM) domains (EF-SAM). More structures, a model could be proposed for metal-dependent recently, we have investigated STIM2, a homologoue of changes in the oligomerization state of the protein. The human STIM1, and identified key structural features which role of both zinc and calcium in target binding by S100A12 contribute to the properties of the Ca2+ sensory function during host parasite responses is confirmed by experiments of STIM proteins. We found that EF-hand and SAM do- with paramyosin from the tropical parasites Onchocerca volvu- mains cooperate in transmitting the luminal Ca2+ signal into lus and Brugia malayi. Possible common features of calcium the activation of SOCE and that structural stability and and zinc-dependent modulation of oligomerisation and Ca2+ affinity of those isoforms are well balanced in order to target interactions for the other S100 family members will execute their respective functions. We provide atomic reso- also be discussed. lution insight into the molecular basis for STIM-mediated References: SOCE initiation and show that the folded/unfolded state 1. Dell’Angelica EC et al. (1994) J Biol Chem 269: 28929–28936. of the Ca2+ sensing region of STIM is crucial to SOCE 2. Moroz OV et al. (2009) BMC Biochem 10: 1. regulation. 3. Moroz OV et al. (2009) J Mol Biol 391: 536–551. Acknowledgements: Supported by CIHR and CFI. Vol. 57 11th Meeting of the European Calcium Society, 2010 19 L3.3 L3.4 Calmodulin and calmodulin- Structural basis of S100-RAGE interaction binding proteins in cancer Michael Koch1, Christine Betz2, Seth Chatiyat3, Colin D. White, Zhigang Li, David B. Sacks Walter J. Chazin3, Guenter Fritz1,2 Brigham and Women’s Hospital and Harvard Medical School, 1University of Konstanz, Biology, Germany; 2University of Freiburg, Department of Pathology, USA Neuropathology, Germany; 3Vanderbilt University, Center for Structural e-mail: David B. Sacks <[email protected]> Biology, USA e-mail: Günter Fritz <[email protected]> A substantial body of evidence supports a fundamental role for calmodulin in cell proliferation and cell cycle progres- S100 proteins constitute the largest subgroup within the sion [1]. Calmodulin is implicated in neoplastic transfor- family of EF-hand calcium-binding proteins. Most S100 mation, with altered expression levels of calmodulin and proteins form homo- and heterodimers under physiological selected calmodulin-binding proteins documented in hu- conditions but also larger assemblies like tetra-, hexa-, and man malignancy. The steroid hormone estrogen promotes octamers are reported. Several S100 proteins are secreted proliferation of human breast epithelial cells by interacting to the extracellular space where they exert neurotrophic, with the estrogen receptor (ER). ER is routinely measured proinflammatory or antimicrobial functions. Prominent in tissue from patients with breast cancer as it has impor- examples are S100B and S100A12, which act via the recep- tant implications for both therapy and prognosis. We have tor for advanced glycation endprodcuts (RAGE). We inves- shown that calmodulin interacts directly with ER, thereby tigate the interactions between RAGE and S100 proteins enhancing ER stability [2]. Further analysis revealed that by in vitro binding studies and X-ray crystallography. The calmodulin reduces proteasome-dependent degradation X-ray structure of human Ca(II)-loaded S100B revealed of ER. In addition, binding to calmodulin is necessary for that S100B can assemble into large multimers, which were normal transcriptional function of ER. Therefore, the mo- confirmed in human brain and by recombinant expression lecular interaction between calmodulin and ER could be a in E. coli [1]. Such multimers exhibit decreased dissociation target for therapeutic intervention in patients with breast from RAGE resulting most likely in a sustained activation cancer. of the receptor signal cascade. The ErbB family of receptor tyrosine kinases comprises To further characterize S100-RAGE interaction we deter- four members, namely the epidermal growth factor recep- mined the X-ray structure of RAGE ligand-binding do- tor (EGFR), HER2/Neu, ErbB3 and ErbB4/ErbB1 [3]. main at 1.85 Å resolution [2]. The arrangement of RAGE ErbB members are potent mediators of normal cell growth ectodiomain molecules in the crystal and complementary and development, and aberrant receptor expression or biochemical studies suggest a role for self-association in functioning plays a crucial part in the development and ev- RAGE function.The S100 interactiong surface of RAGE olution of cancer [3]. For example, HER2 is overexpressed was mapped onto the structure from titrations with S100B in ~25% of invasive breast cancer and is associated with re- monitored by heteronuclear NMR spectroscopy. These duced survival. Published evidence reveals that calmodulin NMR chemical shift perturbations were used as input for binds EGFR and HER2 [4]. IQGAP1, a scaffold protein restrained docking calculations to generate a model for the that integrates signalling pathways, binds to and regulates VC1-S100B complex [2]. numerous proteins [5]. Accumulating evidence suggest IQ- References: GAP1 is an oncogene that is important for tumourigenesis 1. Ostendorp O. et al. (2007) Embo J 26: 3868–3878. [6]. Calmodulin binds to and regulates the function of IQ- 2. Koch M. et al. (2010) structure (in press). GAP1. Importantly, we have observed that IQGAP1 binds directly to both EGFR and HER2, thereby altering func- tion of the receptors. Analogous to other IQGAP1 bind- ing partners, calmodulin modulates the interaction between IQGAP1 and HER2. Collectively, these data suggest that calmodulin antagonists may be of value in the treatment of breast cancer. References: 1. Lu KP et al. (1993) Endocr Rev 14: 40–58. 2. Li Z et al. (2001) J Biol Chem 276: 17354–17360. 3. Baselga J et al. (2009) Nat Rev Cancer 9: 463–475. 4. Sanchez-Gonzalez P et al. (2010) FEBs J 277: 327–342. 5. Brown MD et al. (2006) Trends Cell Biol 16: 242–249. 6. White CD et al. (2009) FEBs Lett 583: 1817–1824. 20 Abstracts 2010 L3.5 Posters Membrane targetting of the EF-hand P3.1 containing calcium sensing proteins Calneurons I and II (CaBP8 and CaBP7) Fine-tuning of cerebellar Purkinje Hannah V. McCue, Robert D. Burgoyne, Lee P. Haynes cells by calbindin D-28k University of Liverpool, Physiology Department, United Kingdom Jarosław J. Barski1, 2 e-mail: Hannah McCue <[email protected]> 1Medical University of Silesia, Center for Experimental Medicine, Katowice, Poland; 2Medical University of Silesia, Department of Physiology, Katowice, Poland Calneuron I and Calneuron II (also known as CaBP8 and e-mail: Jaroslaw Barski <[email protected]> CaBP7 respectively) make up a subfamily of the calcium binding protein (CaBP) family of calcium sensors. The Long-term depression (LTD) of Purkinje cell-parallel fiber CaBPs are a family of Calmodulin-like, EF-hand contain- synaptic transmission is a critical determinant of normal ing proteins which have recently emerged as regulators of cerebellar function. Impairment of LTD through, for ex- various important