The ubiquitous Ca(2+)-binding protein calmodulin (CaM) is a key protein in Ca2+ homeostasis and activation of eukaryotic cells. CaM is the molecular link between free Ca2+ in the cell and the inhibition, or activation, of numerous enzymes. Many nuclear functions are under Ca2+/CaM control, and some transcriptional activators are known to be Ca2+ modulated indirectly through Ca2+/CaM-dependent protein kinases. But Ca2+/CaM has not yet been found to directly modulate any transcription factor or other DNA-binding protein. Transcription factors of the basic-helix-loop-helix (bHLH) group are important regulators in numerous systems. Here we report that binding of Ca(2+)-loaded CaM to the bHLH domains of several bHLH proteins directly inhibits their DNA binding. Other bHLH proteins are either less sensitive or resistant. Ca2+ ionophore selectively inhibits transcriptional activation by Ca2+/CaM-sensitive bHLH proteins in vivo, implying that Ca2+ can directly influence transcription through differential CaM inhibition of bHLH domains.

Basic helix-loop-helix (bHLH) 1 proteins are a class of transcription factors that are important regulators in numerous systems, often involving the control of cell growth and differentiation (1). Most bHLH proteins (with some exceptions) can be broadly classified into two groups based on their patterns of expression. Class A bHLH transcription factors, also called E proteins, are broadly expressed and include the E2A gene products E12/E47 (2) and products of the E2-2/SEF2-1 gene (3, 4). E proteins are capable of forming both homodimers with themselves and heterodimers with cell type-specific class B proteins. This large group of tissue-restricted proteins includes the myogenic proteins (myogenin (5, 6), MyoD (7), MRF4 (8 -10), and Myf5 (11)) and proteins involved in neurogenesis, including MASH2 (12) and NeuroD (13). Functional activity of a class B protein in vivo requires heterodimerization with an E protein, resulting in the commitment of cells to differentiation pathways (14).Calcium plays a crucial role in many cellular processes (15, 16). Its actions are largely mediated through a family of calcium-binding proteins, of which calmodulin is the major calcium sensor. Calmodulin is a highly conserved, ubiquitously expressed protein that is essential for cell growth (17, 18). Calmodulin has four high affinity calcium binding sites called EF-hands, each composed of two ␣-helices connected by a calcium binding loop (19). Upon calcium binding, calmodulin undergoes a conformational change to expose hydrophobic patches (Ref. 20 and references therein), which allows interaction with numerous target proteins and the subsequent activation of signaling pathways.S-100 proteins are other members of the EF-hand protein family that also modulate the activities of various proteins. At least 17 members of the S-100 family have been identified. They vary in their target specificity, cell type distribution, and cell cycle regulation (21). The best studied members of the S-100 family, S-100␣ and S-100␤, have been shown to exist as both homodimers with themselves and heterodimers with each other. Their expression patterns differ; S-100␣ is predominantly found in muscle, whereas S-100␤ is highly expressed in cells within the nervous system (22-26). S-100 proteins are believed to interact with many proteins, and the identification of several common targets with calmodulin suggests that a common structural domain mediates these interactions (27)(28)(29)(30).We have previously shown that calcium-loaded calmodulin can selectively inhibit the DNA binding of E protein homodimers in vitro and that the calcium ionophore ionomycin inhibits their activity in vivo. In contrast, the heterodimers E12/MASH2 and E12/MyoD were either less sensitive or completely resistant (31).Here we identify the protein sequences within the bHLH proteins that determine the differential inhibition by calmodulin. We show that this inhibition is the result of a physical interaction between the DNA binding basic sequence and calmodulin. Both E12 and MyoD basic seq...

Calmodulin is the predominant intracellular receptor for Ca(2+) signals, mediating the regulation of numerous cellular processes. Previous studies have shown that calcium-loaded calmodulin can bind to and inhibit the activity of certain basic helix-loop-helix (bHLH) transcription factors. The basic sequence within the bHLH domain is the primary target for calmodulin binding, and sequences modulating the calmodulin interaction reside directly N-terminal to the basic sequence. Here we show that the interaction of calmodulin with bHLH proteins is of a novel type, displaying characteristics very different from those of previously characterized calmodulin-target complexes. We show that calmodulin interacts much stronger with a dimeric basic sequence than with the monomeric form. The calmodulin-bHLH protein complex contains equimolar amounts of calmodulin and bHLH chains. The interaction is unusual in being to a large extent polar in nature, and it is highly resistant to tested calmodulin inhibitors. Both the N-terminal and C-terminal domains of calmodulin can independently bind to and inhibit the DNA binding of bHLH proteins. The C-terminal domain preferentially binds to the basic sequence, whereas the N-terminal domain is essential for the effect of the modulatory sequence. We propose a model for the calmodulin-bHLH complex where two calmodulin molecules interact with one bHLH dimer, with one domain of calmodulin preferentially binding to the basic sequence of bHLH proteins and the other domain interacting with the modulatory sequence.

Calmodulin is the predominant intracellular receptor for Ca 2+ signals, mediating the regulation of numerous cellular processes. It can inhibit the DNA binding of basic helix-loop-helix transcription factors by a direct interaction of a novel type. To structurally characterize this novel calmodulin-target interaction, we decided to study the complex of calmodulin with a dimeric peptide corresponding to the DNA-binding domains of the dimeric basic helix-loop-helix transcription factor SEF2-1 (SEF2-1mp) using NMR. Here, we report that the stoichiometry of the calmodulin:SEF2-1mp complex is one dimeric peptide binding two calmodulin molecules. We also report the 1H, 13C, and 15N resonance assignments and the secondary structure of calmodulin in this for NMR large (∼38 kD) complex, as well as the 1H assignments and secondary structure of SEF2-1mp. In addition, we determined the amide proton exchange rates of calmodulin and measured intermolecular calmodulin:SEF2-1mp and calmodulin:calmodulin NOE contacts. The isotope-filtered experiments show a large number of SEF2-1mp to calmodulin NOE contacts indicating that a tight complex is formed, which is confirmed by an intermolecular calmodulin:calmodulin NOE contact. The secondary structure and amide proton exchange data show that the binding does not occur via the classical wraparound binding mode. Instead, the data indicate that calmodulin interacts with SEF2-1mp in a more open conformation, although the hydrophobic surfaces of the N-and C-terminal domains still form the main interaction sites. Interactions involving charged residues are also identified in agreement with the known relatively high sensitivity of the binding to ionic strength. Finally, the peptide does not form an ␣-helix as in the classical wraparound binding mode. Keywords:Dimer; calmodulin; SEF2-1; NMR; NMR assignment; protein complex Calmodulin (CaM) is a small (16.7 kD) ubiquitous and multifunctional Ca 2+ binding protein that plays a central role in the regulation of numerous cellular processes (see Van Eldik and Watterson 1998 and references therein). In accordance with the essential function of calmodulin, the amino acid sequence, and thus the structure, displays an exceptional degree of conservation across species and throughout evolution. The structure of CaM reminds of a dumbbellshaped molecule with two globular domains connected with a 28-amino acid-long ␣-helix, called the central helix (Babu et al. 1988;Chattopadhyaya et al. 1992). NMR studReprint requests to: Sybren Wijmenga, Department of Medical Biosciences, Biophysics, University of Umeå, S-901 87 Umeå, Sweden; e-mail: sybren.wijmenga@chem.umu.se; fax: 46-90-136310. 3 Present address: ICRF Skin Tumour Labs, Centre for Cutaneous Research, 2 Newark street, London E1 2AT, England.4 Present address: Department of Biosciences, Karolinska institutet, Novum, S-141 57 Huddinge, Sweden.Abbreviations: Baa, basic amphipilic ␣-helix; bHLH, basic-helix-loophelix; CaM, Calmodulin; Diamide, Azodicarboxylic acid bis(dimethylamide); NMR, nucle...

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