• Cloning and molecular characterization of the murine S100(beta), a glial-specific neurotrophic factor in the nervous system.

      Jiang, Hao; Hilt, Dana C. (1994)
      The organization, sequence and transcriptional regulation of the murine S100{dollar}\beta{dollar} gene have been studied. The gene is approximately 9 kb in length and is composed of three exons and two introns. The murine S100{dollar}\beta{dollar} gene contains a TATA box (AATAA) and a reverse CCAAT box (ATTGG) located at 30 nucleotides and 92 nucleotides upstream of the transcription start site, respectively. A 149 bp DNA fragment ({dollar}-{dollar}157/{dollar}-{dollar}9) spanning the TATA box and reverse CCAAT box functions as a promoter. The murine S100{dollar}\beta{dollar} promoter drives a 4-fold higher level of transcription in C6 rat glioma cells than in non-glial 3T3 murine fibroblast cells, suggesting the existence of a cell type-specific regulatory element within the promoter region. The 5{dollar}\sp\prime{dollar}-flanking region suppresses transcription from the homologous S100{dollar}\beta{dollar} as well as heterologous SV40 promoters in an orientation-independent fashion. However, the 5{dollar}\sp\prime{dollar}-flanking region exhibits cell-type specificity when suppressing the S100{dollar}\beta{dollar} promoter-dependent transcription, indicating its involvement in the regulation of the cell type-specific expression of the murine S100{dollar}\beta{dollar} gene. In order to map the cell type-specific regulatory elements, transcriptional analyses of various deletions of the 5{dollar}\sp\prime{dollar}-flanking region were carried out in both C6 and 3T3 cells. Two cell type-specific negative regulatory elements, one active in non-glial cells and another active in glial cells, were mapped to the regions {dollar}-{dollar}1552/{dollar}-{dollar}1234 and {dollar}-{dollar}1234/{dollar}-{dollar}551, respectively. A strong negative regulatory element and a relatively weak negative regulatory element were located in the regions {dollar}-{dollar}551/{dollar}-{dollar}157 and {dollar}-{dollar}1669/{dollar}-{dollar}1552, respectively. These results lead to the conclusion that the murine S100{dollar}\beta{dollar} gene is under complex transcriptional regulation involving tonic negative control exerted by a combination of multiple cis-acting regulatory elements including cell type-specific elements. Gel mobility shift assay with the cell type-specific regulatory element III (CSREIII, {dollar}-{dollar}1552/{dollar}-{dollar}1234) showed that a unique trans-acting protein factor may be present in 3T3 nuclear extract but absent in C6 nuclear extract. This factor can specifically bind to a 17 bp DNA fragment ({dollar}-{dollar}1513/{dollar}-{dollar}1497) which contains a consensus silencer element. These findings suggest that the glial-specific expression of the murine S100{dollar}\beta{dollar} gene is probably mediated by both cis-acting silencer elements and trans-acting protein factors.
    • Structural and functional studies on S100A1

      Wright, Nathan Thompson; Weber, David J., Ph.D. (2008)
      S100 proteins are small, dimeric calcium binding proteins that contain two EF-hands per subunit. While these proteins are expressed plentifully in a tissue-specific manner, the cellular functions of most S100 proteins remain understudied. This dissertation describes efforts to elucidate both the structure and the function of one member of this family, S100A1, in various biologically relevant states. Multidimensional NMR spectroscopy techniques were used to solve the solution structure of S100A1 in its Ca 2+-bound form. The protein folds as a symmetric homodimer, with an X-type helix bundle comprising the dimer interface. A large conformational change, involving the reorientation of helix 3, accompanies S100A1 calcium binding. This change exposes a previously hidden hydrophobic pocket which is the general target protein binding site and specifically the binding site for the calmodulin binding domain of the ryanodine receptor. The solution structure of S100A1 bound to a peptide from this region, along with whole-cell calcium transient measurements in S100A1 knockout mice, provide evidence that S100A1 directly increases RyR1-mediated calcium release by binding to a discrete area on the cytoplasmic face of RyR1. This binding event is driven by both hydrophobic and ionic interactions between S100A1 and the RyR peptide. A competition between S100A1 and calmodulin for the same region of RyR1 likely represents a cellular mechanism of modulating SR Ca2+ release in skeletal muscle fibers.;Studies detailing the internal backbone dynamics of the Ca2+-loaded form of another S100 protein, S100B, are also presented in this manuscript. These experiments were conducted to further our understanding of the molecular mechanisms driving S100 protein structure and function. Here, S100B is shown to be a stable protein in the Ca2+-bound form, with significant motions in the hydrophobic pocket. These motions may assist in peptide binding, and support a previously suggested coupling between Ca2+ and target peptide binding in S100 proteins.
    • Structure, Dynamics, and Function of S100B and S100A5 Complexes

      Liriano, Melissa Ana; Weber, David J., Ph.D. (2012)
      The S100 family is a class of small, homodimeric proteins that are often characterized by their calcium-dependent biological effects, which is typically the result of a calcium-dependent conformational change. The majority of S100 proteins have a low μM binding affinity for calcium, but in the presence of a target, this affinity can increase dramatically, as seen with the 5-fold increase in calcium binding affinity when S100B is bound to the capZ-derived TRTK-12 peptide. However, S100A5 is an exception, where the binding affinity of S100A5 for calcium is approximately 50-fold tighter than S100B not bound to a molecular target (Ca EF2KD - 0.25-1 μM). Interestingly, we have discovered that once bound to a molecular target (i.e. TXIP - Truncated eXchanger Inhibitory Peptide from NCX1) the calcium affinity for S100A5 decreases 10-fold, opposite of what is found in most other S100 proteins once bound to target. One possible explanation for the calcium "tightening" effect seen with S100B in the presence of molecular target or with S100A5 in the absence of peptide is that the calcium coordination of the EF-hands may have altered to a more optimal geometry. However, x-ray crystal structures of calcium-loaded S100B (±TRTK-12) and the calcium-bound S100A5 structure presented here, indicate that all complexes have identical calcium coordination in both the S100 (EF1) and canonical (EF2) EF-hands. Therefore a static structural explanation is not sufficient to explain how S100A5 can bind calcium so tightly in the absence of target or how calcium "tightening" occurs with S100B once bound to TRTK-12. An alternative mechanism that could explain the calcium binding properties of S100B and S100A5 may involve dynamics. For S100B, the dynamic properties for residues in the overall protein (i.e. 15N backbone amides) and EF2 (i.e. 15N side chains) could be stabilized upon S100-target complex formation. Indeed, 15N dynamics were measured for S100B in the presence and absence of TRTK-12 and upon TRTK-12 binding, the movements of several backbone amide residues were quenched at fast (ns) and slow (μs - ms) timescales. This decrease of backbone amide exchange was also translated to the EF2-hand of D63NS100B, a mutant that allows reliable detection of 15N exchange in a residue that directly coordinates calcium. For S100A5, the findings were contrary as to what was seen with S100B in the absence and bound to a molecular target. In the absence of target, there was no exchange detected in the terminal amine side chain of Asn61, a ligand that directly coordinates Ca2+ in position 3 of EF2 in S100A5. However with target bound, chemical exchange (μs - ms) and further fast time-scale motion (ns) became apparent in the backbone amides of residues in the EF-hand, helix 4 and the hinge region. These data suggest that an increase of dynamics may explain in part the decrease of Ca2+-affinity seen in the S100A5-target complex.