Synthesis and expression of a gene coding for the calcium-modulated protein S100 beta and designed for cassette-based, site-directed mutagenesis.

Eldik, Linda J. Van; Staecker, Jeffrey L.; Winningham-Major, F

doi:10.1016/s0021-9258(18)68573-4

Cited by 78 publications

(5 citation statements)

References 59 publications

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“…Expression of recombinant human S100b protein in cells containing the plasmid pSS2 was induced via the addition of IPTG, as described under Materials and Methods. Purification of human S100b was a simplified version of a previous method (VanEldik et al, 1988). The purification procedure was monitored by SDS-PAGE (Figure 1).…”

Section: Resultsmentioning

confidence: 99%

Structural Influence of Cation Binding to Recombinant Human Brain S100b: Evidence for Calcium-Induced Exposure of a Hydrophobic Surface

et al. 1996

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The dimeric calcium-binding protein S100b is proposed to undergo a calcium-induced structural change allowing it to interact, via a hydrophobic surface, with other proteins. Previously it has been suggested that calcium binding to S100b leads to the exposure of at least one phenylalanine residue (Mani et al., 1982, 1983). This effect appears to be "reversed" at higher ionic strength, leading to a possible reburying of phenylalanine residues (Mani et al., 1982, 1983). To study these effects, we monitored calcium binding to recombinant human S100b by NMR spectroscopy under different salt (KCI) conditions. 15N-Labeled glycine residues in S100b showed calcium-induced chemical shift changes similar to those reported for the related monomeric protein calbindin D9k, suggesting similar conformational changes are occurring in the calcium-binding loops of these two proteins. Calcium binding to S100b also resulted in a shifting and broadening of several 1H resonances from the Ca-S100b form only including those from the side chains of residues F14, F70, and F73 but not those of residue Y17. This broadening was enhanced with increased ionic strength (KCI). However, small additions ( < 15% v/v) of the hydrophobic solvent trifluoroethanol relieved this phenomenon, leading to narrower line widths. These observations are consistent with the calcium-induced exposure of at least one of these hydrophobic residues, resulting in self-association of the S100b dimer. Trifluoroethanol serves to dissociate these complexes back to the dimeric calcium species. We propose that this cluster of hydrophobic residues which include F14, F73, and F88 may be important for interactions with a target protein.

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Section: Resultsmentioning

confidence: 99%

Structural Influence of Cation Binding to Recombinant Human Brain S100b: Evidence for Calcium-Induced Exposure of a Hydrophobic Surface

et al. 1996

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“…Protein Expression and Purification. The gene for Bos taurus S100B (30) was subcloned into the pSV281 vector, a pET27 derivative with Kanamycin resistance, a His-tag, and an rTEV cleavage site. The resultant plasmid was transformed into Escherichia coli BL21 Codon plus (DE3) RIL competent cells (Stratagene), which were then overexpressed in isotopically enriched minimal media to get 13 C, 15 N-and 15 N-labeled protein.…”

Section: Methodsmentioning

confidence: 99%

Structure of the Ca²⁺/S100B/NDR Kinase Peptide Complex: Insights into S100 Target Specificity and Activation of the Kinase

et al. 2003

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NDR, a nuclear serine/threonine kinase, belongs to the subfamily of Dbf2 kinases that is critical to the morphology and proliferation of cells. The activity of NDR kinase is modulated in a Ca(2+)/S100B-dependent manner by phosphorylation of Ser281 in the catalytic domain and Thr444 in the C-terminal regulatory domain. S100B, which is a member of the S100 subfamily of EF-hand proteins, binds to a basic/hydrophobic sequence at the junction of the N-terminal regulatory and catalytic domains (NDR(62-87)). Unlike calmodulin-dependent kinases, regulation of NDR by S100B is not associated with direct autoinhibition of the active site, but rather involves a conformational change in the catalytic domain triggered by Ca(2+)/S100B binding to the junction region. To gain further insight into the mechanism of activation of the kinase, studies have been carried out on Ca(2+)/S100B in complex with the intact N-terminal regulatory domain, NDR(1-87). Multidimensional heteronuclear NMR analysis showed that the binding mode and stoichiometry of a peptide fragment of NDR (NDR(62-87)) is the same as for the intact N-terminal regulatory domain. The solution structure of Ca(2+)/S100B and NDR(62-87) has been determined. One target molecule is found to associate with each subunit of the S100B dimer. The peptide adopts three turns of helix in the bound state, and the complex is stabilized by both hydrophobic and electrostatic interactions. These structural studies, in combination with available biochemical data, have been used to develop a model for calcium-induced activation of NDR kinase by S100B.

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“…The F87A mutant contains a Phe (TTC) to Ala (GCC) mutation at positions 326-328 of pSS2, and F8788A has both mutations. The bovine S100B mutants S100B83stop and S100B85stop were constructed and expressed in the pVUSB-1 vector (37). In the truncation mutants S100B85stop and S100B83stop, residues His85 and Ala83, respectively, have been changed to stop codons (TAA), resulting in mutant proteins lacking the C-terminal seven and nine residues, respectively.…”

Section: Methodsmentioning

confidence: 99%

The C-Terminus and Linker Region of S100B Exert Dual Control on Protein−Protein Interactions with TRTK-12

2002

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S100B, an EF-hand calcium-binding protein composed of two S100beta monomers, undergoes a calcium-dependent conformational change that provides a surface for target interactions. In this study, the calcium-sensitive S100B-binding epitope TRTK-12 has been used to probe the contributions of the linker and C-terminal regions of S100B to protein-protein interactions. These contributions were quantified using C-terminal mutant S100B proteins lacking the C-terminal seven (S100B85stop) or nine (S100B83stop) residues or containing alanine substitutions at Phe87 (F87A), Phe88 (F88A), or both (F8788A). Both F8788A and F88A bound TRTK-12 less tightly (K(d) = 1.85 +/- 0.02 and 0.97 +/- 0.08 microM, respectively) than the wild-type protein (K(d) = 0.27 +/- 0.03 microM, DeltaG = -37.2 kJ/mol), indicating these residues are important for TRTK-12 interaction. The truncated S100B proteins bound TRTK-12 much more weakly (K(d) = 659.7 +/- 119.3 microM, DeltaG = -17.9 kJ/mol), indicating the linker region contributed about 50% to the binding of TRTK-12, while the C-terminus contributed the remaining 50% of the binding energy. Based on mutagenesis and NMR chemical shift studies, a comparison with known S100-target protein complexes showed the S100B-TRTK-12 complex has the strongest resemblance to the S100A10-annexin II interaction.

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Synthesis and expression of a gene coding for the calcium-modulated protein S100 beta and designed for cassette-based, site-directed mutagenesis.

Cited by 78 publications

References 59 publications

Structural Influence of Cation Binding to Recombinant Human Brain S100b: Evidence for Calcium-Induced Exposure of a Hydrophobic Surface

Structural Influence of Cation Binding to Recombinant Human Brain S100b: Evidence for Calcium-Induced Exposure of a Hydrophobic Surface

Structure of the Ca²⁺/S100B/NDR Kinase Peptide Complex: Insights into S100 Target Specificity and Activation of the Kinase

The C-Terminus and Linker Region of S100B Exert Dual Control on Protein−Protein Interactions with TRTK-12

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Synthesis and expression of a gene coding for the calcium-modulated protein S100 beta and designed for cassette-based, site-directed mutagenesis.

Cited by 78 publications

References 59 publications

Structural Influence of Cation Binding to Recombinant Human Brain S100b: Evidence for Calcium-Induced Exposure of a Hydrophobic Surface

Structural Influence of Cation Binding to Recombinant Human Brain S100b: Evidence for Calcium-Induced Exposure of a Hydrophobic Surface

Structure of the Ca2+/S100B/NDR Kinase Peptide Complex: Insights into S100 Target Specificity and Activation of the Kinase

The C-Terminus and Linker Region of S100B Exert Dual Control on Protein−Protein Interactions with TRTK-12

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Structure of the Ca²⁺/S100B/NDR Kinase Peptide Complex: Insights into S100 Target Specificity and Activation of the Kinase