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Shibani Bhattacharya

Publications and source records attributed to Shibani Bhattacharya.

6 recordsLinked to original sources
PubMed2005

The modular structure of SIP facilitates its role in stabilizing multiprotein assemblies.

Siah-interacting protein (SIP) was identified as a novel adaptor that physically links the E3 ubiquitin ligase activity of Siah-1 with Skp1 and Ebi F-Box protein in the degradation of beta-catenin, a transcriptional activator of TCF/LEF genes. In this study, we have used solution NMR spectroscopy to characterize the domain structure of SIP, which includes a novel helical hairpin domain at the N-terminus flexibly linked to a CS domain and an unstructured carboxy terminal SGS domain. These studies have been complemented by mapping the sites of functionally important protein-protein interactions involving Siah-1 and Skp1 to individual domains of SIP. NMR-based chemical shift perturbation assays show that Siah-1 interacts with the flexible linker between SIP N and CS domains. This site for interaction in the linker does not perturb residues in the structured region at the N-terminus but does appear to restrict the rotational freedom of the SIP CS domain in the context of the full-length protein. In contrast, Skp1 engages the SIP CS domain exclusively through weak interactions that are not coupled to the other domains. The principal role of the modular structure of SIP appears to be in bringing these two proteins into physical proximity and orchestrating the orientation required for polyubiquitination of beta-catenin in the intact SCF-type complex.

Amino Acid Motifs↗
PubMed2004

Target selectivity in EF-hand calcium binding proteins.

EF-hand calcium binding proteins have remarkable sequence homology and structural similarity, yet their response to binding of calcium is diverse and they function in a wide range of biological processes. Knowledge of the fine-tuning of EF-hand protein sequences to optimize specific biochemical properties has been significantly advanced over the past 10 years by determination of atomic resolution structures. These data lay the foundation for addressing how functional selectivity is generated from a generic ionic signal. This review presents current ideas about the structural mechanisms that provide the selectivity of different EF-hand proteins for specific cellular targets, using S100 and calmodulin family proteins to demonstrate the critical concepts. Three factors contribute significantly to target selectivity: molecular architecture, response to binding of Ca(2+) ions, and the characteristics of target binding surfaces. Comparisons of calmodulin and S100 proteins provide insights into the role these factors play in facilitating the variety of binding configurations necessary for recognizing a diverse set of targets.

Amino Acid Sequence↗
PubMed2003

Structure of the Ca2+/S100B/NDR kinase peptide complex: insights into S100 target specificity and activation of the kinase.

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.

Amino Acid Sequence↗
PubMed2002

Characterization of binding-induced changes in dynamics suggests a model for sequence-nonspecific binding of ssDNA by replication protein A.

Single-stranded-DNA-binding proteins (SSBs) are required for numerous genetic processes ranging from DNA synthesis to the repair of DNA damage, each of which requires binding with high affinity to ssDNA of variable base composition. To gain insight into the mechanism of sequence-nonspecific binding of ssDNA, NMR chemical shift and (15)N relaxation experiments were performed on an isolated ssDNA-binding domain (RPA70A) from the human SSB replication protein A. The backbone (13)C, (15)N, and (1)H resonances of RPA70A were assigned for the free protein and the d-CTTCA complex. The binding-induced changes in backbone chemical shifts were used to map out the ssDNA-binding site. Comparison to results obtained for the complex with d-C(5) showed that the basic mode of binding is independent of the ssDNA sequence, but that there are differences in the binding surfaces. Amide nitrogen relaxation rates (R(1) and R(2)) and (1)H-(15)N NOE values were measured for RPA70A in the absence and presence of d-CTTCA. Analysis of the data using the Model-Free formalism and spectral density mapping approaches showed that the structural changes in the binding site are accompanied by some significant changes in flexibility of the primary DNA-binding loops on multiple timescales. On the basis of these results and comparisons to related proteins, we propose that the mechanism of sequence-nonspecific binding of ssDNA involves dynamic remodeling of the binding surface.

DNA, Single-Stranded↗