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Biomedical subjects

Christopher G Bunick

Publications and source records attributed to Christopher G Bunick.

5 recordsLinked to original sources
PubMed2006

Biochemical and structural domain analysis of xeroderma pigmentosum complementation group C protein.

XPC is a 940-residue multidomain protein critical for the sensing of aberrant DNA and initiation of global genome nucleotide excision repair. The C-terminal portion of XPC (residues 492-940; XPC-C) has critical interactions with DNA, RAD23B, CETN2, and TFIIH, whereas functional roles have not yet been assigned to the N-terminal portion (residues 1-491; XPC-N). In order to analyze the molecular basis for XPC function and mutational defects associated with xeroderma pigmentosum (XP) disease, a series of stable bacterially expressed N- and C-terminal fragments were designed on the basis of sequence analysis and produced for biochemical characterization. Limited proteolysis experiments combined with mass spectrometry revealed that the full XPC-C is stable but XPC-N is not. However, a previously unrecognized folded helical structural domain was found within XPC-N, XPC(156-325). Pull-down and protease protection assays demonstrated that XPC(156-325) physically interacts with the DNA repair factor XPA, establishing the first functional role for XPC-N. XPC-C exhibits binding characteristics of the full-length protein, including stimulation of DNA binding by physical interaction with RAD23B and CETN2. Analysis of an XPC missense mutation (Trp690Ser) found in certain patients with XP disease revealed that this mutation is associated with a diminished ability to bind DNA. Evidence of contributions to protein interactions from regions in both XPC-N and XPC-C along with recently recognized homologies to yeast PNGase prompted construction of a structural model of a folded XPC core. This model offers key insights into how domains from the two portions of the protein may cooperate in generating specific XPC functions.

Binding Sites↗
PubMed2005

Structure of the N-terminal calcium sensor domain of centrin reveals the biochemical basis for domain-specific function.

Centrin is an essential component of microtubule-organizing centers in organisms ranging from algae and yeast to humans. It is an EF-hand calcium-binding protein with homology to calmodulin but distinct calcium binding properties. In a previously proposed model, the C-terminal domain of centrin serves as a constitutive anchor to target proteins, and the N-terminal domain serves as the sensor of calcium signals. The three-dimensional structure of the N-terminal domain of Chlamydomonas rheinhardtii centrin has been determined in the presence of calcium by solution NMR spectroscopy. The domain is found to occupy an open conformation typical of EF-hand calcium sensors. Comparison of the N- and C-terminal domains of centrin reveals a structural and biochemical basis for the domain specificity of interactions with its cellular targets and the distinct nature of centrin relative to other EF-hand proteins. An NMR titration of the centrin N-terminal domain with a fragment of the known centrin target Sfi1 reveals binding of the peptide to a discrete site on the protein, which supports the proposal that the N-terminal domain serves as a calcium sensor in centrin.

Animals↗
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↗
PubMed2004

Designing sequence to control protein function in an EF-hand protein.

The extent of conformational change that calcium binding induces in EF-hand proteins is a key biochemical property specifying Ca(2+) sensor versus signal modulator function. To understand how differences in amino acid sequence lead to differences in the response to Ca(2+) binding, comparative analyses of sequence and structures, combined with model building, were used to develop hypotheses about which amino acid residues control Ca(2+)-induced conformational changes. These results were used to generate a first design of calbindomodulin (CBM-1), a calbindin D(9k) re-engineered with 15 mutations to respond to Ca(2+) binding with a conformational change similar to that of calmodulin. The gene for CBM-1 was synthesized, and the protein was expressed and purified. Remarkably, this protein did not exhibit any non-native-like molten globule properties despite the large number of mutations and the nonconservative nature of some of them. Ca(2+)-induced changes in CD intensity and in the binding of the hydrophobic probe, ANS, implied that CBM-1 does undergo Ca(2+) sensorlike conformational changes. The X-ray crystal structure of Ca(2+)-CBM-1 determined at 1.44 A resolution reveals the anticipated increase in hydrophobic surface area relative to the wild-type protein. A nascent calmodulin-like hydrophobic docking surface was also found, though it is occluded by the inter-EF-hand loop. The results from this first calbindomodulin design are discussed in terms of progress toward understanding the relationships between amino acid sequence, protein structure, and protein function for EF-hand CaBPs, as well as the additional mutations for the next CBM design.

Amino Acid Sequence↗