C-Terminal Half of Human Centrin 2 Behaves like a Regulatory EF-Hand Domain

Matei, Elena; Miron, Simona; Blouquit, Y.; Duchambon, Patricia; Durussel, Isabelle; Cox, Jos A.; Craescu, Constantin T.

doi:10.1021/bi0269714

Cited by 69 publications

(151 citation statements)

References 38 publications

(60 reference statements)

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“…Full length human centrin-2 (HsCen-2), and the carboxyl-terminal domains of HsCen-2 fragments, bind calcium at one high affinity site, probably in the most carboxyl-terminal EF-hand [19,20]. Previous studies suggested that HsCen-2 is dimeric and binds only two calciums per dimer, likely to site IV in each monomer [19].…”

mentioning

confidence: 99%

Metal-binding properties of human centrin-2 determined by micro-electrospray ionization mass spectrometry and UV spectroscopy

Craig

Benson

Bergen

et al. 2006

J. Am. Soc. Mass Spectrom.

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We analyzed the metal-binding properties of human centrin-2 (HsCen-2) and followed the changes in HsCen-2 structure upon metal-binding using micro-electrospray ionization mass spectrometry (ESI-MS). Apo-HsCen-2 is mostly monomeric. The ESI spectra of HsCen-2 show two charge-state distributions, representing two conformations of the protein. HsCen-2 binds four moles calcium/mol protein: one mol of calcium with high affinity, one additional mol of calcium with lower affinity, and two moles of calcium at low affinity sites. HsCen-2 binds four moles of magnesium/mol protein. The conformation giving the lower charge-state HsCen-2 by ESI, binds calcium and magnesium more readily than does the higher charge-state HsCen-2. Both conformations of HsCen-2 bind calcium more readily than magnesium. Calcium was more effective in displacing magnesium bound to HsCen-2 than vice versa. Binding of a peptide from a known binding partner, the xeroderma pigmentosum complementation group protein C (XPC), to apo-HsCen-2, occurs in the presence or the absence of calcium. Near and far-UV CD spectra of HsCen-2 show little difference with addition of calcium or magnesium. Minor changes in secondary structure are noted. Melting curves derived from temperature dependence of molar ellipticity at 222 nm for HsCen-2 show that calcium increases protein stability whereas magnesium does not. ⌬25 HsCen-2 behaves similarly to HsCen-2. We conclude that HsCen-2 binds calcium and magnesium and that calcium modulates HsCen-2 structure and function by increasing its stability without undergoing significant changes in secondary or tertiary

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confidence: 99%

Metal-binding properties of human centrin-2 determined by micro-electrospray ionization mass spectrometry and UV spectroscopy

Craig

Benson

Bergen

et al. 2006

J. Am. Soc. Mass Spectrom.

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“…In agreement with this, only the C-terminal half of centrin 2 may be required for interaction with XPC (10,31,46). Although the primary amino acid sequence of the centrin 2-binding domain of XPC is classified as a 1-14 motif, the structural basis of complex formation may be different than that for typical calmodulin complexes like smMLCK and skMLCK.…”

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confidence: 76%

“…In the absence of both HR23 proteins, XPC is markedly destabilized in vivo as well as in vitro, and thus, the HR23 proteins are effectively essential for efficient GG-NER (2,5,40,43). A recombinant XPC-HR23B heterodimer exhibits specific binding affinities for various types of lesions, including 6-4PP, and has been successfully used to reconstitute a cell-free NER reaction (1,3,31). Centrin 2 is thus dispensable for NER at least in vitro, so the biological significance of its presence in the XPC complex remains to be elucidated, although the physical stability of XPC-HR23B may increase slightly upon binding to centrin 2 (2).…”

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confidence: 99%

Centrin 2 Stimulates Nucleotide Excision Repair by Interacting with Xeroderma Pigmentosum Group C Protein

Nishi

Okuda

Watanabe

et al. 2005

Molecular and Cellular Biology

231

212

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Xeroderma pigmentosum group C (XPC) protein plays a key role in DNA damage recognition in global genome nucleotide excision repair (NER). The protein forms in vivo a heterotrimeric complex involving one of the two human homologs of Saccharomyces cerevisiae Rad23p and centrin 2, a centrosomal protein. Because centrin 2 is dispensable for the cell-free NER reaction, its role in NER has been unclear. Binding experiments with a series of truncated XPC proteins allowed the centrin 2 binding domain to be mapped to a presumed ␣-helical region near the C terminus, and three amino acid substitutions in this domain abrogated interaction with centrin 2. Human cell lines stably expressing the mutant XPC protein exhibited a significant reduction in global genome NER activity. Furthermore, centrin 2 enhanced the cell-free NER dual incision and damaged DNA binding activities of XPC, which likely require physical interaction between XPC and centrin 2. These results reveal a novel vital function for centrin 2 in NER, the potentiation of damage recognition by XPC.

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“…The region is of interest, because it is not present in any other members of the calmodulin-parvalbumin superfamily. Titrating HsCen-2 (26,43) and other centrins (44) to Ca 2ϩ saturation is complicated by aggregation of high order oligomers, an aggregation of reversible nature. Deletion of amino acids 1-24 has little effect on protein stability, global secondary structure, or metal binding (29) but does prevent aggregation; thus, these residues must be involved in the self-assembly (45).…”

Section: Disordered Hscen-2 N-terminalmentioning

confidence: 99%

The Structure of the Human Centrin 2-Xeroderma Pigmentosum Group C Protein Complex

Thompson

Ryan

Salisbury

et al. 2006

Journal of Biological Chemistry

117

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Human centrin-2 plays a key role in centrosome function and stimulates nucleotide excision repair by binding to the xeroderma pigmentosum group C protein. To determine the structure of human centrin-2 and to develop an understanding of molecular interactions between centrin and xeroderma pigmentosum group C protein, we characterized the crystal structure of calcium-loaded full-length centrin-2 complexed with a xeroderma pigmentosum group C peptide. Our structure shows that the carboxyl-terminal domain of centrin-2 binds this peptide and two calcium atoms, whereas the amino-terminal lobe is in a closed conformation positioned distantly by an ordered ␣-helical linker. A stretch of the amino-terminal domain unique to centrins appears disordered. Two xeroderma pigmentosum group C peptides both bound to centrin-2 also interact to form an ␣-helical coiled-coil. The interface between centrin-2 and each peptide is predominantly nonpolar, and key hydrophobic residues of XPC have been identified that lead us to propose a novel binding motif for centrin.Human centrin-2 (HsCen-2) 2 is a Ca 2ϩ -binding protein of the calmodulin-parvalbumin EF-hand superfamily (2, 3). HsCen-2 and two other human centrins (4) are best known for functions outside the nucleus. Centrins have essential roles in the duplication and segregation of microtubule organizing centers (5, 6). Much research has focused on these functions, because abnormal centrosome duplication may lead to chromosomal instability and then cancer, an idea supported by discovery of supernumerary abnormal centrosomes in different human tumor cells (7-10). In addition to its role in centrosome function, HsCen-2 is found as a stabilizing component of xeroderma pigmentosum complement protein C (XPC) and HRad23B complexes (11,12). The XPCcontaining heterotrimer is involved in recognition of DNA lesions and initiation of global genome nucleotide excision repair. Global genome nucleotide excision repair is an important DNA repair pathway for damage caused by UV radiation, carcinogens, and chemotherapeutic agents, and impairment of XPC function is associated with the genetic disorder xeroderma pigmentosum. HsCen-2 appears to promote DNA binding by XPC both in vivo and in vitro and increases the specificity of the heterotrimer for damaged DNA (12, 13). The mechanism by which HsCen-2 binds to XPC is not understood.Centrins are also involved in cilia function (14). Moreover, Ca 2ϩ -triggered assembly of HsCen-1 and transducin appears to regulate transducin translocation through the connecting cilium of vertebrate photoreceptors (15)(16)(17)(18). Whereas the first centrin, also known as caltractin (Cdc31p), was found in fibers linking the flagellar apparatus to the nuclei of Tetraselmis striata green alga (19), homologs have since been identified in many organisms, including fungi, plants, and higher eukaryotes. Functional similarities between centrins from various species seem likely. For example, Ca 2ϩ -induced contractions of a fiber formed by caltractin and Sfi1p are thought to play ...

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C-Terminal Half of Human Centrin 2 Behaves like a Regulatory EF-Hand Domain

Cited by 69 publications

References 38 publications

Metal-binding properties of human centrin-2 determined by micro-electrospray ionization mass spectrometry and UV spectroscopy

Metal-binding properties of human centrin-2 determined by micro-electrospray ionization mass spectrometry and UV spectroscopy

Centrin 2 Stimulates Nucleotide Excision Repair by Interacting with Xeroderma Pigmentosum Group C Protein

The Structure of the Human Centrin 2-Xeroderma Pigmentosum Group C Protein Complex

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