Structural Independence of the Two EF-hand Domains of Caltractin

Veeraraghavan, Sudha; Fagan, Patricia A.; Hu, Haitao; Lee, Vincent; Harper, Jeffrey F.; Huang, Bessie; Chazin, Walter J.

doi:10.1074/jbc.m112232200

Cited by 56 publications

(83 citation statements)

References 30 publications

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“…Additional Ca 2ϩ binding sites were of low affinity. Other centrins for which Ca 2ϩ -binding was reported include Chlamydomonas centrin, which binds four moles of calcium/mol protein [22]. No data were previously reported concerning the Mg 2ϩ -binding stoichiometry of HsCen-2 or Chlamydomonas centrin other than to note that the uptake of Mg 2ϩ reduces the affinity of calcium in both proteins [19,80].…”

Section: Spectroscopymentioning

confidence: 95%

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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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Section: Spectroscopymentioning

confidence: 95%

“…Studies of an N-terminal 25 amino acid truncation of HsCen-2 suggest that the N-terminus is involved in the calcium-dependent dimerization of the protein [21]. On the other hand, Veeraraghavan et al showed that Chlamydomonas centrin-2 binds 4 mol of calcium/mol protein [22].…”

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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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“…HsCen-2 Metal Binding and EF-hands-The affinities of Ca 2ϩ -binding sites within HsCen-2 and other centrins are lower than for calmodulin (43,(47)(48)(49) Table 2 herein. 27-fold higher affinity for the XPC peptide used herein compared with Ca 2ϩ -free centrin-2 (11).…”

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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confidence: 99%

“…More recently, novel centrin functions have been discovered such as involvement in recombinational DNA repair through modulation of the nucleotide excision repair pathway in humans and plants (2, 47), a role in the nuclear mRNA export machinery in yeast (11), regulation of calcium-dependent ciliary activities in ciliates (14, 15), and signal transduction in vertebrate photoreceptors (53). How centrin exerts these different functions at the molecular level is, however, still unknown although considerable progress has recently been made through in vitro studies of centrin and its interaction with target peptides (8,19,20,40,75).In some organisms, the functional diversity of centrin may be related to the presence of different centrin isotypes, e.g., four centrin proteins (centrin1p to centrin4p), which are differentially expressed in different tissues and cell types, have been identified so far in mammals (10,12,29,34,35,45). In the ciliate Paramecium, three centrin genes have been characterized to date (38) and in another ciliate (Paraurostyla) evidence has been presented for differential localization of two centrin isotypes (36).…”

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confidence: 99%

Centrin Scaffold in Chlamydomonas reinhardtii Revealed by Immunoelectron Microscopy

Geimer

Melkonian

2005

Eukaryot Cell

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In the flagellate green alga Chlamydomonas reinhardtii the Ca 2؉ -binding EF-hand protein centrin is encoded by a single-copy gene. Previous studies have localized the protein to four distinct structures in the flagellar apparatus: the nucleus-basal body connector, the distal connecting fiber, the flagellar transitional region, and the axoneme. To explain the disjunctive distribution of centrin, the interaction of centrin with as yet unknown specific centrin-binding proteins has been implied. Here, we demonstrate using serial section postembedding immunoelectron microscopy of isolated cytoskeletons that centrin is located in additional structures (transitional fibers and basal body lumen) and that the centrin-containing structures of the basal apparatus are likely part of a continuous filamentous scaffold that extends from the nucleus to the flagellar bases. In addition, we show that centrin is located in the distal lumen of the basal body in a rotationally asymmetric structure, the V-shaped filament system. This novel centrin-containing structure has also been detected near the distal end of the probasal bodies. Taken together, these results suggest a role for a rotationally asymmetric centrin "seed" in the growth and development of the centrin scaffold following replication of the basal apparatus.Centrin (also known as caltractin) is a member of the highly conserved superfamily of calcium-binding EF-hand proteins and occurs in all eukaryotic cells, being mostly associated with centrosomes (58, 64). It was first discovered in flagellate green algae, where it is located in the basal apparatus (the part of the cytoskeleton anchoring the flagella) and involved in the contraction of calcium-sensitive filaments (42, 60) which, in general, play an important role in the dynamic behavior of centrosomes by controlling the position and orientation of centrosomal structures (58, 64). Analysis of centrin mutants and RNA interference strains deficient in centrin in the green algal model system Chlamydomonas reinhardtii suggested that centrin is essential for basal body duplication and segregation during cell division (28,39,73), functions also deduced for centrin homologues in vertebrates (46, 62) and yeast spp. (3,50,69). More recently, novel centrin functions have been discovered such as involvement in recombinational DNA repair through modulation of the nucleotide excision repair pathway in humans and plants (2, 47), a role in the nuclear mRNA export machinery in yeast (11), regulation of calcium-dependent ciliary activities in ciliates (14, 15), and signal transduction in vertebrate photoreceptors (53). How centrin exerts these different functions at the molecular level is, however, still unknown although considerable progress has recently been made through in vitro studies of centrin and its interaction with target peptides (8,19,20,40,75).In some organisms, the functional diversity of centrin may be related to the presence of different centrin isotypes, e.g., four centrin proteins (centrin1p to centrin4p), which are d...

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Structural Independence of the Two EF-hand Domains of Caltractin

Abstract: Caltractin (centrin) is a member of the calmodulin subfamily of EF-hand

Cited by 56 publications

References 30 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

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

Centrin Scaffold in Chlamydomonas reinhardtii Revealed by Immunoelectron Microscopy

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