Binding of human centrin 2 to the centrosomal protein hSfi1

hSfi1, a human centrosomal protein with homologs in other eukaryotic organisms, includes 23 repeats, each of 23 amino acids, separated by 10 residue linkers. The main molecular partner in the centrosome is a small, calcium-binding EF-hand protein, the human centrin 2. Using isothermal titration calorimetry experiments, we characterized the centrin-binding capacity of three isolated hSfi1 repeats, two exhibiting the general consensus motif and the third being the unique Pro-containing human repeat. The two standard peptides bind human centrin 2 and its isolated C-terminal domain with high affinity (approximately 10(7) M(-1)) by an enthalpy-driven mechanism, with a moderate Ca2+ dependence. The Pro-containing repeat shows a binding affinity that is two orders of magnitude lower. The target binding site is localized within the C-terminal domain of human centrin 2. Fluorescence titration and NMR spectroscopy show that the well-conserved Trp residue situated in the C-terminus of each repeat is deeply embedded in a protein hydrophobic cavity, indicating that the peptide direction is reversed relative to previously studied centrin targets. The present results suggest that almost all of the repeats of the Sfi1 protein may independently bind centrin molecules. On the basis of this hypothesis and previous studies on centrin self-assembly, we propose a working model for the role of centrin-Sfi1 interactions in the dynamic structure of centrosome-associated contractile fibers.     

Sfi1p has conserved centrin-binding sites and an essential function in budding yeast spindle pole body duplication

Centrins are calmodulin-like proteins present in microtubule-organizing centers. The Saccharomyces cerevisiae centrin, Cdc31p, was functionally tagged with a single Z domain of protein A, and used in pull-down experiments to isolate Cdc31p-binding proteins. One of these, Sfi1p, localizes to the half-bridge of the spindle pole body (SPB), where Cdc31p is also localized. Temperature-sensitive mutants in SFI1 show a defect in SPB duplication and genetic interactions with cdc31-1. Sfi1p contains multiple internal repeats that are also present in a Schizosaccharomyces pombe protein, which also localizes to the SPB, and in several human proteins, one of which localizes close to the centriole region. Cdc31p binds directly to individual Sfi1 repeats in a 1:1 ratio, so a single molecule of Sfi1p binds multiple molecules of Cdc31p. The centrosomal human protein containing Sfi1 repeats also binds centrin in the repeat region, showing that this centrin-binding motif is conserved.     

An Sfi1p-like centrin-binding protein mediates centrin-based Ca2+ -dependent contractility in Paramecium tetraurelia

The previous characterization and structural analyses of Sfi1p, a Saccharomyces cerevisiae centrin-binding protein essential for spindle pole body duplication, have suggested molecular models to account for centrin-mediated, Ca2+-dependent contractility processes (S. Li, A. M. Sandercock, P. Conduit, C. V. Robinson, R. L. Williams, and J. V. Kilmartin, J. Cell Biol. 173:867-877, 2006). Such processes can be analyzed by using Paramecium tetraurelia, which harbors a large Ca2+ -dependent contractile cytoskeletal network, the infraciliary lattice (ICL). Previous biochemical and genetic studies have shown that the ICL is composed of diverse centrin isoforms and a high-molecular-mass centrin-associated protein, whose reduced size in the démaillé (dem1) mutant correlates with defective organization of the ICL. Using sequences derived from the high-molecular-mass protein to probe the Paramecium genome sequence, we characterized the PtCenBP1 gene, which encodes a 460-kDa protein. PtCenBP1p displays six almost perfect repeats of ca. 427 amino acids (aa) and harbors 89 potential centrin-binding sites with the consensus motif LLX11F/LX2WK/R, similar to the centrin-binding sites of ScSfi1p. The smaller (260-kDa) protein encoded by the dem1 mutant PtCenBP1 allele comprises only two repeats of 427 aa and 46 centrin-binding sites. By using RNA interference and green fluorescent protein fusion experiments, we showed that PtCenBP1p forms the backbone of the ICL and plays an essential role in its assembly and contractility. This study provides the first in vivo demonstration of the role of Sfi1p-like proteins in centrin-mediated Ca2+-dependent contractile processes.     

Kar1 binding to Sfi1 C-terminal regions anchors the SPB bridge to the nuclear envelope

The yeast spindle pole body (SPB) is the functional equivalent of the mammalian centrosome. The half bridge is a SPB substructure on the nuclear envelope (NE), playing a key role in SPB duplication. Its cytoplasmic components are the membrane-anchored Kar1, the yeast centrin Cdc31, and the Cdc31-binding protein Sfi1. In G1, the half bridge expands into the bridge through Sfi1 C-terminal (Sfi1-CT) dimerization, the licensing step for SPB duplication. We exploited photo-activated localization microscopy (PALM) to show that Kar1 localizes in the bridge center. Binding assays revealed direct interaction between Kar1 and C-terminal Sfi1 fragments. kar1Δ cells whose viability was maintained by the dominant CDC31-16 showed an arched bridge, indicating Kar1’s function in tethering Sfi1 to the NE. Cdc31-16 enhanced Cdc31–Cdc31 interactions between Sfi1–Cdc31 layers, as suggested by binding free energy calculations. In our model, Kar1 binding is restricted to Sfi1-CT and Sfi1 C-terminal centrin-binding repeats, and centrin and Kar1 provide cross-links, while Sfi1-CT stabilizes the bridge and ensures timely SPB separation.     

Structural role of Sfi1p-centrin filaments in budding yeast spindle pole body duplication

Centrins are calmodulin-like proteins present in centrosomes and yeast spindle pole bodies (SPBs) and have essential functions in their duplication. The Saccharomyces cerevisiae centrin, Cdc31p, binds Sfi1p on multiple conserved repeats; both proteins localize to the SPB half-bridge, where the new SPB is assembled. The crystal structures of Sfi1p-centrin complexes containing several repeats show Sfi1p as an alpha helix with centrins wrapped around each repeat and similar centrin-centrin contacts between each repeat. Electron microscopy (EM) shadowing of an Sfi1p-centrin complex with 15 Sfi1 repeats and 15 centrins bound showed filaments 60 nm long, compatible with all the Sfi1 repeats as a continuous alpha helix. Immuno-EM localization of the Sfi1p N and C termini showed Sfi1p-centrin filaments spanning the length of the half-bridge with the Sfi1p N terminus at the SPB. This suggests a model for SPB duplication where the half-bridge doubles in length by association of the Sfi1p C termini, thereby providing a new Sfi1p N terminus to initiate SPB assembly.     

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.     

The mode of action of centrin. Binding of Ca2+ and a peptide fragment of Kar1p to the C-terminal domain

Centrin is an EF-hand calcium-binding protein closely related to the prototypical calcium sensor protein calmodulin. It is found in microtubule-organizing centers of organisms ranging from algae and yeast to man. In vitro, the C-terminal domain of centrin binds to the yeast centrosomal protein Kar1p in a calcium-dependent manner, whereas the N-terminal domain does not show any appreciable affinity for Kar1p. To obtain deeper insights into the structural basis for centrin's function, we have characterized the affinities of the C-terminal domain of Chlamydomonas reinhardtii centrin for calcium and for a peptide fragment of Kar1p using CD, fluorescence, and NMR spectroscopy. Calcium binding site IV in C. reinhardtii centrin was found to bind Ca2+ approximately 100-fold more strongly than site III. In the absence of Ca2+, the protein occupies a mixture of closed conformations. Binding of a single ion in site IV is sufficient to radically alter the conformational equilibrium, promoting occupancy of an open conformation. However, an exchange between closed and open conformations remains even at saturating levels of Ca2+. The population of the open conformation is substantially stabilized by the presence of the target peptide Kar1p-(239-257) to a point where a single ion bound in site IV is sufficient to completely shift the conformational equilibrium to the open conformation. This is reflected in the enhancement of the Ca2+ affinity in this site by more than an order of magnitude. These data confirm the direct coupling of the Ca2+ binding-induced shift in the equilibrium between the closed and open conformations to the binding of the peptide. Combined with the common localization of the two proteins in the microtubule organizing center, our results suggest that centrin is constitutively bound to Kar1p through its C-terminal domain and that centrin's calcium sensor activities are mediated by the N-terminal domain.     

Structure, Dynamics and Thermodynamics of the Human Centrin 2/hSfi1 Complex

Centrin, an EF-hand calcium-binding protein, has been shown to be involved in the duplication of centrosomes, and Sfi1 (Suppressor of fermentation-induced loss of stress resistance protein 1) is one of its centrosomal targets. There are three isoforms of human centrin, but here we only considered centrin 2 (HsCen2). This protein has the ability to bind to any of the ∼ 25 repeats of human Sfi1 (hSfi1) with more or less affinity. In this study, we mainly focused on the 17th repeat (R17-hSfi1-20), which presents the highest level of similarity with a well-studied 17-residue peptide (P17-XPC) from human xeroderma pigmentosum complementation group C protein, another centrin target for DNA repair. The only known structure of HsCen2 was resolved in complex with P17-XPC. The 20-residue peptide R17-hSfi1-20 exhibits the motif L8L4W1, which is the reverse of the XPC motif, W1L4L8. Consequently, the dipole of the helix formed by this motif has a reverse orientation. We wished to ascertain the impact of this reversal on the structure, dynamics and affinity of centrin. To address this question, we determined the structure of C-HsCen2 [the C-terminal domain of HsCen2 (T94-Y172)] in complex with R17-hSfi1-20 and monitored its dynamics by NMR, after having verified that the N-terminal domain of HsCen2 does not interact with the peptide. The structure shows that the binding mode is similar to that of P17-XPC. However, we observed a 2 -Å translation of the R17-hSfi1-20 helix along its axis, inducing less anchorage in the protein and the disruption of a hydrogen bond between a tryptophan residue in the peptide and a well-conserved nearby glutamate in C-HsCen2. NMR dynamic studies of the complex strongly suggested the existence of an unusual calcium secondary binding mode in calcium-binding loop III, made possible by the uncommon residue composition of this loop. The secondary metal site is only populated at high calcium concentration and depends on the type of bound ligand.     

Calcium-dependent assembly of centrin-G-protein complex in photoreceptor cells

Photoexcitation of rhodopsin activates a heterotrimeric G-protein cascade leading to cyclic GMP hydrolysis in vertebrate photoreceptors. Light-induced exchanges of the visual G-protein transducin between the outer and inner segment of rod photoreceptors occur through the narrow connecting cilium. Here we demonstrate that transducin colocalizes with the Ca(2+)-binding protein centrin 1 in a specific domain of this cilium. Coimmunoprecipitation, centrifugation, centrin overlay, size exclusion chromatography, and kinetic light-scattering experiments indicate that Ca(2+)-activated centrin 1 binds with high affinity and specificity to transducin. The assembly of centrin-G-protein complex is mediated by the betagamma-complex. The Ca(2+)-dependent assembly of a G protein with centrin is a novel aspect of the supply of signaling proteins in sensory cells and a potential link between molecular translocations and signal transduction in general.     

Cloning,localization, and axonemal function of Tetrahymena centrin

Centrin, an EF hand Ca(2+) binding protein, has been cloned in Tetrahymena thermophila. It is a 167 amino acid protein of 19.4 kDa with a unique N-terminal region, coded by a single gene containing an 85-base pair intron. It has > 80% homology to other centrins and high homology to Tetrahymena EF hand proteins calmodulin, TCBP23, and TCBP25. Specific cellular localizations of the closely related Tetrahymena EF hand proteins are different from centrin. Centrin is localized to basal bodies, cortical fibers in oral apparatus and ciliary rootlets, the apical filament ring and to inner arm (14S) dynein (IAD) along the ciliary axoneme. The function of centrin in Ca(2+) control of IAD activity was explored using in vitro microtubule (MT) motility assays. Ca(2+) or the Ca(2+)-mimicking peptide CALP1, which binds EF hand proteins in the absence of Ca(2+), increased MT sliding velocity. Antibodies to centrin abrogated this increase. This is the first demonstration of a specific centrin function associated with axonemal dynein. It suggests that centrin is a key regulatory protein for Tetrahymena axonemal Ca(2+) responses, including ciliary reversal or chemotaxis.     

Expression of a mutant form of Leishmania donovani centrin reduces the growth of the parasite.

Leishmania donovani, a protozoan parasite, causes visceral disease in humans. To identify genes that control growth, we have isolated for the first time in the order Kinetoplastida a gene encoding for centrin from L. donovani. Centrin is a calcium-binding cytoskeletal protein essential for centrosome duplication or segregation. Protein sequence similarity and immunoreactivity confirmed that Leishmania centrin is a homolog of human centrin 2. Immunofluorescence analysis localized the protein in the basal body. Calcium binding analysis revealed that its C-terminal Ca(2+) binding domain binds 16-fold more calcium than the N-terminal domain. Electrophoretic mobility shift of centrin treated with EGTA and abrogation of the shift in its mutants lacking a Ca(2+) binding site suggest that Ca(2+) binding to these regions may have a role in the protein conformation. The levels of centrin mRNA and protein were high during the exponential growth of the parasite in culture and declined to a low level in the stationary phase. Expression of N-terminal-deleted centrin in the parasite significantly reduces its growth rate, and it was found that significantly more cells are arrested in the G(2)/M stage than in control cells. These studies indicate that centrin may have a functional role in Leishmania growth.     

Novel sfi1 alleles uncover additional functions for Sfi1p in bipolar spindle assembly and function

A variety of spindle and kinetochore defects have been shown to induce a mitotic delay through activation of the spindle checkpoint. With the aim of identifying novel mitotic defects we carried out a mad1 synthetic lethal screen in budding yeast. In this screen, four novel alleles of sfi1 were isolated. SFI1 is an essential gene, previously identified through its interaction with centrin/CDC31 and shown to be required for spindle pole body (SPB) duplication. The new mutations were all found in the C-terminal domain of Sfi1p, which has no known function, but it is well conserved among budding yeasts. Analysis of the novel sfi1 mutants, through a combination of light and electron microscopy, revealed duplicated SPBs <0.3 microm apart. Importantly, these SPBs have completed duplication, but they are not separated, suggesting a possible defect in splitting of the bridge. We discuss possible roles for Sfi1p in this step in bipolar spindle assembly.     

The N-terminal domain of human centrin 2 has a closed structure, binds calcium with a very low affinity, and plays a role in the protein self-assembly

Centrins are well-conserved calcium binding proteins from the EF-hand superfamily implicated in various cellular functions, such as centrosome duplication, DNA repair, and nuclear mRNA export. The intrinsic molecular flexibility and the self-association tendency make difficult the structural characterization of the integral protein. In this paper we report the solution structure, the Ca2+ binding properties, and the intermolecular interactions of the N-terminal domain of two human centrin isoforms, HsCen1 and HsCen2. In the absence of Ca2+, the N-terminal construct of HsCen2 revealed a compact core conformation including four almost antiparallel alpha-helices and a short antiparallel beta-sheet, very similar to the apo state structure of other calcium regulatory EF-hand domains. The first 25 residues show a highly irregular and dynamic structure. The three-dimensional model for the N-terminal domain of HsCen1, based on the high sequence conservation and NMR spectroscopic data, shows very close structural properties. Ca2+ titration of the apo-N-terminal domain of HsCen1 and HsCen2, monitored by NMR spectroscopy, revealed a very weak affinity (10(2)-10(3) M(-1)), suggesting that the cellular role of this domain is not calcium dependent. Isothermal calorimetric titrations showed that an 18-residue peptide, derived from the N-terminal unstructured fragment, has a significant affinity (approximately 10(5) M(-1)) for the isolated C-terminal domain, suggesting an active role in the self-assembly of centrin molecules.     

The two human centrin homologues have similar but distinct functions at Tetrahymena basal bodies

Centrins are a ubiquitous family of small Ca²⁺-binding proteins found at basal bodies that are placed into two groups based on sequence similarity to the human centrins 2 and 3. Analyses of basal body composition in different species suggest that they contain a centrin isoform from each group. We used the ciliate protist Tetrahymena thermophila to gain a better understanding of the functions of the two centrin groups and to determine their potential redundancy. We have previously shown that the Tetrahymena centrin 1 (Cen1), a human centrin 2 homologue, is required for proper basal body function. In this paper, we show that the Tetrahymena centrin 2 (Cen2), a human centrin 3 homologue, has functions similar to Cen1 in basal body orientation, maintenance, and separation. The two are, however, not redundant. A further examination of human centrin 3 homologues shows that they function in a manner distinct from human centrin 2 homologues. Our data suggest that basal bodies require a centrin from both groups in order to function correctly.     

Rapid downregulation of the Ca2+-signal after exocytosis stimulation in Paramecium cells: essential role of a centrin-rich filamentous cortical network, the infraciliary lattice

We analysed in Paramecium tetraurelia cells the role of the infraciliary lattice, a cytoskeletal network containing numerous centrin isoforms tightly bound to large binding proteins, in the re-establishment of Ca2+ homeostasis following exocytosis stimulation. The wild type strain d4-2 has been compared with the mutant cell line Delta-PtCenBP1 which is devoid of the infraciliary lattice ("Delta-PtCenBP1" cells). Exocytosis is known to involve the mobilization of cortical Ca2+-stores and a superimposed Ca2+-influx and was analysed using Fura Red ratio imaging. No difference in the initial signal generation was found between wild type and Delta-PtCenBP1 cells. In contrast, decay time was greatly increased in Delta-PtCenBP1 cells particularly when stimulated, e.g., in presence of 1mM extracellular Ca2+, [Ca2+]o. Apparent halftimes of f/f0 decrease were 8.5 s in wild type and approximately 125 s in Delta-PtCenBP1 cells, requiring approximately 30 s and approximately 180 s, respectively, to re-establish intracellular [Ca2+] homeostasis. Lowering [Ca2+]o to 0.1 and 0.01 mM caused an acceleration of intracellular [Ca2+] decay to t(1/2)=33 s and 28 s, respectively, in Delta-PtCenBP1 cells as compared to 8.1 and 5.6, respectively, for wild type cells. We conclude that, in Paramecium cells, the infraciliary lattice is the most efficient endogenous Ca2+ buffering system allowing the rapid downregulation of Ca2+ signals after exocytosis stimulation.     

Flexibility and plasticity of human centrin 2 binding to the xeroderma pigmentosum group C protein (XPC) from nuclear excision repair

Human centrin 2 is a component of the nucleotide excision repair system, as a subunit of the heterotrimer including xeroderma pigmentosum group C protein (XPC) and hHR23B. The C-terminal domain of centrin (C-HsCen2) binds strongly a peptide from the XPC protein (P1-XPC: N(847)-R(863)). Here, we characterize the solution Ca(2+)-dependent structural and molecular features of the C-HsCen2 in complex with P1-XPC, mainly using NMR spectroscopy and molecular modeling. The N-terminal half of the peptide, organized as an alpha helix is anchored into a deep hydrophobic cavity of the protein, because of three bulky hydrophobic residues in position 1-4-8 and electrostatic contacts with the centrin helix E. Investigation of the whole centrin interactions shows that the N-terminal domain of the protein is not involved in the complex formation and is structurally independent from the peptide-bound C-terminal domain. The complex may exist in three different binding conformations corresponding to zero, one, and two Ca(2+)-bound states, which may exchange with various rates and have distinct structural stability. The various features of the intermolecular interaction presented here constitute a centrin-specific mode for the target binding.     

Structural independence of the two EF-hand domains of caltractin

Caltractin (centrin) is a member of the calmodulin subfamily of EF-hand Ca2+-binding proteins that is an essential component of microtubule-organizing centers in many organisms ranging from yeast and algae to humans. The protein contains two homologous EF-hand Ca2+-binding domains linked by a flexible tether; each domain is capable of binding two Ca2+ ions. In an effort to search for domain-specific functional properties of caltractin, the two isolated domains were subcloned and expressed in Escherichia coli. Ca2+ binding affinities and the Ca2+ dependence of biophysical properties of the isolated domains were monitored by UV, CD, and NMR spectroscopy. Comparisons to the corresponding results for the intact protein showed that the two domains function independently of each other in these assays. Titration of a peptide fragment from the yeast Kar1p protein to the isolated domains and intact caltractin shows that the two domains interact in a Ca2+-dependent manner, with the C-terminal domain binding much more strongly than the N-terminal domain. Measurements of the macroscopic Ca2+ binding constants show that only the N-terminal domain has sufficient apparent Ca2+ affinity in vitro (1-10 microm) to be classified as a traditional calcium sensor in signal transduction pathways. However, investigation of the microscopic Ca2+ binding events in the C-terminal domain by NMR spectroscopy revealed that the observed macroscopic binding constant likely results from binding to two sites with very different affinities, one in the micromolar range and the other in the millimolar range. Thus, the C-terminal domain appears to also be capable of sensing Ca2+ signals but is activated by the binding of a single ion.     

The N-terminus of Sfi1 and yeast centrin Cdc31 provide the assembly site for a new spindle pole body

The spindle pole body (SPB) provides microtubule-organizing functions in yeast and duplicates exactly once per cell cycle. The first step in SPB duplication is the half-bridge to bridge conversion via the antiparallel dimerization of the centrin (Cdc31)-binding protein Sfi1 in anaphase. The bridge, which is anchored to the old SPB on the proximal end, exposes free Sfi1 N-termini (N-Sfi1) at its distal end. These free N-Sfi1 promote in G₁ the assembly of the daughter SPB (dSPB) in a yet unclear manner. This study shows that N-Sfi1 including the first three Cdc31 binding sites interacts with the SPB components Spc29 and Spc42, triggering the assembly of the dSPB. Cdc31 binding to N-Sfi1 promotes Spc29 recruitment and is essential for satellite formation. Furthermore, phosphorylation of N-Sfi1 has an inhibitory effect and delays dSPB biogenesis until G₁. Taking these data together, we provide an understanding of the initial steps in SPB assembly and describe a new function of Cdc31 in the recruitment of dSPB components.     

The two domains of centrin have distinct basal body functions in Tetrahymena

The basal body is a microtubule-organizing center responsible for organizing the cilium, a structure important for cell locomotion and sensing of the surrounding environment. A widely conserved basal body component is the Ca(2+)-binding protein centrin. Analyses of centrin function suggest a role in basal body assembly and stability; however, its molecular mechanisms remain unclear. Here we describe a mutagenic strategy to study the function and essential nature of the various structural features of Cen1 in the ciliate Tetrahymena. We find that the two domains of Cen1 are both essential, and examination of strains containing mutant CEN1 alleles indicates that there are two predominant basal body phenotypes: misorientation of newly assembled basal bodies and stability defects. The results also show that the two domains of Cen1 are able to bind Ca(2+) and that perturbation of Ca(2+) binding affects Cen1 function. In all, the data suggest that the two domains of Cen1 have distinct functions.