Structural, thermodynamic, and cellular characterization of human centrin 2 interaction with xeroderma pigmentosum group C protein

Human centrin 2 (HsCen2), an EF-hand calcium binding protein, plays a regulatory role in the DNA damage recognition during the first steps of the nucleotide excision repair. This biological action is mediated by the binding to a short fragment (N847-R863) from the C-terminal region of xeroderma pigmentosum group C (XPC) protein. This work presents a detailed structural and energetic characterization of the HsCen2/XPC interaction. Using a truncated form of HsCen2 we obtained a high resolution (1.8 A) X-ray structure of the complex with the peptide N847-R863 from XPC. Structural and thermodynamic analysis of the interface revealed the existence of both electrostatic and apolar inter-molecular interactions, but the binding energy is mainly determined by the burial of apolar bulky side-chains into the hydrophobic pocket of the HsCen2 C-terminal domain. Binding studies with various peptide variants showed that XPC residues W848 and L851 constitute the critical anchoring side-chains. This enabled us to define a minimal centrin binding peptide variant of five residues, which accounts for about 75% of the total free energy of interaction between the two proteins. Immunofluorescence imaging in HeLa cells demonstrated that HsCen2 binding to the integral XPC protein may be observed in living cells, and is determined by the same interface residues identified in the X-ray structure of the complex. Overexpression of XPC perturbs the cellular distribution of HsCen2, by inducing a translocation of centrin molecules from the cytoplasm to the nucleus. The present data confirm that the in vitro structural features of the centrin/XPC peptide complex are highly relevant to the cellular context.     

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.     

Centrin: its secondary structure in the presence and absence of cations

Centrin is a low molecular mass (20 kDa) protein that belongs to the EF-hand superfamily of calcium-binding proteins. Local and overall changes were investigated for interactions between cations and Chlamydomonas centrin using Fourier transform infrared (FT-IR) and circular dichroic (CD) spectroscopies. FT-IR spectral features studied included the amide I' band and the side-chain absorbances for aspartate residues located almost exclusively at the calcium-binding sites in the spectral region of 1700-1500 cm(-1). The amide I' band is exquisitely sensitive to changes in protein secondary structure and is observed to shift from 1626.5 to 1642.7 cm(-1) in the presence and absence of calcium. These spectral bands are complex and were further studied using two-dimensional Fourier transform infrared (2D-FT-IR) correlation along with curve-fitting routines. Using these methods the secondary structure contributions were determined for holocentrin and apocentrin. The alpha-helical content in centrin was determined to be 60%-53% in the presence and absence of cations, respectively. Furthermore, the beta-strand content was determined to be 12%-36%, while the random coil component remained almost constant at 7%-13.5% in the presence and absence of cations, respectively. Changes in the side-chain band are mostly due to the monodentate coordination of aspartate to the cation. A shift of approximately 4 cm(-1) (for the COO- antisymmetric stretch in Asp) from 1565 to 1569 cm(-1) is observed for apocentrin and holocentrin, respectively. Thermal dependence revealed reversible conformational transition temperatures for apocentrin at 37 degrees C and holocentrin at 45 degrees C, suggesting greater stability for holocentrin.     

Binding Proteins from Animals with Possible Transport Function

Several proteins from various animal tissues with possible transport function have been briefly described, with emphasis given to a vitamin D-induced calcium-binding protein (CaBP) implicated in calcium translocation across epithelial membranes. The latter protein was shown to be present in the small intestine, colon, kidney, and the uterus (shell gland) of the chicken. CaBP was also found in the small intestine of the rat, dog, bovine, and monkey. This protein has been isolated in high purity from chick intestinal mucosa and some of its properties determined. Its molecular weight is about 28,000, its formation constant, about 2.6 x 10⁵ M^-1, and its binding capacity, 1 calcium atom per protein molecule. Correlative studies have shown that CaBP concentration in intestinal mucosa varies with the calcium absorptive capacity of the gut, thereby suggesting that CaBP is intimately involved in the process of calcium absorption. CaBP has been localized in the brush border region of the intestinal absorptive cell and within goblet cells. Among other proteins mentioned were the intrinsic factor required for vitamin B₁₂ absorption and the protein(s) associated with iron translocation.     

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.     

Interaction of two brain annexins, CaBP33 and CaBP37, with membrane-skeleton proteins

CaPB33 and CaPB37, two annexins purified from bovine brain, interact with a Triton X-100-resistant fraction (cytoskeleton) from bovine brain membranes in a Ca2(+)-dependent way in vitro. The binding is saturable with respect to the CaBP33-CaBP37 concentration, half-maximal binding occurring at approximately 15 micrograms of the CaBP33-CaBP37 mixture/ml. The binding of these two annexins to the crude cytoskeleton preparation as a function of free Ca2+ concentration is biphasic, with half-maximal binding at approximately 50 microM and approximately 400 microM free Ca2+ for the first and the second component, respectively. By an overlay technique, CaBP33 and CaBP37 bind to a set of low Mr polypeptides (10-20 kDa) in the crude cytoskeleton preparation, with formation of an 85-90 kDa complex as investigated in cross-linking experiments. No binding of the CaBP33-CaBP37 mixture to either G- or F-actin has been observed. Identification of the CaBP33-CaBP37-binding proteins in cytoskeletons would help elucidating the function(s) of these annexins in the brain.     

The carboxy-terminal domain of xeroderma pigmentosum complementation group C protein, involved in TFIIH and centrin binding, is highly disordered

Xeroderma pigmentousum group C protein (XPC) is involved in the first step of nucleotide excision repair, with multiple functional roles including DNA damage recognition and recruitment of the repair machinery. This human protein of 940 residues forms a strong heterotrimeric complex with Rad23B and centrin 2. The structure of XPC is actually not known, and lack of significant sequence homology with proteins from structural data bases precludes any relevant prediction. Here, we present the molecular and structural characterization of a C-terminal fragment of XPC (C-XPC: 126 residues, 815-940), which was shown to be involved in centrin 2 and TFIIH binding. C-XPC may be highly expressed in E. coli, but because of its limited solubility it was purified under 6 M urea. Using bioinformatics tools, and a combination of several experimental methods (circular dichroism, fluorescence, nuclear magnetic resonance, and small-angle X-ray scattering), we show that C-XPC has a highly flexible structure under native physiological conditions, with a propensity to form helical secondary structures. Isothermal titration calorimetry experiments show that the C-XPC fragment binds human centrin 2 with high affinity and a 1:1 stoichiometry. NMR analysis indicates that the physical interaction between C-XPC and centrin 2 induces only minor conformational changes into XPC, localized around the 17-mer segment (847-863), showed to be critically involved in the centrin binding.     

Purification and characterization of a novel 12,000-Da calcium binding protein from smooth muscle.

A new low molecular weight calcium binding protein, designated 12-kDa CaBP, has been isolated from chicken gizzard using a phenyl-Sepharose affinity column followed by ion-exchange and gel filtration chromatographies. The isolated protein was homogeneous and has a molecular weight of 12,000 based on sodium dodecyl sulfate-gel electrophoresis. The amino acid composition of this protein is similar to but distinct from other known low molecular weight Ca2+ binding proteins. Ca2+ binding assays using Arsenazo III (Sigma) indicated that the protein binds 1 mol of Ca2+/mol of protein. The 12-kDa CaBP underwent a conformational change upon binding Ca2+, as revealed by uv difference spectroscopy and circular dichroism studies in the aromatic and far-ultraviolet range. Addition of Ca2+ to the 12-kDa CaBP labeled with 2-p-toluidinylnaphthalene-6-sulfonate (TNS) resulted in a sevenfold increase in fluorescence intensity, accompanied by a blue shift of the emission maximum from 463 to 445 nm. Hence, the probe in the presence of Ca2+ moves to a more nonpolar microenvironment. Like calmodulin and other related Ca2+ binding proteins, this protein also exposes a hydrophobic site upon binding calcium. Fluorescence titration with Ca2+ using TNS-labeled protein revealed the presence of a single high affinity calcium binding site (kd approximately 1 x 10(-6) M).     

Lead-binding properties of intestinal calcium-binding proteins

The bovine and chick vitamin D-induced intestinal calcium-binding proteins (CaBP) bind lead. Bovine CaBP binds 2 atoms of lead/molecule, and chick CaBP binds 4 atoms of lead per molecule and these values are identical to those for calcium binding. 45Calcium-displacement studies indicate significantly higher affinities for lead than for calcium for both proteins. All evidence indicates that lead is bound to the 4 high affinity calcium-binding sites on chick CaBP and to the corresponding 2 high affinity sites on bovine CaBP, and that binding of lead to sulfhydryl groups is, relatively, not significant. Calmodulin, troponin C, and oncomodulin also bind lead with high affinities and in preference to calcium, indicating that lead binding is a general property of proteins belonging to the troponin C superfamily of calcium-binding proteins.     

Structural and thermodynamic studies of two centrin isoforms from Blastocladiella emersonii upon calcium binding

Centrins are calcium-binding proteins associated with microtubules organizing centers. Members of two divergent subfamilies of centrins were found in the aquatic fungus Blastocladiella emersonii, contrasting with the occurrence of only one member known for the better explored terrestrial fungi. BeCen1 shows greatest identity with human centrins HsCen1, HsCen2 and green algae centrin CrCenp, while BeCen3 records largest identity with human centrin HsCen3 and yeast centrin Cdc31p. Following the discovery of this unique feature, BeCen1 and BeCen3 centrins were produced to study whether these proteins had distinct features upon calcium binding. Circular dichroism showed opposite calcium binding effects on the α-helix arrangement of the secondary structure. The spectra indicated a decrease in α-helix signal for holo-BeCen1 contrasting with an increase for holo-BeCen3. In addition, only BeCen1 refolds after being de-natured. The fluorescence emission of the hydrophobic probe ANS increases for both proteins likely due to hydrophobic exposure, however, only BeCen1 presents a clear blue shift when calcium is added. ITC experiments identified four calcium binding sites for both proteins. In contrast to calcium binding to BeCen1, which is mainly endothermic, binding to BeCen3 is mainly exothermic. Light-scattering evidenced the formation of large particles in solution for BeCen1 and BeCen3 at temperatures above 30 °C and 40 °C, respectively. Atomic force microscopy confirmed the presence of supramolecular structures, which differ in the compactness and branching degree. Binding of calcium leads to different structural changes in BeCen1 and BeCen3 and the thermodynamic characteristics of the interaction also differ.     

Centrin 2 stimulates nucleotide excision repair by interacting with xeroderma pigmentosum group C protein

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 alpha-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.     

Structural adaptation of extreme halophilic proteins through decrease of conserved hydrophobic contact surface

Background

Disrupting protein-protein interactions by small organic molecules is nowadays a promising strategy employed to block protein targets involved in different pathologies. However, structural changes occurring at the binding interfaces make difficult drug discovery processes using structure-based drug design/virtual screening approaches. Here we focused on two homologous calcium binding proteins, calmodulin and human centrin 2, involved in different cellular functions via protein-protein interactions, and known to undergo important conformational changes upon ligand binding.

Results

In order to find suitable protein conformations of calmodulin and centrin for further structure-based drug design/virtual screening, we performed in silico structural/energetic analysis and molecular docking of terphenyl (a mimicking alpha-helical molecule known to inhibit protein-protein interactions of calmodulin) into X-ray and NMR ensembles of calmodulin and centrin. We employed several scoring methods in order to find the best protein conformations. Our results show that docking on NMR structures of calmodulin and centrin can be very helpful to take into account conformational changes occurring at protein-protein interfaces.

Conclusions

NMR structures of protein-protein complexes nowadays available could efficiently be exploited for further structure-based drug design/virtual screening processes employed to design small molecule inhibitors of protein-protein interactions.     

Identification of two intestinal vitamin D-dependent calcium-binding proteins in the X-linked hypophosphataemic mouse

Two vitamin D-dependent calcium binding proteins (CaBP) of molecular weight approximately 10,000 and 30,000 daltons have been identified in the intestinal mucosal cell cytosol of genetically hypophosphataemic (Hyp) male mice and their normal littermates. Similar amounts of vitamin D-dependent CaBP were found in the two groups of animals. The possible significance of this observation is discussed.     

Interaction of S100A13 with C2 domain of receptor for advanced glycation end products (RAGE)

S100A13 is involved in several key biological functions like angiogenesis, tumor formation and cell apoptosis. It is a homodimeric protein that belongs to the S100 protein family. S100A13 is co-expressed with acidic fibroblast growth factor (FGF1) and interleukin-1α which are key angiogenesis inducers. The S100 proteins have been shown to be involved in several cellular functions such as calcium homeostasis, cell growth and differentiation dynamic of cytoskeleton. Its biological functions are mainly mediated through the receptor for advanced glycation end products (RAGE) signaling. RAGE is involved in inflammatory processes and is associated with diabetic complications, tumor outgrowth, and neurodegenerative disorders. RAGE induces cellular signaling upon binding of different ligands, such as S100 proteins, glycated proteins, and HMGB1. RAGE signaling is complex, and it depends on the cell type and concentration of the ligand. Molecular level interactions of RAGE and S100 proteins are useful to understand the RAGE signaling diversity. In this report we focus on the molecular level interactions of S100A13 and RAGE C2 domain. The binding between RAGE C2 and S100A13 is moderately strong (K_d ~ 1.3 μM). We have solved the solution structure of the S100A13–RAGE C2 complex and pronounce the interface regions in S100A13–RAGE C2 complex which are helpful for drug development of RAGE induced diseases.     

Expression of Calcium Binding Proteins in Mouse Type II Taste Cells

It is well established that calcium is a critical signaling molecule in the transduction of taste stimuli within the peripheral taste system. However, little is known about the regulation and termination of these calcium signals in the taste system. The authors used Western blot, immunocytochemical, and RT-PCR analyses to evaluate the expression of multiple calcium binding proteins in mouse circumvallate taste papillae, including parvalbumin, calbindin D28k, calretinin, neurocalcin, NCS-1 (or frequenin), and CaBP. They found that all of the calcium binding proteins they tested were expressed in mouse circumvallate taste cells with the exception of NCS-1. The authors correlated the expression patterns of these calcium binding proteins with a marker for type II cells and found that neurocalcin was expressed in 80% of type II cells, whereas parvalbumin was found in less than 10% of the type II cells. Calretinin, calbindin, and CaBP were expressed in about half of the type II cells. These data reveal that multiple calcium binding proteins are highly expressed in taste cells and have distinct expression patterns that likely reflect their different roles within taste receptor cells.     

Cloning and expression pattern of EPAS1 in the chicken embryo. Colocalization with tyrosine hydroxylase

The thermodynamics of interaction of two model peptides melittin and mastoparan with bovine brain calmodulin (CAM) and a smaller CAM analogue, a calcium binding protein from Entamoeba histolytica (CaBP) in 10 mM MOPS buffer (pH 7.0) was examined using isothermal titration calorimetry (ITC). These data show that CAM binds to both the peptides and the enthalpy of binding is endothermic for melittin and exothermic for mastoparan at 25 degrees C. CaBP binds to the longer peptide melittin, but does not bind to mastoparan, the binding enthalpy being endothermic in nature. Concurrently, we also observe a larger increase in alpha-helicity upon the binding of melittin to CAM when compared to CaBP. The role of hydrophobic interactions in the binding process has also been examined using 8-anilino-1-naphthalene-sulphonic acid (ANS) binding monitored by ITC. These results have been employed to rationalize the energetic consequences of the binding reaction.     

Mapping the transition state for a binding reaction between ancient intrinsically disordered proteins

Intrinsically disordered protein domains often have multiple binding partners. It is plausible that the strength of pairing with specific partners evolves from an initial low affinity to a higher affinity. However, little is known about the molecular changes in the binding mechanism that would facilitate such a transition. We previously showed that the interaction between two intrinsically disordered domains, NCBD and CID, likely emerged in an ancestral deuterostome organism as a low-affinity interaction that subsequently evolved into a higher-affinity interaction before the radiation of modern vertebrate groups. Here we map native contacts in the transition states of the low-affinity ancestral and high-affinity human NCBD/CID interactions. We show that the coupled binding and folding mechanism is overall similar but with a higher degree of native hydrophobic contact formation in the transition state of the ancestral complex and more heterogeneous transient interactions, including electrostatic pairings, and an increased disorder for the human complex. Adaptation to new binding partners may be facilitated by this ability to exploit multiple alternative transient interactions while retaining the overall binding and folding pathway.     

An EF-hands protein, centrin-1, is an EGTA-sensitive SUMO-interacting protein in mouse testis

A multifunctional calcium‐binding protein, centrin‐1, is specifically expressed in male germ cells, certain neurons and ciliated cells. We identified centrin‐1 as a protein interacting with SUMO‐2/3 using yeast two‐hybrid screening of a mouse testicular cDNA library. In bead halo assays, the interaction between centrin‐1 and SUMO‐2/3 was reduced in the presence of EGTA and facilitated by the addition of CaCl_2. immunostaining of seminiferous tubules in 35‐day‐old mouse testes revealed that cells in the layer containing spermatogonia showed colocalization of SUMO‐2/3 with centrin‐1 in cytoplasmic spots. Identification of centrin‐1 as the EGTA‐sensitive SUMO‐2/3‐interacting protein indicates the possible role of calcium in modulating the centrin‐1–SUMO‐2/3 interaction and suggests the importance of this interaction in mouse testis. Copyright © 2010 John Wiley & Sons, Ltd.     

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.     

Caldendrin, a neuron-specific modulator of Cav/1.2 (L-type) Ca2+ channels

EF-hand Ca2+-binding proteins such as calmodulin and CaBP1 have emerged as important regulatory subunits of voltage-gated Ca2+ channels. Here, we show that caldendrin, a variant of CaBP1 enriched in the brain, interacts with and distinctly modulates Cav1.2 (L-type) voltage-gated Ca2+ channels relative to other Ca2+-binding proteins. Caldendrin binds to the C-terminal IQ-domain of the pore-forming alpha1-subunit of Cav1.2 (alpha(1)1.2) and competitively displaces calmodulin and CaBP1 from this site. Compared with CaBP1, caldendrin causes a more modest suppression of Ca2+-dependent inactivation of Cav1.2 through a different subset of molecular determinants. Caldendrin does not bind to the N-terminal domain of alpha11.2, a site that is critical for functional interactions of the channel with CaBP1. Deletion of the N-terminal domain inhibits CaBP1, but spares caldendrin modulation of Cav1.2 inactivation. In contrast, mutations of the IQ-domain abolish physical and functional interactions of caldendrin and Cav1.2, but do not prevent channel modulation by CaBP1. Using antibodies specific for caldendrin and Cav1.2, we show that caldendrin coimmunoprecipitates with Cav1.2 from the brain and colocalizes with Cav1.2 in somatodendritic puncta of cortical neurons in culture. Our findings reveal functional diversity within related Ca2+-binding proteins, which may enhance the specificity of Ca2+ signaling by Cav1.2 channels in different cellular contexts.