Structural, Thermodynamic, and Cellular Characterization of Human Centrin 2 Interaction with Xeroderma Pigmentosum Group C Protein

Charbonnier, Jean‐Baptiste; Renaud, Emilie; Miron, Simona; Du, M.H. Le; Blouquit, Y.; Duchambon, Patricia; Christova, P.; Shosheva, Alexandra; Rose, Thierry; Angulo, Jaime F.; Craescu, Constantin T.

doi:10.1016/j.jmb.2007.08.046

Cited by 56 publications

(68 citation statements)

References 48 publications

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“…Thus, the equilibrium distribution of centrin-2 between the cytoplasm and the nucleus is most probably determined by its binding to interaction partners in the two compartments. This interpretation is supported by the finding that overexpression of the XPC protein leads to enhanced nuclear accumulation of centrin-2 (Charbonnier et al, 2007).…”

Section: Discussionmentioning

confidence: 71%

SUMO-dependent regulation of centrin-2

Klein

Nigg

2009

Journal of Cell Science

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2+-binding proteins that are highly conserved from yeast to humans. Centrin-2 is a core component of the centrosome of higher eukaryotes. In addition, it is present within the nucleus, in which it is part of the xeroderma pigmentosum group C (XPC) complex, which controls nucleotide excision repair (NER). Regulation of the subcellular distribution of centrin-2 has so far remained elusive. Here we show that centrin-2 is a substrate of SUMOylation in vitro and in vivo, and that it is preferentially modified by SUMO2/3. Moreover, we identify the SUMO E3-like ligase human polycomb protein 2 (PC2; also known as hPC2) as essential for centrin-2 modification. Interference with the SUMOylation pathway leads to a striking defect in nuclear localization of centrin-2 and accumulation in the cytoplasm, whereas centrosomal recruitment of centrin-2 is unaffected. Depletion of the XPC protein mimics this situation and we provide evidence that SUMO conjugation of centrin-2 enhances its binding to the XPC protein. These data show that the nucleocytoplasmic shuttling of centrin-2 depends on the SUMO system and indicates that localization of centrin-2 within the nucleus depends on its ability to bind to the XPC protein.Supplementary material available online at

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Section: Discussionmentioning

confidence: 71%

SUMO-dependent regulation of centrin-2

Klein

Nigg

2009

Journal of Cell Science

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“…Our observations indicate that this glutamate may play a significant role in the interaction with the peptide. Indeed, in the crystal 25,26 and NMR 34 structures of the complex HsCen2/P17-XPC, the carboxyl group of E148 faces the positive pole of the peptide helix and establishes a hydrogen bond with the tryptophan of XPC (either with the N ɛ1 atom of its side chain or with the amide N of its backbone through a water molecule), whereas in the complex with R17-hSfi1, E148 faces the negative pole of the peptide helix that pushes the Glu side chain away from Trp, leading to a disruption of the H-bond. This observation is corroborated by the presence of several medium and weak nuclear Overhauser enhancement (NOE) effects between the tryptophan of P17-XPC and E148 of HsCen2, whereas no corresponding effect was observed between E148 and the tryptophan of R17-hSfi1-20 (the lists of the NMR restraints are available in the PDB, linked to structures 2A4J and 2K2I of complexes HsCen2/P17-XPC and HsCen2/R17-hSfi1-20, respectively).…”

Section: Consequences Of the Helix Dipole Orientationmentioning

confidence: 99%

“…Two crystal structures of the integral centrin are presently available: human centrin 2 (HsCen2) in complex with a 17-residue peptide derived from the human xeroderma pigmentosum complementation group C protein (XPC) [P17-XPC (peptide N847-R863 from the human XPC protein)] 25,26 and yeast (Saccharomyces cerevisiae) Cdc31 in complex with two or three repeats of ScSfi1. 27 The global shape of the domains is similar for the two centrins: the N-terminal domain is in a closed conformation, where the helices of the EF hand are anti-parallel, whereas the Cterminal domain is in an open conformation, with perpendicular helices.…”

Section: Introductionmentioning

confidence: 99%

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

Martinez-Sanz

Kateb

Assairi

et al. 2010

Journal of Molecular Biology

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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 approximately 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 -A 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.

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“…This appears to confirm earlier observations that these helices might not be completely structured. 7,36 Finally, the lowest order parameters 0.5 < S < 0.6 are observed for the Cγ 1 H 2 resonances of isoleucine 106, 121, and 146 due to enhanced mobility of their side-chains. Combining site-specific dipolar order parameters with relaxation measurements and molecular dynamics should allow one to obtain deeper insight into the mobility of backbone and side-chains.…”

Section: Partial Site-specific Assignments Ofmentioning

confidence: 94%

Probing Structural and Motional Features of the C-Terminal Part of the Human Centrin 2/P17-XPC Microcrystalline Complex by Solid-State NMR Spectroscopy

et al. 2012

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Insight into structural and motional features of the Cterminal part of the Human Centrin 2 in complex with the peptide P17-XPC was obtained by using complementary solid-state NMR methods. We demonstrate that the experimental conditions and procedures of sample crystallization determine the quality of solid-state NMR spectra and the internal mobility of the protein. 13 C− 13 C and 15 N− 15 N correlation spectra reveal intra-and inter-residue dipolar connectivities and provide partial, site-specific assignments of 13 C and 15 N resonance signals. The secondary structure of the C-ter HsCen2/P17-XPC complex in a microcrystalline state appears similar to that found in solution. Conformational flexibility is probed through relaxation-compensated measurements of dipolar order parameters that exploit the dynamics of cross-polarization in multidimensional experiments. The extracted dipolar coupling constants and relevant order parameters reveal increased backbone flexibility of the loops except for residues involved in coordination with the Ca 2+ cation that stabilizes the hydrophobic pocket containing the peptide P17-XPC.

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Structural, Thermodynamic, and Cellular Characterization of Human Centrin 2 Interaction with Xeroderma Pigmentosum Group C Protein

Cited by 56 publications

References 48 publications

SUMO-dependent regulation of centrin-2

SUMO-dependent regulation of centrin-2

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

Probing Structural and Motional Features of the C-Terminal Part of the Human Centrin 2/P17-XPC Microcrystalline Complex by Solid-State NMR Spectroscopy

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