A key interaction with RPA orients XPA in NER complexes.

The XPA protein functions together with the single-stranded DNA (ssDNA) binding protein RPA as the central scaffold to ensure proper positioning of repair factors in multi-protein nucleotide excision repair (NER) machinery. We previously determined the structure of a short motif in the disordered XPA N-terminus bound to the RPA32C domain. However, a second contact between the XPA DNA-binding domain (XPA DBD) and the RPA70AB tandem ssDNA-binding domains, which is likely to influence the orientation of XPA and RPA on the damaged DNA substrate, remains poorly characterized. NMR was used to map the binding interfaces of XPA DBD and RPA70AB. Combining NMR and X-ray scattering data with comprehensive docking and refinement revealed how XPA DBD and RPA70AB orient on model NER DNA substrates. The structural model enabled design of XPA mutations that inhibit the interaction with RPA70AB. These mutations decreased activity in cell-based NER assays, demonstrating the functional importance of XPA DBD-RPA70AB interaction. Our results inform ongoing controversy about where XPA is bound within the NER bubble, provide structural insights into the molecular basis for malfunction of disease-associated XPA missense mutations, and contribute to understanding of the structure and mechanical action of the NER machinery.

Analysis of DNA binding by human factor xeroderma pigmentosum complementation group A (XPA) provides insight into its interactions with nucleotide excision repair substrates.

Xeroderma pigmentosum (XP) complementation group A (XPA) is an essential scaffolding protein in the multiprotein nucleotide excision repair (NER) machinery. The interaction of XPA with DNA is a core function of this protein; a number of mutations in the DNA-binding domain (DBD) are associated with XP disease. Although structures of the central globular domain of human XPA and data on binding of DNA substrates have been reported, the structural basis for XPA's DNA-binding activity remains unknown. X-ray crystal structures of the central globular domain of yeast XPA (Rad14) with lesion-containing DNA duplexes have provided valuable insights, but the DNA substrates used for this study do not correspond to the substrates of XPA as it functions within the NER machinery. To better understand the DNA-binding activity of human XPA in NER, we used NMR to investigate the interaction of its DBD with a range of DNA substrates. We found that XPA binds different single-stranded/double-stranded junction DNA substrates with a common surface. Comparisons of our NMR-based mapping of binding residues with the previously reported Rad14-DNA crystal structures revealed similarities and differences in substrate binding between XPA and Rad14. This includes direct evidence for DNA contacts to the residues extending C-terminally from the globular core, which are lacking in the Rad14 construct. Moreover, mutation of the XPA residue corresponding to Phe-262 in Rad14, previously reported as being critical for DNA binding, had only a moderate effect on the DNA-binding activity of XPA. The DNA-binding properties of several disease-associated mutations in the DBD were investigated. These results suggest that for XPA mutants exhibiting altered DNA-binding properties, a correlation exists between the extent of reduction in DNA-binding affinity and the severity of symptoms in XP patients.

MAP kinase p38 is a novel target of CacyBP/SIP phosphatase.

Mitogen-activated protein (MAP) kinases are important players in cellular signaling pathways. Recently, it has been shown that CacyBP/SIP serves as a phosphatase for one of the MAP kinases, ERK1/2. Through dephosphorylation of this kinase CacyBP/SIP modulates the transcriptional activity of Elk-1 and the activity of the CREB-BDNF pathway. In this work, using NB2a cell lysate and recombinant proteins, we show that CacyBP/SIP binds and dephosphorylates another member of the MAP kinase family, p38. Analysis of recombinant full-length CacyBP/SIP and its three major domains, N-terminal, middle CS and C-terminal SGS, indicates that the middle CS domain is responsible for p38 dephosphorylation. Moreover, we show that CacyBP/SIP might be implicated in response to oxidative stress. Dephosphorylation of phospho-p38 by CacyBP/SIP in NB2a cells treated with hydrogen peroxide is much more effective than in control ones. In conclusion, involvement of CacyBP/SIP in the regulation of p38 kinase activity, in addition to that of ERK1/2, might point to the function of CacyBP/SIP in pro-survival and pro-apoptotic pathways.

CacyBP/SIP--Structure and variety of functions.

BACKGROUND: CacyBP/SIP (Calcyclin-Binding Protein and Siah-1 Interacting Protein) is a small modular protein implicated in a wide range of cellular processes. It is expressed in different tissues of mammals but homologs are also found in some lower organisms. In mammals, a high level of CacyBP/SIP is present in tumor cells and in neurons. CacyBP/SIP binds several target proteins such as members of the S100 family, components of a ubiquitin ligase complex, and cytoskeletal proteins. SCOPE OF REVIEW: CacyBP/SIP has been shown to be involved in protein de-phosphorylation, ubiquitination, cytoskeletal dynamics, regulation of gene expression, cell proliferation, differentiation, and tumorigenesis. This review focuses on very recent reports on CacyBP/SIP structure and function in these important cellular processes. MAJOR CONCLUSIONS: CacyBP/SIP is a multi-domain and multi-functional protein. Altered levels of CacyBP/SIP in several cancers implicate its involvement in the maintenance of cell homeostasis. Changes in CacyBP/SIP subcellular localization in neurons of AD brains suggest that this protein is strongly linked to neurodegenerative diseases. Elucidation of CacyBP/SIP structure and cellular function is leading to greater understanding of its role in normal physiology and disease pathologies. GENERAL SIGNIFICANCE: The available results suggest that CacyBP/SIP is a key player in multiple biological processes. Detailed characterization of the physical, biochemical and biological properties of CacyBP/SIP will provide better insight into the regulation of its diverse functions in vivo, and given the association with specific diseases, will help clarify the potential of therapeutic targeting of this protein.

Dimerization and phosphatase activity of calcyclin-binding protein/Siah-1 interacting protein: the influence of oxidative stress.

CacyBP/SIP [calcyclin-binding protein/Siah-1 [seven in absentia homolog 1 (Siah E3 ubiquitin protein ligase 1)] interacting protein] is a multifunctional protein whose activity includes acting as an ERK1/2 phosphatase. We analyzed dimerization of mouse CacyBP/SIP in vitro and in mouse neuroblastoma cell line (NB2a) cells, as well as the structure of a full-length protein. Moreover, we searched for the CacyBP/SIP domain important for dimerization and dephosphorylation of ERK2, and we analyzed the role of dimerization in ERK1/2 signaling in NB2a cells. Cell-based assays showed that CacyBP/SIP forms a homodimer in NB2a cell lysate, and biophysical methods demonstrated that CacyBP/SIP forms a stable dimer in vitro. Data obtained using small-angle X-ray scattering supported a model in which CacyBP/SIP occupies an anti-parallel orientation mediated by the N-terminal dimerization domain. Site-directed mutagenesis established that the N-terminal domain is indispensable for full phosphatase activity of CacyBP/SIP. We also demonstrated that the oligomerization state of CacyBP/SIP as well as the level of post-translational modifications and subcellular distribution of CacyBP/SIP change after activation of the ERK1/2 pathway in NB2a cells due to oxidative stress. Together, our results suggest that dimerization is important for controlling phosphatase activity of CacyBP/SIP and for regulating the ERK1/2 signaling pathway.