SMC/kleisin complexes form elongated annular structures, which are critical for chromosome segregation, genome maintenance, and the regulation of gene expression. We describe marked structural similarities between bacterial and eukaryotic SMC/kleisin partner proteins (designated here as "kite" proteins for kleisin interacting tandem winged-helix (WH) elements of SMC complexes). Kite proteins are integral parts of all prokaryotic SMC complexes and Smc5/6 but not cohesin and condensin. They are made up of tandem WH domains, form homo- or heterodimers via their amino-terminal WH domain, and they associate with the central part of a kleisin subunit. In placental mammals, the kite subunit NSE3 gave rise to several (>60) kite-related proteins, named MAGE, many of which encode tumor- and testis-specific antigens. Based on architectural rather than sequence similarity, we propose an adapted model for the evolution of the SMC protein complexes and discuss potential functional similarities between bacterial Smc/ScpAB and eukaryotic Smc5/6.

• MeSH
• archeální proteiny chemie   genetika   metabolismus   MeSH
• bakteriální proteiny chemie   genetika   metabolismus   MeSH
• kineziny chemie   metabolismus   MeSH
• konzervovaná sekvence MeSH
• molekulární sekvence - údaje MeSH
• proteiny buněčného cyklu chemie   genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

The SMC (Structural Maintenance of Chromosomes) complexes are composed of SMC dimers, kleisin and kleisin-interacting (HAWK or KITE) subunits. Mutual interactions of these subunits constitute the basal architecture of the SMC complexes. In addition, binding of ATP molecules to the SMC subunits and their hydrolysis drive dynamics of these complexes. Here, we developed new systems to follow the interactions between SMC5/6 subunits and the relative stability of the complex. First, we show that the N-terminal domain of the Nse4 kleisin molecule binds to the SMC6 neck and bridges it to the SMC5 head. Second, binding of the Nse1 and Nse3 KITE proteins to the Nse4 linker increased stability of the ATP-free SMC5/6 complex. In contrast, binding of ATP to SMC5/6 containing KITE subunits significantly decreased its stability. Elongation of the Nse4 linker partially suppressed instability of the ATP-bound complex, suggesting that the binding of the KITE proteins to the Nse4 linker constrains its limited size. Our data suggest that the KITE proteins may shape the Nse4 linker to fit the ATP-free complex optimally and to facilitate opening of the complex upon ATP binding. This mechanism suggests an important role of the KITE subunits in the dynamics of the SMC5/6 complexes.

• MeSH
• adenosintrifosfatasy metabolismus   MeSH
• jaderné proteiny genetika   metabolismus   MeSH
• makromolekulární látky metabolismus   MeSH
• mutageneze cílená MeSH
• proteiny buněčného cyklu genetika   metabolismus   MeSH
• Schizosaccharomyces pombe - proteiny genetika   metabolismus   MeSH
• Schizosaccharomyces genetika   metabolismus   MeSH
• sekvenční seřazení MeSH
• techniky dvojhybridového systému MeSH
• transportní proteiny genetika   metabolismus   MeSH
• vazba proteinů genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Analysis of protein-protein interactions (PPI) is key for the understanding of most protein assemblies including structural maintenance of chromosomes (SMC) complexes. SMC complexes are composed of SMC proteins, kleisin, and kleisin-interacting subunits. These subunits interact in specific ways to constitute and regulate the closed structure of the complexes. Specifically, kleisin molecules bridge the SMC dimers and the kleisin-interacting subunits modulate stability of the bridge. Here we describe a multicomponent version of a yeast two-hybrid (Y2H) method and its application for analysis of the bridging role of the Nse4 kleisin in the SMC5/6 complex. Using this technique, we also show a stabilizing effect of KITE (kleisin-interacting tandem winged-helix element) proteins on SMC5/6.

• MeSH
• chromozomy hub fyziologie   MeSH
• mapy interakcí proteinů fyziologie   MeSH
• multiproteinové komplexy metabolismus   MeSH
• podjednotky proteinů metabolismus   MeSH
• proteiny buněčného cyklu metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny metabolismus   MeSH
• Saccharomyces cerevisiae metabolismus   MeSH
• techniky dvojhybridového systému MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

A family of Structural Maintenance of Chromosome (SMC) complexes is essential for key cellular processes ensuring proper cohesion, condensation and replication. They share a common SMC-kleisin architecture allowing them to embrace DNA. In SMC5/6, the NSE1 and NSE3 KITE and NSE4 kleisin subunits form a stable subcomplex that binds DNA and regulates essential processes. In addition, NSE5 and NSE6 subunits associate with the core SMC5/6 complex and recruit it to DNA repair sites. The architecture of the SMC5/6 complex is crucial for its proper functioning, and mutations within the human SMC5/6 subunits result in severe syndromes. Therefore, we aimed to analyze interactions within the human SMC5/6 complex and determine its detailed architecture. Firstly, we analyzed different parts of SMC5/6 by crosslinking and MS/MS analysis. Our data suggested domain arrangements of hNSE1-hNSE3 and orientation of hNSE4 within the hNSE1-hNSE3-hNSE4 subcomplex. The crosslinking and electron microscopic analysis of the SMC5/6 core complex showed its rod-like architecture with juxtaposed hSMC5-hSMC6 arms. Additionally, we observed fully or partially opened hSMC5-hSMC6 shapes with the hNSE1-hNSE3-hNSE4 trimer localized in the SMC head domains. To complete mapping of the human SMC5/6 complex architecture, we analyzed positions of hNSE5-hNSE6 at the hSMC5-hSMC6 arms. We showed that hNSE6 binding to hNSE5 and the coiled-coil arm of hSMC6 is mediated by a conserved FAM178 domain, which we therefore renamed CANIN (Coiled-coil SMC6 And NSE5 INteracting) domain. Interestingly, hNSE6 bound both hSMC5 and hSMC6 arms, suggesting that hNSE6 may lock the arms and regulate the dynamics of the human SMC5/6 complex.

• MeSH
• chromozomální proteiny, nehistonové genetika   MeSH
• lidé MeSH
• multimerizace proteinu genetika   MeSH
• multiproteinové komplexy genetika   MeSH
• mutace MeSH
• oprava DNA genetika   MeSH
• proteinové domény genetika   MeSH
• proteiny buněčného cyklu genetika   MeSH
• transportní proteiny genetika   MeSH
• vazba proteinů genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: The Nse1, Nse3 and Nse4 proteins form a tight sub-complex of the large SMC5-6 protein complex. hNSE3/MAGEG1, the mammalian ortholog of Nse3, is the founding member of the MAGE (melanoma-associated antigen) protein family and the Nse4 kleisin subunit is related to the EID (E1A-like inhibitor of differentiation) family of proteins. We have recently shown that human MAGE proteins can interact with NSE4/EID proteins through their characteristic conserved hydrophobic pocket. METHODOLOGY/PRINCIPAL FINDINGS: Using mutagenesis and protein-protein interaction analyses, we have identified a new Nse3/MAGE-binding domain (NMBD) of the Nse4/EID proteins. This short domain is located next to the Nse4 N-terminal kleisin motif and is conserved in all NSE4/EID proteins. The central amino acid residues of the human NSE4b/EID3 domain were essential for its binding to hNSE3/MAGEG1 in yeast two-hybrid assays suggesting they form the core of the binding domain. PEPSCAN ELISA measurements of the MAGEC2 binding affinity to EID2 mutant peptides showed that similar core residues contribute to the EID2-MAGEC2 interaction. In addition, the N-terminal extension of the EID2 binding domain took part in the EID2-MAGEC2 interaction. Finally, docking and molecular dynamic simulations enabled us to generate a structure model for EID2-MAGEC2. Combination of our experimental data and the structure modeling showed how the core helical region of the NSE4/EID domain binds into the conserved pocket characteristic of the MAGE protein family. CONCLUSIONS/SIGNIFICANCE: We have identified a new Nse4/EID conserved domain and characterized its binding to Nse3/MAGE proteins. The conservation and binding of the interacting surfaces suggest tight co-evolution of both Nse4/EID and Nse3/MAGE protein families.

• MeSH
• interakční proteinové domény a motivy MeSH
• intracelulární signální peptidy a proteiny chemie   genetika   metabolismus   MeSH
• jaderné proteiny chemie   MeSH
• konzervovaná sekvence MeSH
• lidé MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• mutageneze cílená MeSH
• peptidové fragmenty chemie   genetika   metabolismus   MeSH
• počítačová simulace MeSH
• proteiny buněčného cyklu genetika   metabolismus   MeSH
• rekombinantní proteiny chemie   genetika   metabolismus   MeSH
• Schizosaccharomyces pombe - proteiny chemie   MeSH
• Schizosaccharomyces MeSH
• sekvence aminokyselin MeSH
• substituce aminokyselin MeSH
• techniky dvojhybridového systému MeSH
• transportní proteiny chemie   genetika   metabolismus   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

During mitotic prophase, chromosomes of the pathogenic unicellular eukaryote Giardia intestinalis condense in each of the cell's two nuclei. In this study, Giardia chromosomes were investigated using light microscopy, high-resolution field emission scanning electron microscopy, and in situ hybridization. For the first time, we describe the overall morphology, condensation stages, and mitotic segregation of these chromosomes. Despite the absence of several genes involved in the cohesion and condensation pathways in the Giardia genome, we observed chromatin organization similar to those found in eukaryotes, i.e., 10-nm nucleosomal fibrils, 30-nm fibrils coiled to chromomeres or in parallel arrangements, and closely aligned sister chromatids. DNA molecules of Giardia terminate with telomeric repeats that we visualized on each of the four chromatid endings of metaphase chromosomes. Giardia chromosomes lack primary and secondary constrictions, thus preventing their classification based on the position of the centromere. The anaphase poleward segregation of sister chromatids is atypical in orientation and tends to generate lagging chromatids between daughter nuclei. In the Giardia genome database, we identified two putative members of the kleisin family thought to be responsible for condensin ring establishment. Thus far, Giardia chromosomes (300 nm to 1.5 μm) are the smallest chromosomes that were analyzed at the ultrastructural level. This study complements the existing molecular and sequencing data on Giardia chromosomes with cytological and ultrastructural information.

• MeSH
• adenosintrifosfatasy analýza   MeSH
• buněčné jádro ultrastruktura   MeSH
• chromozomy fyziologie   ultrastruktura   MeSH
• DNA vazebné proteiny analýza   MeSH
• Giardia lamblia genetika   ultrastruktura   MeSH
• mitóza MeSH
• multiproteinové komplexy analýza   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH