multimerization
Dotaz
Zobrazit nápovědu
G-quadruplexes can multimerize under certain conditions, but the sequence requirements of such structures are not well understood. In this study, we investigated the ability of all possible variants of the central tetrad in a monomeric, parallel-strand G-quadruplex to form higher-order structures. Although most of these 256 variants existed primarily as monomers under the conditions of our screen, ∼10% formed dimers or tetramers. These structures could form in a wide range of monovalent and divalent metal ions, and folding was highly cooperative in both KCl and MgCl2. As was previously shown for G-quadruplexes that bind GTP and promote peroxidase reactions, G-quadruplexes that form dimers and tetramers have distinct sequence requirements. Some mutants could also form heteromultimers, and a second screen was performed to characterize the sequence requirements of these structures. Taken together, these experiments provide new insights into the sequence requirements and structures of both homomultimeric and heteromultimeric G-quadruplexes.
G-Quadruplexes are noncanonical nucleic acid structures made up of stacked guanosine tetrads connected by short loops. They are frequently used building blocks in synthetic biology and thought to play widespread biological roles. Multimerization can change the functional properties of G-quadruplexes, and understanding the factors that modulate this process remains an important goal. Here, we report the discovery of a novel mechanism by which the formation of multimeric G-quadruplexes can be controlled using GTP. We show that GTP likely inhibits multimer formation by becoming incorporated into a tetrad in the monomeric form of the structure and define the sequence requirements of G-quadruplexes that form GTP-dependent structures. These experiments provide new insights into the small molecule control of G-quadruplex multimerization. They also suggest possible roles for GTP-dependent multimeric G-quadruplexes in both synthetic and natural biological systems.
- MeSH
- biochemické jevy MeSH
- DNA genetika metabolismus MeSH
- G-kvadruplexy * MeSH
- guanosintrifosfát metabolismus MeSH
- lidé MeSH
- mutace MeSH
- Pan troglodytes MeSH
- Pongo MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Mason-Pfizer monkey virus (M-PMV) Gag protein contains a domain p12 that is unique to this virus (simian retrovirus-3) and its close relatives. The alpha-helical N-terminal half of p12, which contains a leucine zipper-like region, forms ordered structures in E. coli and the C-terminal half can form SDS-resistant oligomers in vitro. Together these properties suggest that p12 is a strong protein-protein interaction domain that facilitates Gag-Gag oligomerization. We have analyzed the oligomerization potential of a panel of p12 mutants, including versions containing substituted dimer, trimer, and tetramer leucine zippers, expressed in bacteria and in the context of the Gag precursor expressed in vitro and in cells. Purified recombinant p12 and its mutants could form various oligomers as shown by chemical cross-linking experiments. Within Gag these same mutants could assemble when overexpressed in cells. In contrast, all the mutants, including the leucine zipper mutants, were assembly defective in a cell-free system. These data highlight the importance of a region containing alternating leucines and isoleucines within p12, but also indicate that this domain's scaffold-like function is more complex than small number oligomerization.
- MeSH
- Cercopithecus aethiops MeSH
- COS buňky MeSH
- financování organizované MeSH
- genové produkty gag genetika metabolismus MeSH
- leucinové zipy MeSH
- Masonův-Pfizerův opičí virus fyziologie genetika ultrastruktura MeSH
- molekulární sekvence - údaje MeSH
- mutace MeSH
- rekombinantní proteiny genetika izolace a purifikace metabolismus MeSH
- terciární struktura proteinů MeSH
- transmisní elektronová mikroskopie MeSH
- vazba proteinů MeSH
- virion ultrastruktura MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
Several peripheral membrane proteins are known to form membrane pores through multimerization. In many cases, in biochemical reconstitution experiments, a complex distribution of oligomeric states has been observed that may, in part, be irrelevant to their physiological functions. This phenomenon makes it difficult to identify the functional oligomeric states of membrane lipid interacting proteins, for example, during the formation of transient membrane pores. Using fibroblast growth factor 2 (FGF2) as an example, we present a methodology applicable to giant lipid vesicles by which functional oligomers can be distinguished from nonspecifically aggregated proteins without functionality. Two distinct populations of fibroblast growth factor 2 were identified with (i) dimers to hexamers and (ii) a broad population of higher oligomeric states of membrane-associated FGF2 oligomers significantly distorting the original unfiltered histogram of all detectable oligomeric species of FGF2. The presented statistical approach is relevant for various techniques for characterizing membrane-dependent protein oligomerization.
G-quadruplexes are noncanonical nucleic acid structures formed from stacked guanine tetrads. They are frequently used as building blocks and functional elements in fields such as synthetic biology and also thought to play widespread biological roles. G-quadruplexes are often studied as monomers, but can also form a variety of higher-order structures. This increases the structural and functional diversity of G-quadruplexes, and recent evidence suggests that it could also be biologically important. In this review, we describe the types of multimeric topologies adopted by G-quadruplexes and highlight what is known about their sequence requirements. We also summarize the limited information available about potential biological roles of multimeric G-quadruplexes and suggest new approaches that could facilitate future studies of these structures.
The European Clinical Laboratory and Molecular (ECLM) classification of von Willebrand disease (vWD) is based on the splitting approach which uses sensitive and specific von Willebrand factor (vWF) assays with regard to the updated molecular data on structure and function of vWF gene and protein defects. A complete set of FVIII:C and vWF ristocetine cofactor, collagen binding, and antigen, vWF multimeric analysis in low- and medium-resolution gels, and responses to desmopressin (DDAVP) of FVIII:C and vWF parameters are mandatory. The ECLM classification distinguishes recessive types 1 and 3 vWD from recessive vWD 2C due to mutations in the D1 and D2 domains and vWD 2N due to mutations in the D'-FVIII-binding domain of vWF. The ECLM classification differentiates between mild vWD type 1 with variable penetrance of bleedings from symptomatic dominant type 1 vWD secretion defect and/or clearance defect with normal vWF multimers versus vWD 1M and 2M with normal or smeary vWF multimers in low- and medium-resolution gels. High-quality multimeric analysis of vWF in medium-resolution gels based on a DDAVP challenge test clearly delineates and distinguishes each of the dominant type 2 vWDs 1/2E, 2M, 2B, 2A, and 2D caused by vWF gene mutations in the D3 multimerization domain, loss or gain-of-function mutations in the glycoprotein Ib receptor A1 domain, gene mutations in the A2 proteolytic domain, and the C-terminal dimerization domain, respectively.
- MeSH
- desmopresin farmakologie MeSH
- diferenciální diagnóza * MeSH
- lidé MeSH
- multimerizace proteinu MeSH
- mutace MeSH
- von Willebrandova nemoc, typ 1 diagnóza MeSH
- von Willebrandova nemoc, typ 2 diagnóza MeSH
- von Willebrandův faktor analýza genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Phage tail fibres are elongated protein assemblies capable of specific recognition of bacterial surfaces during the first step of viral infection1-4. The folding of these complex trimeric structures often requires a phage-encoded tail fibre assembly (Tfa) protein5-7. Despite the wide occurrence of Tfa proteins, their functional mechanism has not been elucidated. Here, we investigate the tail fibre and Tfa of Escherichia coli phage Mu. We demonstrate that Tfa forms a stable complex with the tail fibre, and present a 2.1 Å resolution X-ray crystal structure of this complex. We find that Tfa proteins are comprised of two domains: a non-conserved N-terminal domain that binds to the C-terminal region of the fibre and a conserved C-terminal domain that probably mediates fibre oligomerization and assembly. Tfa forms rapidly exchanging multimers on its own, but not a stable trimer, implying that Tfa does not specify the trimeric state of the fibre. We propose that the key conserved role of Tfa is to ensure that fibre assembly and multimerization initiates at the C terminus, ensuring that the intertwined and repetitive structural elements of fibres come together in the correct sequence. The universal importance of correctly aligning the C termini of phage fibres is highlighted by our work.
- MeSH
- bakteriofágy klasifikace fyziologie MeSH
- Escherichia coli metabolismus virologie MeSH
- krystalografie rentgenová MeSH
- molekulární modely MeSH
- multimerizace proteinu MeSH
- proteiny virových bičíků chemie genetika metabolismus MeSH
- sbalování proteinů MeSH
- sekvence aminokyselin MeSH
- sekvenční seřazení MeSH
- vazba proteinů MeSH
- virové proteiny chemie genetika metabolismus MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Activation of the Wnt/β-catenin pathway crucially depends on the polymerization of dishevelled 2 (DVL2) into biomolecular condensates. However, given the low affinity of known DVL2 self-interaction sites and its low cellular concentration, it is unclear how polymers can form. Here, we detect oligomeric DVL2 complexes at endogenous protein levels in human cell lines, using a biochemical ultracentrifugation assay. We identify a low-complexity region (LCR4) in the C-terminus whose deletion and fusion decreased and increased the complexes, respectively. Notably, LCR4-induced complexes correlated with the formation of microscopically visible multimeric condensates. Adjacent to LCR4, we mapped a conserved domain (CD2) promoting condensates only. Molecularly, LCR4 and CD2 mediated DVL2 self-interaction via aggregating residues and phenylalanine stickers, respectively. Point mutations inactivating these interaction sites impaired Wnt pathway activation by DVL2. Our study discovers DVL2 complexes with functional importance for Wnt/β-catenin signaling. Moreover, we provide evidence that DVL2 condensates form in two steps by pre-oligomerization via high-affinity interaction sites, such as LCR4, and subsequent condensation via low-affinity interaction sites, such as CD2.
- MeSH
- beta-katenin metabolismus genetika MeSH
- HEK293 buňky MeSH
- lidé MeSH
- multimerizace proteinu MeSH
- protein dishevelled * metabolismus genetika MeSH
- signální dráha Wnt * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Sedimentation equilibrium and size-exclusion chromatography experiments on Mycobacterium tuberculosis hypoxanthine-guanine phosphoribosyltransferase (MtHGPRT) have established the existence of this enzyme as a reversibly associating mixture of dimeric and tetrameric species in 0.1 M Tris-HCl-0.012 M MgCl2, pH 7.4. Displacement of the equilibrium position towards the larger oligomer by phosphate signifies the probable existence of MtHGPRT as a tetramer in the biological environment. These data thus add credibility to the relevance of considering enzyme function in the light of a published tetrameric structure deduced from X-ray crystallography. Failure of 5-phospho-α-d-ribosyl-1-pyrophosphate (PRib-PP) to perturb the dimer-tetramer equilibrium position indicates the equivalence and independence of binding for this substrate (the first to bind in an ordered sequential mechanism) to the two oligomers. By virtue of the displacement of the equilibrium position towards dimer that is affected by removing MgCl2 from the Tris-HCl buffer, it can be concluded that divalent metal ions, as well as phosphate, can affect the oligomerization. These characteristics of MtHGPRT in solution are correlated with published crystal structures of four enzyme-ligand complexes.
Von Willebrandov faktor (vWF) je multimerický proteín, ktorý plní dve kľúčové funkcie v hemostáze. Podieľa sa na interakcii medzi doštičkami a subendotelom v mieste cievneho poškodenia a interakcii medzi doštičkami navzájom. vWF tvorí komplex s faktorom VIII v pomere (1: 1), čím ho chráni pred proteolýzou aktivovaným proteínom C. vWF je proteín, ktorý je zostavený z identických podjednotiek do lineárnych reťazcov rôznej veľkosti označovaných ako multiméry. Tieto multiméry môžu mať hmotnosť > 20 miliónov daltonov a dĺžku > 2 mikrometre. Klinicky významné a najúčinnejšie pri interakcii s kolagénom a trombocytovými receptormi sú multiméry s vysokou molekulovou hmotnosťou (HMW). V článku uvádzame porovnanie dvoch dostupných metód analýzy multimérov von Willebrandovho faktora - klasickú manuálnu metódu a novú semiautomatickú metódu prístroja Hydrasys. Nová metóda využíva vopred pripravený 2% agarózový gél, výsledky sú dostupné do 6 h, metóda je štandardizovaná a reprodukovateľná. Avšak abnormality multimérov sa musia ďalej podrobnejšie skúmať pomocou klasickej - manuálnej metódy s rôznymi koncentráciami agarózových gélov.
Von Willebrand factor (vWF) is a multimeric protein that plays a principal role in two key functions in hemostasis. It participates in the interaction between the platelets and the subendothelium at the site of the vascular damage and if is also important in the interaction between the platelets. vWF is complexed with factor VIII (1: 1) to protect it from proteolysis by activated Protein C. vWF is a protein that is assembled from identical subunits into linear strings of varying size referred to as multimers. These multimers can be > 20 million daltons in mass and > 2 micrometers in length. Clinically significant and most effective in interacting with collagen and thrombocyte receptors are high molecular weight multimers. In our article we present comparing of two available methods of von Willebrand factor multimer analysis - the classical manual method and the new semi-automatic method of the Hydrasys instrument. The new method uses a prepared 2% agarose gel, the results are available within 6 h, the method is standardized and reproducible. However, abnormalities of multimers must be further investigated in more detail using the classical-manual method with different concentrations of agarose gels.
