Oligomeric assembly and interactions within the human RuvB-like RuvBL1 and RuvBL2 complexes

2010 ◽  
Vol 429 (1) ◽  
pp. 113-125 ◽  
Author(s):  
Andrew Niewiarowski ◽  
Alison S. Bradley ◽  
Jayesh Gor ◽  
Adam R. McKay ◽  
Stephen J. Perkins ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Structural Model ◽  
Analytical Ultracentrifugation ◽  
Protein Complexes ◽  
Adenine Nucleotides ◽  
Size Exclusion ◽  
Molecular Architecture ◽  
Essential Components ◽  
Exclusion Chromatography

The two closely related eukaryotic AAA+ proteins (ATPases associated with various cellular activities), RuvBL1 (RuvB-like 1) and RuvBL2, are essential components of large multi-protein complexes involved in diverse cellular processes. Although the molecular mechanisms of RuvBL1 and RuvBL2 function remain unknown, oligomerization is likely to be important for their function together or individually, and different oligomeric forms might underpin different functions. Several experimental approaches were used to investigate the molecular architecture of the RuvBL1–RuvBL2 complex and the role of the ATPase-insert domain (domain II) for its assembly and stability. Analytical ultracentrifugation showed that RuvBL1 and RuvBL2 were mainly monomeric and each monomer co-existed with small proportions of dimers, trimers and hexamers. Adenine nucleotides induced hexamerization of RuvBL2, but not RuvBL1. In contrast, the RuvBL1–RuvBL2 complexes contained single- and double-hexamers together with smaller forms. The role of domain II in complex assembly was examined by size-exclusion chromatography using deletion mutants of RuvBL1 and RuvBL2. Significantly, catalytically competent dodecameric RuvBL1–RuvBL2, complexes lacking domain II in one or both proteins could be assembled but the loss of domain II in RuvBL1 destabilized the dodecamer. The composition of the RuvBL1–RuvBL2 complex was analysed by MS. Several species of mixed RuvBL1/2 hexamers with different stoichiometries were seen in the spectra of the RuvBL1–RuvBL2 complex. A number of our results indicate that the architecture of the human RuvBL1–RuvBL2 complex does not fit the recent structural model of the yeast Rvb1–Rvb2 complex.

scholarly journals Enhanced oligomerization of full-length RAGE by synergy of the interaction of its domains

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Alexander Moysa ◽  
Dietmar Hammerschmid ◽  
Roman H. Szczepanowski ◽  
Frank Sobott ◽  
Michal Dadlez
Keyword(s):  
Analytical Ultracentrifugation ◽  
Vascular Diseases ◽  
Native Mass Spectrometry ◽  
Full Length ◽  
Size Exclusion ◽  
Glycation End Products ◽  
Exclusion Chromatography ◽  
Hydrogen Deuterium

AbstractThe pattern recognition receptor RAGE (receptor for advanced glycation end-products) transmits proinflammatory signals in several inflammation-related pathological states, including vascular diseases, cancer, neurodegeneration and diabetes. Its oligomerization is believed to be important in signal transduction, but RAGE oligomeric structures and stoichiometries remain unclear. Different oligomerization modes have been proposed in studies involving different truncated versions of the extracellular parts of RAGE. Here, we provide basic characterization of the oligomerization patterns of full-length RAGE (including the transmembrane (TM) and cytosolic regions) and compare the results with oligomerization modes of its four truncated fragments. For this purpose, we used native mass spectrometry, analytical ultracentrifugation, and size-exclusion chromatography coupled with multi-angle light scattering. Our results confirm known oligomerization tendencies of separate domains and highlight the enhanced oligomerization properties of full-length RAGE. Mutational analyses within the GxxxG motif of the TM region show sensitivity of oligomeric distributions to the TM sequence. Using hydrogen–deuterium exchange, we mapped regions involved in TM-dependent RAGE oligomerization. Our data provide experimental evidence for the major role of the C2 and TM domains in oligomerization, underscoring synergy among different oligomerization contact regions along the RAGE sequence. These results also explain the variability of obtained oligomerization modes in RAGE fragments.

scholarly journals A Mechanically Weak Extracellular Membrane-Adjacent Domain Induces Dimerization of Protocadherin-15

2018 ◽  
Author(s):  
P. De-la-Torre ◽  
D. Choudhary ◽  
R. Araya-Secchi ◽  
Y. Narui ◽  
M. Sotomayor
Keyword(s):  
Protein Interactions ◽  
Analytical Ultracentrifugation ◽  
Size Exclusion ◽  
Molecular Architecture ◽  
Protein Protein Interactions ◽  
Tip Link ◽  
Exclusion Chromatography ◽  
Protocadherin 15 ◽  
Cadherin 23 ◽  
Dynamics Simulations

ABSTRACTThe cadherin superfamily of proteins is defined by the presence of extracellular cadherin (EC) repeats that engage in protein-protein interactions to mediate cell-cell adhesion, cell signaling, and mechanotransduction. The extracellular domains of non-classical cadherins often have a large number of EC repeats along with other subdomains of various folds. Protocadherin-15 (PCDH15), a protein component of the inner-ear tip link filament essential for mechanotransduction, has eleven EC repeats and a membrane adjacent domain (MAD12) of atypical fold. Here we report the crystal structure of a pig PCDH15 fragment including EC10, EC11, and MAD12 in a parallel dimeric arrangement. MAD12 has a unique molecular architecture and folds as a ferredoxin-like domain similar to that found in the nucleoporin protein Nup54. Analytical ultracentrifugation experiments along with size exclusion chromatography coupled to multi-angle laser light scattering and small-angle X-ray scattering corroborate the crystallographic dimer and show that MAD12 induces parallel dimerization of PCDH15 near its membrane insertion point. In addition, steered molecular dynamics simulations suggest that MAD12 is mechanically weak and may unfold before tip-link rupture. Sequence analyses and structural modeling predict the existence of similar domains in cadherin-23, protocadherin-24, and the “giant” FAT and CELSR cadherins, indicating that some of them may also exhibit MAD-induced parallel dimerization.

scholarly journals Studies of recombinant TWA1 reveal constitutive dimerization

2017 ◽  
Vol 37 (1) ◽  
Author(s):  
Ore Francis ◽  
Genevieve E. Baker ◽  
Paul R. Race ◽  
Josephine C. Adams
Keyword(s):  
Protein Complexes ◽  
Carbon Sources ◽  
Gel Filtration ◽  
Cd Spectroscopy ◽  
Cysteine Residue ◽  
Size Exclusion ◽  
Native Page ◽  
Exclusion Chromatography ◽  
Key Enzyme

The mammalian muskelin/RanBP9/C-terminal to LisH (CTLH) complex and the Saccharomyces cerevisiae glucose-induced degradation (GID) complex are large, multi-protein complexes that each contain a RING E3 ubiquitin ligase. The yeast GID complex acts to degrade a key enzyme of gluconeogenesis, fructose 1,6-bisphosphatase, under conditions of abundant fermentable carbon sources. However, the assembly and functions of the mammalian complex remain poorly understood. A striking feature of these complexes is the presence of multiple proteins that contain contiguous lissencephaly-1 homology (LisH), CTLH and C-terminal CT11-RanBP9 (CRA) domains. TWA1/Gid8, the smallest constituent protein of these complexes, consists only of LisH, CTLH and CRA domains and is highly conserved in eukaryotes. Towards better knowledge of the role of TWA1 in these multi-protein complexes, we established a method for bacterial expression and purification of mouse TWA1 that yields tag-free, recombinant TWA1 in quantities suitable for biophysical and biochemical studies. CD spectroscopy of recombinant TWA1 indicated a predominantly α-helical protein. Gel filtration chromatography, size-exclusion chromatography (SEC) with multi-angle light scattering (SEC-MALS) and native PAGE demonstrated a propensity of untagged TWA1 to form stable dimers and, to a lesser extent, higher order oligomers. TWA1 has a single cysteine residue, Cys139, yet the dimeric form was preserved when TWA1 was purified in the presence of the reducing agent tris(2-carboxyethyl)phosphine (TCEP). These findings have implications for understanding the molecular role of TWA1 in the yeast GID complex and related multi-protein E3 ubiquitin ligases identified in other eukaryotes.

scholarly journals Metagenomic Profiling of Fecal-Derived Bacterial Membrane Vesicles in Crohn’s Disease Patients

Cells ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 2795
Author(s):  
Nader Kameli ◽  
Heike E. F. Becker ◽  
Tessa Welbers ◽  
Daisy M. A. E. Jonkers ◽  
John Penders ◽  
...  
Keyword(s):  
Crohn’S Disease ◽  
Crohn's Disease ◽  
Membrane Vesicles ◽  
Amplicon Sequencing ◽  
Size Exclusion ◽  
Bacterial Membrane ◽  
Microbial Composition ◽  
Fecal Samples ◽  
Exclusion Chromatography

Background: In the past, many studies suggested a crucial role for dysbiosis of the gut microbiota in the etiology of Crohn’s disease (CD). However, despite being important players in host–bacteria interaction, the role of bacterial membrane vesicles (MV) has been largely overlooked in the pathogenesis of CD. In this study, we addressed the composition of the bacterial and MV composition in fecal samples of CD patients and compared this to the composition in healthy individuals. Methods: Fecal samples from six healthy subjects (HC) in addition to twelve CD patients (six active, six remission) were analyzed in this study. Fecal bacterial membrane vesicles (fMVs) were isolated by a combination of ultrafiltration and size exclusion chromatography. DNA was obtained from the fMV fraction, the pellet of dissolved feces as bacterial DNA (bDNA), or directly from feces as fecal DNA (fDNA). The fMVs were characterized by nanoparticle tracking analysis and cryo-electron microscopy. Amplicon sequencing of 16s rRNA V4 hypervariable gene regions was conducted to assess microbial composition of all fractions. Results: Beta-diversity analysis showed that the microbial community structure of the fMVs was significantly different from the microbial profiles of the fDNA and bDNA. However, no differences were observed in microbial composition between fDNA and bDNA. The microbial richness of fMVs was significantly decreased in CD patients compared to HC, and even lower in active patients. Profiling of fDNA and bDNA demonstrated that Firmicutes was the most dominant phylum in these fractions, while in fMVs Bacteroidetes was dominant. In fMV, several families and genera belonging to Firmicutes and Proteobacteria were significantly altered in CD patients when compared to HC. Conclusion: The microbial alterations of MVs in CD patients particularly in Firmicutes and Proteobacteria suggest a possible role of MVs in host-microbe symbiosis and induction or progression of inflammation in CD pathogenesis. Yet, the exact role for these fMV in the pathogenesis of the disease needs to be elucidated in future studies.

scholarly journals The Two ADF-H Domains of Twinfilin Play Functionally Distinct Roles in Interactions with Actin Monomers

2002 ◽  
Vol 13 (11) ◽  
pp. 3811-3821 ◽  
Author(s):  
Pauli J. Ojala ◽  
Ville O. Paavilainen ◽  
Maria K. Vartiainen ◽  
Roman Tuma ◽  
Alan G. Weeds ◽  
...  
Keyword(s):  
Binding Site ◽  
Binding Activity ◽  
Size Exclusion ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Actin Monomer ◽  
Wild Type ◽  
Native Page ◽  
Exclusion Chromatography

Twinfilin is a ubiquitous and abundant actin monomer–binding protein that is composed of two ADF-H domains. To elucidate the role of twinfilin in actin dynamics, we examined the interactions of mouse twinfilin and its isolated ADF-H domains with G-actin. Wild-type twinfilin binds ADP-G-actin with higher affinity (K D = 0.05 μM) than ATP-G-actin (K D = 0.47 μM) under physiological ionic conditions and forms a relatively stable (k off = 1.8 s−1) complex with ADP-G-actin. Data from native PAGE and size exclusion chromatography coupled with light scattering suggest that twinfilin competes with ADF/cofilin for the high-affinity binding site on actin monomers, although at higher concentrations, twinfilin, cofilin, and actin may also form a ternary complex. By systematic deletion analysis, we show that the actin-binding activity is located entirely in the two ADF-H domains of twinfilin. Individually, these domains compete for the same binding site on actin, but the C-terminal ADF-H domain, which has >10-fold higher affinity for ADP-G-actin, is almost entirely responsible for the ability of twinfilin to increase the amount of monomeric actin in cosedimentation assays. Isolated ADF-H domains associate with ADP-G-actin with rapid second-order kinetics, whereas the association of wild-type twinfilin with G-actin exhibits kinetics consistent with a two-step binding process. These data suggest that the association with an actin monomer induces a first-order conformational change within the twinfilin molecule. On the basis of these results, we propose a kinetic model for the role of twinfilin in actin dynamics and its possible function in cells.

scholarly journals Biochemical and biophysical characterization of purified native CD20 alone and in complex with rituximab and obinutuzumab

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Morgane Agez ◽  
Elodie Desuzinges Mandon ◽  
Thomas Iwema ◽  
Reto Gianotti ◽  
Florian Limani ◽  
...  
Keyword(s):  
B Cell ◽  
B Lymphocyte ◽  
Analytical Ultracentrifugation ◽  
B Cell Lymphoma ◽  
Integral Membrane Protein ◽  
Size Exclusion ◽  
Hek293 Cells ◽  
Extraction And Purification ◽  
Exclusion Chromatography ◽  
Specificity Of Binding

Abstract CD20 is a B-lymphocyte specific integral membrane protein, an activated-glycosylated phosphoprotein expressed on the surface of B-cells and a clinically validated target of monoclonal antibodies such as rituximab, ocrelizumab, ofatumumab and obinutuzumab in the treatment of all B cell lymphomas and leukemias as well as autoimmune diseases. Here, we report the extraction and purification of native CD20 from SUDHL4 and RAMOS cell lines. To improve the protein yield, we applied a calixarene-based detergent approach to solubilize, stabilize and purify native CD20 from HEK293 cells. Size Exclusion Chromatography (SEC) and Analytical Ultracentrifugation show that purified CD20 was non-aggregated and that CD20 oligomerization is concentration dependent. Negative stain electron microscopy and atomic force microscopy revealed homogenous populations of CD20. However, no defined structure could be observed. Interestingly, micellar solubilized and purified CD20 particles adopt uniformly confined nanodroplets which do not fuse and aggregate. Finally, purified CD20 could bind to rituximab and obinutuzumab as demonstrated by SEC, and Surface Plasmon Resonance (SPR). Specificity of binding was confirmed using CD20 antibody mutants to human B-cell lymphoma cells. The strategy described in this work will help investigate CD20 binding with newly developed antibodies and eventually help to optimize them. This approach may also be applicable to other challenging membrane proteins.

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