Alternative Oligomeric States of the Yeast Rvb1/Rvb2 Complex Induced by Histidine Tags

Cheung, Kevin L.Y.; Huen, Jennifer; Kakihara, Yoshito; Houry, Walid; Ortega, Joaquı́n

doi:10.1016/j.jmb.2010.10.003

Cited by 40 publications

(52 citation statements)

References 26 publications

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“…In that study, however, ExbD carried a StrepII affinity tag at its carboxy terminus and ExbB carried a His 6 tag. Epitope tags have resulted in artifactual protein interactions or prevented native interactions, described in multiple protein systems where histidine tags were used (8,19,47). Within the TonB system, ExbD oligomerization states are affected by epitope tagging.…”

Section: Discussionmentioning

confidence: 99%

The Same Periplasmic ExbD Residues Mediate In Vivo Interactions between ExbD Homodimers and ExbD-TonB Heterodimers

Ollis

Postle

2011

J Bacteriol

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The TonB system couples cytoplasmic membrane proton motive force to TonB-gated outer membrane transporters for active transport of nutrients into the periplasm. In Escherichia coli, cytoplasmic membrane proteins ExbB and ExbD promote conformational changes in TonB, which transmits this energy to the transporters. The only known energy-dependent interaction occurs between the periplasmic domains of TonB and ExbD. This study identified sites of in vivo homodimeric interactions within ExbD periplasmic domain residues 92 to 121. ExbD was active as a homodimer (ExbD 2 ) but not through all Cys substitution sites, suggesting the existence of conformationally dynamic regions in the ExbD periplasmic domain. A subset of homodimeric interactions could not be modeled on the nuclear magnetic resonance (NMR) structure without significant distortion. Most importantly, the majority of ExbD Cys substitutions that mediated homodimer formation also mediated ExbD-TonB heterodimer formation with TonB A150C. Consistent with the implied competition, ExbD homodimer formation increased in the absence of TonB. Although ExbD D25 was not required for their formation, ExbD dimers interacted in vivo with ExbB. ExbD-TonB interactions required ExbD transmembrane domain residue D25. These results suggested a model where ExbD 2 assembled with ExbB undergoes a transmembrane domain-dependent transition and exchanges partners in localized homodimeric interfaces to form an ExbD 2 -TonB heterotrimer. The findings here were also consistent with our previous hypothesis that ExbD guides the conformation of the TonB periplasmic domain, which itself is conformationally dynamic.The TonB system of Gram-negative bacteria couples the proton motive force (PMF) of the cytoplasmic membrane (CM) to the active transport of a diverse range of large, scarce, or important nutrients across the unenergized outer membrane (1,24,25). Active transport across the outer membrane requires TonB-gated transporters, which are 22-stranded ␤-barrels with lumens occluded by an amino-terminal globular domain called the cork (37).In addition to the TonB-gated transporters in the outer membrane, the TonB system consists of 3 integral CM proteins, TonB, ExbB, and ExbD. TonB and ExbD have identical topologies, each with a single transmembrane domain (TMD) and more than two-thirds of each protein in the periplasm (15,20,43). ExbB has 3 TMDs and significant cytoplasmic domains (21,22). ExbB and ExbD appear to couple the energy of the CM PMF to conformational changes in the TonB carboxy terminus (33). The TonB carboxy terminus directly contacts the TonB-gated transporters and somehow transmits energy for active transport of substrates. Evidence suggests all three proteins form a complex in the CM, but the stoichiometry of this complex is unknown (2, 38). The cellular TonB/ExbD/ ExbB ratio is 1:2:7 (16).The only step in TonB energization currently known to require the PMF is characterized by a formaldehyde-cross-linkable interaction between the TonB and ExbD periplasmic domains. While Ton...

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Section: Discussionmentioning

confidence: 99%

The Same Periplasmic ExbD Residues Mediate In Vivo Interactions between ExbD Homodimers and ExbD-TonB Heterodimers

Ollis

Postle

2011

J Bacteriol

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“…However, the presence of an N-terminal histidine tag in yeast Rvb1 or Rvb2 induces the formation of double hexameric ring structures [19,22]. Recently, the structure of the yeast SWR1 [23] and INO80 [24] chromatin-remodeling complexes have been obtained by cryo-EM.…”

Section: Accepted Manuscriptmentioning

confidence: 99%

Yeast Rvb1 and Rvb2 Proteins Oligomerize As a Conformationally Variable Dodecamer with Low Frequency

Jeganathan

Leong

Zhao

et al. 2015

Journal of Molecular Biology

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“…+ proteins typically assemble into single hexameric rings (34), but examples exist where such proteins form double-hexameric ring structures (35)(36)(37). The SEC analysis suggested that Pch2 assembles into a single hexameric ring.…”

Section: Aaamentioning

confidence: 99%

“…Importantly, no additional higher molecular bands that could represent a dodecameric or higher-order complex of Pch2 were observed. As a control, we loaded a mixture of RuvBlike (Rvb)1 and Rvb2 AAA + proteins under conditions where they oligomerize both as single (∼300 kDa) and double (∼600 kDa) hexameric ring structures (36). These oligomers have theoretical molecular masses similar to the expected mass of Pch2 hexamers and dodecamers.…”

Section: Aaamentioning

confidence: 99%

Pch2 is a hexameric ring ATPase that remodels the chromosome axis protein Hop1

Chen

Jomaa

Ortega

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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In budding yeast the pachytene checkpoint 2 (Pch2) protein regulates meiotic chromosome axis structure by maintaining the domain-like organization of the synaptonemal complex proteins homolog pairing 1 (Hop1) and molecular zipper 1 (Zip1). Pch2 has also been shown to modulate meiotic double-strand break repair outcomes to favor recombination between homologs, play an important role in the progression of meiotic recombination, and maintain ribosomal DNA stability. Pch2 homologs are present in fruit flies, worms, and mammals, however the molecular mechanism of Pch2 function is unknown. In this study we provide a unique and detailed biochemical analysis of Pch2. We find that purified Pch2 is an AAA+ (ATPases associated with diverse cellular activities) protein that oligomerizes into single hexameric rings in the presence of nucleotides. In addition, we show Pch2 binds to Hop1, a critical axial component of the synaptonemal complex that establishes interhomolog repair bias, in a nucleotide-dependent fashion. Importantly, we demonstrate that Pch2 displaces Hop1 from large DNA substrates and that both ATP binding and hydrolysis by Pch2 are required for Pch2–Hop1 transactions. Based on these and previous cell biological observations, we suggest that Pch2 impacts meiotic chromosome function by directly regulating Hop1 localization.

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Alternative Oligomeric States of the Yeast Rvb1/Rvb2 Complex Induced by Histidine Tags

Cited by 40 publications

References 26 publications

The Same Periplasmic ExbD Residues Mediate In Vivo Interactions between ExbD Homodimers and ExbD-TonB Heterodimers

The Same Periplasmic ExbD Residues Mediate In Vivo Interactions between ExbD Homodimers and ExbD-TonB Heterodimers

Yeast Rvb1 and Rvb2 Proteins Oligomerize As a Conformationally Variable Dodecamer with Low Frequency

Pch2 is a hexameric ring ATPase that remodels the chromosome axis protein Hop1

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