Subunit Exchange of Multimeric Protein Complexes

Sobott, Frank; Benesch, Justin L. P.; Vierling, Elizabeth; Robinson, Carol V.

doi:10.1074/jbc.m206060200

Cited by 182 publications

(93 citation statements)

References 40 publications

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“…The dynamics of the L7/L12 proteins were investigated using an MS strategy that has proven successful to elucidate the subunit exchange dynamics of other protein complexes (30)(31)(32)(33). Specifically, ribosomes are uniformly labeled with stable isotopes ( 13 C and 15 N) to provide sufficient mass differences to resolve heterocomplexes formed with wild-type ribosomes.…”

Section: Isotopically Labeled Ribosomes Maintain Interactionsmentioning

confidence: 99%

Mechanism and Rates of Exchange of L7/L12 between Ribosomes and the Effects of Binding EF-G

et al. 2012

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The ribosomal stalk complex binds and recruits translation factors to the ribosome during protein biosynthesis. In Escherichia coli the stalk is composed of protein L10 and four copies of L7/L12. Despite the crucial role of the stalk, mechanistic details of L7/L12 subunit exchange are not established. By incubating isotopically labeled intact ribosomes with their unlabeled counterparts we monitored the exchange of the labile stalk proteins by recording mass spectra as a function of time. Based on kinetic analysis we proposed a mechanism whereby exchange proceeds via L7/L12 monomers and dimers. We also compared exchange of L7/L12 from free ribosomes with exchange from ribosomes in complex with elongation factor G (EF-G), trapped in the posttranslocational state by fusidic acid. Results showed that binding of EF-G reduces the L7/L12 exchange reaction of monomers by ~27% and of dimers by ~47% compared with exchange from free ribosomes. This is consistent with a model in which binding of EF-G does not modify interactions between the L7/L12 monomers but rather one of the four monomers, and as a result one of the two dimers, become anchored to the ribosome-EF-G complex preventing their free exchange. Overall therefore our results not only provide mechanistic insight into the exchange of L7/L12 monomers and dimers and the effects of EF-G binding but also have implications for modulating stability in response to environmental and functional stimuli within the cell.Protein biosynthesis is carried out by the ribosomal machinery and requires interaction of several translation factors with the ribosomal stalk complex. In bacteria the base of the stalk region, interacting with the rRNA of the 50S large ribosomal subunit, is composed of adjacent proteins L11 and L10 (1). The C-terminal domain (CTD) of L10 is a long and mobile helix which interacts with two dimers of protein L12 (2). Three pairs of L12 are observed in thermophilic bacteria and archaea (3-6) and L12 is the only protein present as multiple copies on the ribosome. The N-terminal domain (NTD) of L12 is responsible for dimerization and for anchoring of the protein to the ribosome (7). In Escherichia coli (E. Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts coli), L12 is partially N-acetylated to form protein L7 (8). The ratio of L7 to L12 is known to change throughout the growth phase (8-10) and has been linked to variation in the stability of the stalk complex via hydrogen exchange mass spectrometry (MS) experiments (11). L12 CTD and NTD are connected via a flexible hinge region providing mobility to the highly structured CTD (12, 13). These highly mobile proteins have eluded high resolution X-ray analysis for many years. Only recently the atomic structure of a truncated stalk complex of a thermophilic bacterium revealed the binding mode of the NTD of L12 proteins to L10 (4). The tight antiparallel binding of the L12 NTDs is similar to that observed by NMR for the E. coli L7/L12 dimer in solution (14). The mobility and dynamics o...

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Section: Isotopically Labeled Ribosomes Maintain Interactionsmentioning

confidence: 99%

Mechanism and Rates of Exchange of L7/L12 between Ribosomes and the Effects of Binding EF-G

et al. 2012

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“…However, in many cases the large oligomer exhibited unusual dynamic properties that seem to allow the association and dissociation of single subunits without apparent changes in the size of the oligomer. This has been demonstrated for ␣-crystallin, several sHsps from Bradyrhizobium japanoicum, PsHsp18.1 and TaHsp16.9 from plants, and Hsp16.5 from M. janaschii (28,14,29). It is therefore reasonable to assume that this dynamic behavior is generally required for the chaperone function of sHsps.…”

Section: Stability Of Hsp26⅐substratementioning

confidence: 99%

Analysis of the Regulation of the Molecular Chaperone Hsp26 by Temperature-induced Dissociation

Stromer¹,

Fischer

Richter

et al. 2004

Journal of Biological Chemistry

122

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Small heat shock proteins (sHsps) are molecular chaperones that efficiently bind non-native proteins. All members of this family investigated so far are oligomeric complexes. For Hsp26, an sHsp from the cytosol of Saccharomyces cerevisiae, it has been shown that at elevated temperatures the 24-subunit complex dissociates into dimers. This dissociation seems to be required for the efficient interaction with unfolding proteins that results in the formation of large, regular complexes comprising Hsp26 and the non-native proteins. To gain insight into the molecular mechanism of this chaperone, we analyzed the dynamics and stability of the two oligomeric forms of Hsp 26 (i.e. the 24-mer and the dimer) in comparison to a construct lacking the N-terminal domain (Hsp26⌬N). Furthermore, we determined the stabilities of complexes between Hsp26 and non-native proteins. We show that the temperature-induced dissociation of Hsp26 into dimers is a completely reversible process that involves only a small change in energy. The unfolding of the dissociated Hsp26 dimer or Hsp26⌬N, which is a dimer, requires a much higher energy. Because Hsp26⌬N was inactive as a chaperone, these results imply that the N-terminal domain is of critical importance for both the association of Hsp26 with non-native proteins and the formation of large oligomeric complexes. Interestingly, complexes of Hsp26 with non-native proteins are significantly stabilized against dissociation compared with Hsp26 complexes. Taken together, our findings suggest that the quaternary structure of Hsp26 is determined by two elements, (i) weak, regulatory interactions required to form the shell of 24 subunits and (ii) a strong and stable dimerization of the C-terminal domain.

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“…Fluorescence energy transfer experiments conducted with the polydisperse mammalian ␣A-and ␣B-crystallin and HSP27 revealed fully exchanged oligomers within a few hours at 37°C (19,21). Even the well ordered wheat and Methanococcus sHSP oligomers exhibit rapid subunit dissociation and reassociation (11,22). The dynamic nature of sHSP subunit interactions is further seen in the reversible dissociation of the wheat HSP16.9, yeast HSP26, Synechocystis HSP16.6, and M. tuberculosis HSP16.3 oligomers into a smaller subassembly upon heating (8,18,23,24).…”

Section: Small Heat Shock Proteins (Shsps)mentioning

confidence: 99%

Interactions between Small Heat Shock Protein Subunits and Substrate in Small Heat Shock Protein-Substrate Complexes

Friedrich

Giese

Buan³

et al. 2004

Journal of Biological Chemistry

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106

114

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Small heat shock proteins (sHSPs) are dynamic oligomeric proteins that bind unfolding proteins and protect them from irreversible aggregation. This binding results in the formation of sHSP-substrate complexes from which substrate can later be refolded. Interactions between sHSP and substrate in sHSP-substrate complexes and the mechanism by which substrate is transferred to ATP-dependent chaperones for refolding are poorly defined. We have established C-terminal affinity-tagged sHSPs from a eukaryote (pea HSP18.1) and a prokaryote (Synechocystis HSP16.6) as tools to investigate these issues. We demonstrate that sHSP subunit exchange for HSP18.1 and HSP16.6 is temperature-dependent and rapid at the optimal growth temperature for the organism of origin. Increasing the ratio of sHSP to substrate during substrate denaturation decreased sHSP-substrate complex size, and accordingly, addition of substrate to pre-formed sHSP-substrate complexes increased complex size. However, the size of pre-formed sHSP-substrate complexes could not be reduced by addition of more sHSP, and substrate could not be observed to transfer to added sHSP, although added sHSP subunits continued to exchange with subunits in sHSPsubstrate complexes. Thus, although some number of sHSP subunits within complexes remain dynamic and may be important for complex structure/solubility, association of substrate with the sHSP does not appear to be similarly dynamic. These observations are consistent with a model in which ATP-dependent chaperones associate directly with sHSP-bound substrate to initiate refolding. Small heat shock proteins (sHSPs)1 are ubiquitous stress proteins of 12-42 kDa that share a conserved C-terminal domain of ϳ100 amino acids (the ␣-crystallin domain) (1). The conserved ␣-crystallin domain is flanked by a variable length non-conserved N-terminal arm and a short C-terminal extension. In vivo and in vitro sHSP monomers assemble into native oligomers of 9 to Ͼ30 subunits, depending on the specific sHSP (2). sHSPs and the related vertebrate ␣-crystallins are proposed to function as molecular chaperones by preventing irreversible aggregation and insolubilization of denatured proteins (3-11). Many experiments have shown that sHSPs and ␣-crystallins have a large binding capacity for a variety of heat and chemically denatured model protein substrates (8,10,(12)(13)(14)(15). The models for sHSP function have been extended to suggest that substrates in sHSP-substrate complexes (here after referred to as "complexes") are held in a folding competent state, such that substrates can be reactivated by the ATP-dependent chaperone HSP70 (DnaK) and its respective co-chaperones (6, 9, 13, 14, 16).The x-ray structures of HSP16.5 from Methanococcus jannaschii, a 24-subunit oligomer (17), and HSP16.9 from Triticum aestivum (wheat), a 12-subunit oligomer (17, 18), reveal common features of sHSP structure. Despite limited sequence identity, these structures have a 1.5-Å root mean square difference between C-␣ atoms in the conserved ␣-crystallin domain, whic...

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Subunit Exchange of Multimeric Protein Complexes

Cited by 182 publications

References 40 publications

Mechanism and Rates of Exchange of L7/L12 between Ribosomes and the Effects of Binding EF-G

Mechanism and Rates of Exchange of L7/L12 between Ribosomes and the Effects of Binding EF-G

Analysis of the Regulation of the Molecular Chaperone Hsp26 by Temperature-induced Dissociation

Interactions between Small Heat Shock Protein Subunits and Substrate in Small Heat Shock Protein-Substrate Complexes

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