Chaperone activity and structure of monomeric polypeptide binding domains of GroEL

The chaperonin GroEL is a large complex composed of 14 identical 57-kDa subunits that requires ATP and GroES for some of its activities. We find that a monomeric polypeptide corresponding to residues 191 to 345 has the activity of the tetradecamer both in facilitating the refolding of rhodanese and cyclophilin A in the absence of ATP and in catalyzing the unfolding of native barnase. Its crystal structure, solved at 2.5 A resolution, shows a well-ordered domain with the same fold as in intact GroEL. We have thus isolated the active site of the complex allosteric molecular chaperone, which functions as a "minichaperone." This has mechanistic implications: the presence of a central cavity in the GroEL complex is not essential for those representative activities in vitro, and neither are the allosteric properties. The function of the allosteric behavior on the binding of GroES and ATP must be to regulate the affinity of the protein for its various substrates in vivo, where the cavity may also be required for special functions

Chaperone activity and structure of monomeric polypeptide binding domains of GroEL

The chaperonin GroEL is a large complex composed of 14 identical 57-kDa subunits that requires ATP and GroES for some of its activities. We find that a monomeric polypeptide corresponding to residues 191 to 345 has the activity of the tetradecamer both in facilitating the refolding of rhodanese and cyclophilin A in the absence of ATP and in catalyzing the unfolding of native barnase. Its crystal structure, solved at 2.5 A resolution, shows a well-ordered domain with the same fold as in intact GroEL. We have thus isolated the active site of the complex allosteric molecular chaperone, which functions as a "minichaperone." This has mechanistic implications: the presence of a central cavity in the GroEL complex is not essential for those representative activities in vitro, and neither are the allosteric properties. The function of the allosteric behavior on the binding of GroES and ATP must be to regulate the affinity of the protein for its various substrates in vivo, where the cavity may also be required for special functions

Selective <i>selC</i>-Independent Selenocysteine Incorporation into Formate Dehydrogenases

<div><p></p><p>The formate dehydrogenases (Fdh) Fdh-O, Fdh-N, and Fdh-H, are the only proteins in <i>Escherichia coli</i> that incorporate selenocysteine at a specific position by decoding a UGA codon. However, an excess of selenium can lead to toxicity through misincorporation of selenocysteine into proteins. To determine whether selenocysteine substitutes for cysteine, we grew <i>Escherichia coli</i> in the presence of excess sodium selenite. The respiratory Fdh-N and Fdh-O enzymes, along with nitrate reductase (Nar) were co-purified from wild type strain MC4100 after anaerobic growth with nitrate and either 2 µM or 100 µM selenite. Mass spectrometric analysis of the catalytic subunits of both Fdhs identified the UGA-specified selenocysteine residue and revealed incorporation of additional, ‘non-specific’ selenocysteinyl residues, which always replaced particular cysteinyl residues. Although variable, their incorporation was not random and was independent of the selenite concentration used. Notably, these cysteines are likely to be non-essential for catalysis and they do not coordinate the iron-sulfur cluster. The remaining cysteinyl residues that could be identified were never substituted by selenocysteine. Selenomethionine was never observed in our analyses. Non-random substitution of particular cysteinyl residues was also noted in the electron-transferring subunit of both Fdhs as well as in the subunits of the Nar enzyme. Nar isolated from an <i>E. coli selC</i> mutant also showed a similar selenocysteine incorporation pattern to the wild-type indicating that non-specific selenocysteine incorporation was independent of the specific selenocysteine pathway. Thus, selenide replaces sulfide in the biosynthesis of cysteine and misacylated selenocysteyl-tRNA^Cys decodes either UGU or UGC codons, which usually specify cysteine. Nevertheless, not every UGU or UGC codon was decoded as selenocysteine. Together, our results suggest that a degree of misincorporation of selenocysteine into enzymes through replacement of particular, non-essential cysteines, is tolerated and this might act as a buffering system to cope with excessive intracellular selenium.</p></div

Overview of peptides containing cysteines, selenocysteines, and dehydroalanines at specific positions*.

<p>Modifications confirmed by MS/MS are indicated as X, the subscript defines the respective amino acid position in the sequence.Sec-196 is the naturally occurring amino acid at position 196 and cysteine was never identified at this position (see underline peptide).</p

Proteins obtained in fractions 2, 3, and 5 (see Figs. 1A and B) from anion exchange chromatography that were identified by nano-ESI-LTQ-Orbitrap-MS/MS analysis; NCBI entry numbers are given.

<p>Proteins obtained in fractions 2, 3, and 5 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061913#pone-0061913-g001" target="_blank">Figs. 1A and B</a>) from anion exchange chromatography that were identified by nano-ESI-LTQ-Orbitrap-MS/MS analysis; NCBI entry numbers are given.</p

Figure 3

<p>(A) Nano-ESI-LTQ-Orbitrap-MS data. The signal of the selenopeptide from FdoG at <i>m/z</i> 993.4281 matches the expected mass with a deviation of 1.1 ppm. In the inset the signal is shown enlarged, exhibiting the characteristic isotope pattern of a selenopeptide. (B, C) Nano-ESI-LTQ-Orbitrap-MS/MS data of peptides LPSTu₆₁₈FAEENGSIVNSGR (B, <i>m/z</i> 993.4281) and LPSTa₆₁₈FAEENGSIVNSGR (C, <i>m/z</i> 923.9503). Precursor ions were selected, fragmented, and analyzed in the linear ion trap (LTQ). MS/MS data unambiguously identify carbamidomethylated selenocysteine (u) and dehydroalanine (a) at position 618.</p