Insertion of a Chaperone Domain Converts FKBP12 into a Powerful Catalyst of Protein Folding

Knappe, Thomas A.; Eckert, Barbara; Schaarschmidt, Peter; Schölz, Christian; Schmid, Franz X.

doi:10.1016/j.jmb.2007.02.097

Cited by 61 publications

(148 citation statements)

References 66 publications

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“…Its catalytic efficiency could be strongly improved by grafting the chaperone domain of SlyD into a loop of FKBP12 near the active site to create the chimeric protein FKBP12ϩIF (48). The insertion of this chaperone domain left the activity and the very high specificity of the prolyl isomerase active site of FKBP12 toward Xaa-Pro in the tetrapeptide library virtually unchanged ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…In these experiments, the initial rate of folding was determined as a function of the substrate protein concentration. Catalyzed and uncatalyzed folding occur in the presence of a folding catalyst and were accounted for in the analysis by using a procedure originally developed for prolyl isomerization in peptides (51) and adapted for catalyzed protein folding (48,49). The range of substrate concentrations is restricted to less than 10 M in these experiments, because the uncatalyzed reaction dominates at high substrate concentration.…”

Section: Resultsmentioning

confidence: 99%

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Chaperone domains convert prolyl isomerases into generic catalysts of protein folding

Jakob

Žoldák

Aumüller

et al. 2009

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

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The cis/trans isomerization of peptide bonds before proline (prolyl bonds) is a rate-limiting step in many protein folding reactions, and it is used to switch between alternate functional states of folded proteins. Several prolyl isomerases of the FK506-binding protein family, such as trigger factor, SlyD, and FkpA, contain chaperone domains and are assumed to assist protein folding in vivo. The prolyl isomerase activity of FK506-binding proteins strongly depends on the nature of residue Xaa of the Xaa-Pro bond. We confirmed this in assays with a library of tetrapeptides in which position Xaa was occupied by all 20 aa. A high sequence specificity seems inconsistent with a generic function of prolyl isomerases in protein folding. Accordingly, we constructed a library of protein variants with all 20 aa at position Xaa before a rate-limiting cis proline and used it to investigate the performance of trigger factor and SlyD as catalysts of proline-limited folding. The efficiencies of both prolyl isomerases were higher than in the tetrapeptide assays, and, intriguingly, this high activity was almost independent of the nature of the residue before the proline. Apparently, the almost indiscriminate binding of the chaperone domain to the refolding protein chain overrides the inherently high sequence specificity of the prolyl isomerase site. The catalytic performance of these folding enzymes is thus determined by generic substrate recognition at the chaperone domain and efficient transfer to the active site in the prolyl isomerase domain.folding catalysis ͉ folding helpers ͉ folding mechanism ͉ SlyD ͉ trigger factor T he cis/trans isomerization of peptide bonds before proline (Xaa-Pro or prolyl bonds; Fig. 1A) is an intrinsically slow reaction that depends on the nature of the amino acid Xaa (1-3). It determines the rates of many protein folding reactions (4-6), is used as a molecular switch (7-16), and is catalyzed by prolyl isomerases (17-21). Most of the prolyl isomerases that assist in cellular protein folding contain catalytic domains that are homologous to human FKBP12 (FK-506 binding protein of 12 kDa; Fig. 1B) and chaperone domains, which interact transiently with non-native proteins (Fig. 1B). The trigger factor (22-25) and SlyD [product of the slyD (sensitive-to lysis) gene, a cytosolic Escherichia coli chaperone] (26-29) belong to this family of folding enzymes. The chaperone domain of SlyD (the ''insertin-flap'' or IF domain) shows a unique fold (30) and is structurally not related to other chaperones. The chaperone domain of trigger factor shows similarities with prefoldin (31)and SurA (32). FkpA (periplasmic FKBP of E. coli) (33-35) of prokaryotes and FKBP52 of eukaryotes (36) display similar combinations of prolyl isomerase and chaperone domains.FKBP-type prolyl isomerases are highly specific with regard to residue Xaa before the proline (37-39). An initial characterization of human FKBP12 with various proline-containing tetrapeptides and a protease-coupled assay (17) indicated that it catalyzes isomerizati...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Chaperone domains convert prolyl isomerases into generic catalysts of protein folding

Jakob

Žoldák

Aumüller

et al. 2009

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

Self Cite

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“…These studies do not allow distinction between both enzyme families acting on the same substrate molecule versus one acting on one folding protein and the other on another, but the cyclophilins and FKBPs have not been found in the same resident ER protein complexes (Meunier et al 2002;Kleizen and Braakman 2004;Jansen et al 2012), so perhaps they work through different interactions in different chaperone complexes on different substrates or at different times during substrate folding. Studies on family members have shown that purified FKBPs lose their high sequence specificity and turn into effective broad PPIases when attached to or collaborating with a chaperone (Knappe et al 2007;Jakob and Schmid 2009). …”

Section: Ppismentioning

confidence: 99%

Protein Folding in the Endoplasmic Reticulum

Braakman

Hebert

2013

Cold Spring Harbor Perspectives in Biology

415

349

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In this article, we will cover the folding of proteins in the lumen of the endoplasmic reticulum (ER), including the role of three types of covalent modifications: signal peptide removal, Nlinked glycosylation, and disulfide bond formation, as well as the function and importance of resident ER folding factors. These folding factors consist of classical chaperones and their cochaperones, the carbohydrate-binding chaperones, and the folding catalysts of the PDI and proline cis -trans isomerase families. We will conclude with the perspective of the folding protein: a comparison of characteristics and folding and exit rates for proteins that travel through the ER as clients of the ER machinery.

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“…NMR solution structures of E. coli SlyD revealed that the N-terminal region consists of two well-defined domains (6,7). The FKBP (FK-506 binding protein) domain has a similar structure as other members of the FKBP family of peptidyl-prolyl isomerases (PPIases) and catalyzes the otherwise slow isomerisation of proline peptide bonds during protein folding (8,9). The IF (insert in the flap) domain recognizes and binds to unfolded protein, contributing to the protein folding activity (10,11).…”

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Metal Selectivity of the Escherichia coli Nickel Metallochaperone, SlyD

et al. 2011

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SlyD is a Ni(II)-binding protein that contributes to nickel homeostasis in Escherichia coli. The Cterminal domain of SlyD contains a rich variety of metal-binding amino acids, suggesting broader metal-binding capabilities, and previous work demonstrated that the protein can coordinate several types of first row transition metals. However, the binding of SlyD to metals other than Ni(II) has not been previously characterized. To further our understanding of the in vitro metal-binding activity of SlyD and how it correlates with the in vivo function of this protein, the interactions between SlyD and the series of biologically relevant transition metals Mn(II), Fe(II), Co(II), Cu(I) and Zn(II) were examined by using a combination of optical spectroscopy and mass spectrometry. Tables S1-S3 containing a summary of metal concentrations used for metal toxicity studies, a list of primers used for qRT-PCR analysis and summary of Mn(II), Co(II) and Ni(II) stoichiometries of SlyD; Figure S2 shows representative spectra of a Ni(II) titrations analyzed via ESI-MS; Figures S3-S4 shows a graphical representation of the PAR competition assay used for determining the average K D (Zn(II)-SlyD) and a comparison of CD spectra for Ni(II) and Zn(II) bound SlyD. Figure S5 is a representative data set for a Cu(I) titration of SlyD analyzed via electronic absorption spectroscopy. Figure S6-S7 are competition titrations with Bca and Bcs used for determining copper stoichiometry and affinity, respectively. Figure S8-S10 are a graphical representation of Co(II) titrations analyzed via ESI-MS, competition titrations with Fura2 for determining average K D (Co(II)-SlyD) and titration of a cobalt saturated SlyD sample with Zn(II). Figure S11 is spectroscopy data obtained for titration of Ni(II) compared with a similar titration carried out in the presence of excess Fe(II). Figure S12 depicts representative growth curves of WT and ΔslyD strains conducted in minimal media supplemented with various amounts of metal. Figure S13 is relative mRNA levels measured for various metal transporters under aerobic conditions. Figure S14-S15 are relative mRNA levels measured for various metal transporters under anaerobic conditions. This material is available free of charge via the Internet at http://pubs.acs.org.

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Insertion of a Chaperone Domain Converts FKBP12 into a Powerful Catalyst of Protein Folding

Cited by 61 publications

References 66 publications

Chaperone domains convert prolyl isomerases into generic catalysts of protein folding

Chaperone domains convert prolyl isomerases into generic catalysts of protein folding

Protein Folding in the Endoplasmic Reticulum

Metal Selectivity of the Escherichia coli Nickel Metallochaperone, SlyD

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