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Molecular insights into substrate recognition and catalytic mechanism of the chaperone and FKBP peptidyl-prolyl isomerase SlyD

Quistgaard, Esben M.; Weininger, Ulrich LU ; Ural-Blimke, Yonca; Modig, Kristofer LU ; Nordlund, Pär; Akke, Mikael LU and Löw, Christian (2016) In BMC Biology 14(1).
Abstract

Background: Peptidyl-prolyl isomerases (PPIases) catalyze cis/trans isomerization of peptidyl-prolyl bonds, which is often rate-limiting for protein folding. SlyD is a two-domain enzyme containing both a PPIase FK506-binding protein (FKBP) domain and an insert-in-flap (IF) chaperone domain. To date, the interactions of these domains with unfolded proteins have remained rather obscure, with structural information on binding to the FKBP domain being limited to complexes involving various inhibitor compounds or a chemically modified tetrapeptide. Results: We have characterized the binding of 15-residue-long unmodified peptides to SlyD from Thermus thermophilus (TtSlyD) in terms of binding thermodynamics and enzyme kinetics through the use... (More)

Background: Peptidyl-prolyl isomerases (PPIases) catalyze cis/trans isomerization of peptidyl-prolyl bonds, which is often rate-limiting for protein folding. SlyD is a two-domain enzyme containing both a PPIase FK506-binding protein (FKBP) domain and an insert-in-flap (IF) chaperone domain. To date, the interactions of these domains with unfolded proteins have remained rather obscure, with structural information on binding to the FKBP domain being limited to complexes involving various inhibitor compounds or a chemically modified tetrapeptide. Results: We have characterized the binding of 15-residue-long unmodified peptides to SlyD from Thermus thermophilus (TtSlyD) in terms of binding thermodynamics and enzyme kinetics through the use of isothermal titration calorimetry, nuclear magnetic resonance spectroscopy, and site-directed mutagenesis. We show that the affinities and enzymatic activity of TtSlyD towards these peptides are much higher than for the chemically modified tetrapeptides that are typically used for activity measurements on FKBPs. In addition, we present a series of crystal structures of TtSlyD with the inhibitor FK506 bound to the FKBP domain, and with 15-residue-long peptides bound to either one or both domains, which reveals that substrates bind in a highly adaptable fashion to the IF domain through β-strand augmentation, and can bind to the FKBP domain as both types VIa1 and VIb-like cis-proline β-turns. Our results furthermore provide important clues to the catalytic mechanism and support the notion of inter-domain cross talk. Conclusions: We found that 15-residue-long unmodified peptides can serve as better substrate mimics for the IF and FKBP domains than chemically modified tetrapeptides. We furthermore show how such peptides are recognized by each of these domains in TtSlyD, and propose a novel general model for the catalytic mechanism of FKBPs that involves C-terminal rotation around the peptidyl-prolyl bond mediated by stabilization of the twisted transition state in the hydrophobic binding site.

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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Beta-turn, Chaperone, FK506, FK506-binding protein (FKBP), NMR, Peptidyl-prolyl isomerase (PPIase), Proline, Protein folding, SlyD, X-ray crystal structure
in
BMC Biology
volume
14
issue
1
publisher
BioMed Central
external identifiers
  • scopus:84988596341
  • wos:000383839800001
ISSN
1741-7007
DOI
10.1186/s12915-016-0300-3
language
English
LU publication?
yes
id
986fd875-35aa-403e-8a2f-88a1c545f902
date added to LUP
2016-11-01 10:45:17
date last changed
2017-11-19 04:34:26
@article{986fd875-35aa-403e-8a2f-88a1c545f902,
  abstract     = {<p>Background: Peptidyl-prolyl isomerases (PPIases) catalyze cis/trans isomerization of peptidyl-prolyl bonds, which is often rate-limiting for protein folding. SlyD is a two-domain enzyme containing both a PPIase FK506-binding protein (FKBP) domain and an insert-in-flap (IF) chaperone domain. To date, the interactions of these domains with unfolded proteins have remained rather obscure, with structural information on binding to the FKBP domain being limited to complexes involving various inhibitor compounds or a chemically modified tetrapeptide. Results: We have characterized the binding of 15-residue-long unmodified peptides to SlyD from Thermus thermophilus (TtSlyD) in terms of binding thermodynamics and enzyme kinetics through the use of isothermal titration calorimetry, nuclear magnetic resonance spectroscopy, and site-directed mutagenesis. We show that the affinities and enzymatic activity of TtSlyD towards these peptides are much higher than for the chemically modified tetrapeptides that are typically used for activity measurements on FKBPs. In addition, we present a series of crystal structures of TtSlyD with the inhibitor FK506 bound to the FKBP domain, and with 15-residue-long peptides bound to either one or both domains, which reveals that substrates bind in a highly adaptable fashion to the IF domain through β-strand augmentation, and can bind to the FKBP domain as both types VIa1 and VIb-like cis-proline β-turns. Our results furthermore provide important clues to the catalytic mechanism and support the notion of inter-domain cross talk. Conclusions: We found that 15-residue-long unmodified peptides can serve as better substrate mimics for the IF and FKBP domains than chemically modified tetrapeptides. We furthermore show how such peptides are recognized by each of these domains in TtSlyD, and propose a novel general model for the catalytic mechanism of FKBPs that involves C-terminal rotation around the peptidyl-prolyl bond mediated by stabilization of the twisted transition state in the hydrophobic binding site.</p>},
  articleno    = {82},
  author       = {Quistgaard, Esben M. and Weininger, Ulrich and Ural-Blimke, Yonca and Modig, Kristofer and Nordlund, Pär and Akke, Mikael and Löw, Christian},
  issn         = {1741-7007},
  keyword      = {Beta-turn,Chaperone,FK506,FK506-binding protein (FKBP),NMR,Peptidyl-prolyl isomerase (PPIase),Proline,Protein folding,SlyD,X-ray crystal structure},
  language     = {eng},
  month        = {09},
  number       = {1},
  publisher    = {BioMed Central},
  series       = {BMC Biology},
  title        = {Molecular insights into substrate recognition and catalytic mechanism of the chaperone and FKBP peptidyl-prolyl isomerase SlyD},
  url          = {http://dx.doi.org/10.1186/s12915-016-0300-3},
  volume       = {14},
  year         = {2016},
}