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Dynamics of Aromatic Side Chains in the Active Site of FKBP12.
- Source :
-
Biochemistry [Biochemistry] 2017 Jan 10; Vol. 56 (1), pp. 334-343. Date of Electronic Publication: 2016 Dec 22. - Publication Year :
- 2017
-
Abstract
- FKBP12, a small human enzyme, aids protein folding by catalyzing cis-trans isomerization of peptidyl-prolyl bonds, and is involved in cell signaling pathways, calcium regulation, and the immune response. The underlying molecular mechanisms are not fully understood, but it is well-known that aromatic residues in the active site and neighboring loops are important for substrate binding and catalysis. Here we report micro- to millisecond exchange dynamics of aromatic side chains in the active site region of ligand-free FKBP12, involving a minor state population of 0.5% and an exchange rate of 3600 s <superscript>-1</superscript> , similar to previous results for the backbone and methyl-bearing side chains. The exchange process involves tautomerization of H87. In the major state H87 is highly flexible and occupies the common HNε2 tautomer, while in the minor state it occupies the rare HNδ1 tautomer, which typically requires stabilization by specific interactions, such as hydrogen bonds. This finding suggests that the exchange process is coupled to a rearrangement of the hydrogen bond network around H87. Upon addition of the active-site inhibitor FK506 the exchange of all aromatic residues is quenched, with exception of H87. The H87 resonances are broadened beyond detection, suggesting that interconversion between tautomers prevail in the FK506-bound state. While key active-site residues undergo conformational exchange in the apo state, the exchange rate is considerably faster than the catalytic turnover, as determined herein by Michaelis-Menten type analysis of NMR line shapes and chemical shifts. We discuss alternative interpretations of this observation in terms of FKBP12 function.
- Subjects :
- Amino Acids, Aromatic metabolism
Binding Sites genetics
Histidine chemistry
Histidine metabolism
Humans
Hydrogen Bonding
Isomerism
Kinetics
Magnetic Resonance Spectroscopy
Models, Molecular
Mutation
Protein Binding
Tacrolimus chemistry
Tacrolimus metabolism
Tacrolimus Binding Protein 1A genetics
Tacrolimus Binding Protein 1A metabolism
Amino Acids, Aromatic chemistry
Catalytic Domain
Protein Conformation
Tacrolimus Binding Protein 1A chemistry
Subjects
Details
- Language :
- English
- ISSN :
- 1520-4995
- Volume :
- 56
- Issue :
- 1
- Database :
- MEDLINE
- Journal :
- Biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 27936610
- Full Text :
- https://doi.org/10.1021/acs.biochem.6b01157