Mapping the Suramin-Binding Sites of Human Neutrophil Elastase: Investigation by Fluorescence Resonance Energy Transfer and Molecular Modeling
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- 作者：Yves Mé ; ly ; Martine Cadè ; ne ; Ingebrigt Sylte ; and Joseph G. Bieth
- 刊名：Biochemistry
- 出版年：1997
- 出版时间：December 16, 1997
- 年：1997
- 卷：36
- 期：50
- 页码：15624 - 15631
- 全文大小：211K
- 年卷期：v.36,no.50(December 16, 1997)
- ISSN：1520-4995
文摘
Neutrophil elastase (NE), a mediator of inflammation,binds with high affinity numerous anionicmolecules including suramin, a polysulfated naphthylurea, whichinhibits it with a Ki of 0.2 
M and a4:1suramin:NE stoichiometry and thus constitutes a potential therapeuticagent. In an attempt to locate thesuramin molecules on NE, we investigated the NE-suramin interactionusing steady-state and time-resolved fluorescence spectroscopy. The time-resolved intensitydecay of NE, a protein with three Trpresidues, in positions 27, 141, and 237 (chymotrypsin numbering system)was best described by a three-exponential function with lifetimes ranging from 0.22 to 2.28 ns.Comparison of the accessibility of thethree lifetime classes to the fluorescence quenchers acrylamide andiodide with the computed solventaccessibility of the three Trp residues in the crystal structure of NEindicates that the main, if not the sole,contribution to the 2.28 ns lifetime class is brought about by thefully buried Trp 141 residue. The additionof suramin to NE induces a sharp decrease in NE fluorescence and acorresponding increase in suraminfluorescence due to an efficient fluorescence resonance energy transfer(FRET) between the Trp residuesof NE, acting as donors, and the naphthalene rings of suramin, behavingas acceptors. From the fate ofthe longest lifetime class in the presence of variable suraminconcentrations, we deduce that two suraminsare bound at less than 17 Å from Trp 141, whereas the two others arelocated at least 29 Å from Trp 141.Moreover, neither the binding of suramin to NE nor the FRETprocess was modified when NE wascomplexed with a peptide chloromethylketone inhibitor, suggesting thatsuramin does not directly interferewith the substrate binding site of NE. These data were used asconstraints to model the NE-suramincomplex.
M and a4:1suramin:NE stoichiometry and thus constitutes a potential therapeuticagent. In an attempt to locate thesuramin molecules on NE, we investigated the NE-suramin interactionusing steady-state and time-resolved fluorescence spectroscopy. The time-resolved intensitydecay of NE, a protein with three Trpresidues, in positions 27, 141, and 237 (chymotrypsin numbering system)was best described by a three-exponential function with lifetimes ranging from 0.22 to 2.28 ns.Comparison of the accessibility of thethree lifetime classes to the fluorescence quenchers acrylamide andiodide with the computed solventaccessibility of the three Trp residues in the crystal structure of NEindicates that the main, if not the sole,contribution to the 2.28 ns lifetime class is brought about by thefully buried Trp 141 residue. The additionof suramin to NE induces a sharp decrease in NE fluorescence and acorresponding increase in suraminfluorescence due to an efficient fluorescence resonance energy transfer(FRET) between the Trp residuesof NE, acting as donors, and the naphthalene rings of suramin, behavingas acceptors. From the fate ofthe longest lifetime class in the presence of variable suraminconcentrations, we deduce that two suraminsare bound at less than 17 Å from Trp 141, whereas the two others arelocated at least 29 Å from Trp 141.Moreover, neither the binding of suramin to NE nor the FRETprocess was modified when NE wascomplexed with a peptide chloromethylketone inhibitor, suggesting thatsuramin does not directly interferewith the substrate binding site of NE. These data were used asconstraints to model the NE-suramincomplex.