The effect of substrate binding on the conformation and structural stability of Herpes simplex virus type 1 thymidine kinase.
The structure of Herpes simplex virus type 1 thymidine kinase (TK(HSV1)) is known at high resolution in complex with a series of ligands and exhibits important structural similarities to the nucleoside monophosphate (NMP) kinase family, which are known to show large conformational changes upon binding of substrates. The effect of substrate binding on the conformation and structural stability of TK(HSV1), measured by thermal denaturation experiments, far-UV circular dichroism (CD) and fluorescence is described, and the results indicate that the conformation of the ligand-free TK(HSV1) is less ordered and less stable compared to the ligated enzyme. Furthermore, two crystal structures of TK(HSV1) in complex with two new ligands, HPT and HMTT, refined to 2.2 A are presented. Although TK(HSV1):HPT does not exhibit any significant deviations from the model of TK(HSV1):dT, the TK(HSV1):HMTT complex displays a unique conformationally altered active site resulting in a lowered thermal stability of this complex. Moreover, we show that binding affinity and binding mode of the ligand correlate with thermal stability of the complex. We use this correlation to propose a method to estimate binding constants for new TK(HSV1)substrates using thermal denaturation measurements monitored by CD spectroscopy. The kinetic and structural results of both test substrates HPT and HMTT show that the CD thermal denaturation system is very sensitive to conformational changes caused by unusual binding of a substrate analog.
Department of Applied BioSciences, Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology, CH-8057 Zürich, Switzerland.
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Wurth
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Kessler
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Vogt
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Schulz
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Folkers
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Scapozza
D000954:Antigens, Surface; D001665:Binding Sites; D014408:Biomarkers, Tumor; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D018259:Herpesvirus 1, Human / Q000201:enzymology*; D008024:Ligands; D011487:Protein Conformation; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013937:Thymidine Kinase / Q000737:chemistry*; D000951:Antigens, Neoplasm
D000954:Antigens, Surface; D014408:Biomarkers, Tumor; D008024:Ligands; D013937:Thymidine Kinase; C115012:human epithelial antigen-125; D000951:Antigens, Neoplasm