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 (TKHSV1) is k nown at high resolution in complex with a series of ligands and exhibits im portant structural similarities to the nucleoside monophosphate (NMP) kinas e family, which are known to show large conformational changes upon binding of substrates. The effect of substrate binding on the conformation and str uctural stability of TKHSV1, measured by thermal denaturation experiments, far-UV circular dichroism (CD) and fluorescence is described, and the resul ts indicate that the conformation of the ligand-free TKHSV1 is less ordered and less stable compared to the ligated enzyme. Furthermore, two crystal s tructures of TKHSV1 in complex with two new ligands, HPT and HMTT, refined to 2.2 Angstrom are presented. Although TKHSV1:HPT does not exhibit any sig nificant deviations from the model of TKHSV1:dT, the TKHSV1:HMTT complex di splays 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 com plex. We use this correlation to propose a method to estimate binding const ants for new TK(HSV1)substrates using thermal denaturation measurements mon itored by CD spectroscopy. The kinetic and structural results of both test substrates HPT and HMTT show that the CD thermal denaturation system is ver y sensitive to conformational changes caused by unusual binding of a substr ate analog.