The effect of substrate binding on the conformation and structural stability of Herpes simplex virus type 1 thymidine kinase

Abstract 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 Å 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.