Kinetics and crystal structure of the wild-type and the engineered Y101F mutant of Herpes simplex virus type 1 thymidine kinase interacting with (North)-methanocarba-thymidine.
Kinetic and crystallographic analyses of wild-type Herpes simplex virus type 1 thymidine kinase (TK(HSV1)) and its Y101F-mutant [TK(HSV1)(Y101F)] acting on the potent antiviral drug 2'-exo-methanocarba-thymidine (MCT) have been performed. The kinetic study reveals a 12-fold K(M) increase for thymidine processed with Y101F as compared to the wild-type TK(HSV1). Furthermore, MCT is a substrate for both wild-type and mutant TK(HSV1). Its binding affinity for TK(HSV1) and TK(HSV1)(Y101F), expressed as K(i), is 11 microM and 51 microM, respectively, whereas the K(i) for human cytosolic thymidine kinase is as high as 1.6 mM, rendering TK(HSV1) a selectivity filter for antiviral activity. Moreover, TK(HSV1)(Y101F) shows a decrease in the quotient of the catalytic efficiency (k(cat)/K(M)) of dT over MCT corresponding to an increased specificity for MCT when compared to the wild-type enzyme. Crystal structures of wild-type and mutant TK(HSV1) in complex with MCT have been determined to resolutions of 1.7 and 2.4 A, respectively. The thymine moiety of MCT binds like the base of dT while the conformationally restricted bicyclo[3.1.0]hexane, mimicking the sugar moiety, assumes a 2'-exo envelope conformation that is flatter than the one observed for the free compound. The hydrogen bond pattern around the sugar-like moiety differs from that of thymidine, revealing the importance of the rigid conformation of MCT with respect to hydrogen bonds. These findings make MCT a lead compound in the design of resistance-repellent drugs for antiviral therapy, and mutant Y101F, in combination with MCT, opens new possibilities for gene therapy.
Department of Applied BioSciences, Swiss Federal Institute of Technology (ETH), Zürich, Switzerland.
A
A
Prota
J
J
Vogt
B
B
Pilger
R
R
Perozzo
C
C
Wurth
V E
VE
Marquez
P
P
Russ
G E
GE
Schulz
G
G
Folkers
L
L
Scapozza
D019943:Amino Acid Substitution / Q000235:genetics; D001667:Binding, Competitive / Q000235:genetics; D001643:Bridged Bicyclo Compounds / Q000737:chemistry*; D003460:Crystallization; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000737:chemistry; D018259:Herpesvirus 1, Human / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000235:genetics*; D010766:Phosphorylation; D011487:Protein Conformation; D013936:Thymidine / Q000031:analogs & derivatives* / Q000737:chemistry*; D013937:Thymidine Kinase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics*; D014443:Tyrosine / Q000235:genetics*
D001643:Bridged Bicyclo Compounds; D004791:Enzyme Inhibitors; D046911:Macromolecular Substances; C046059:bicyclo(3.1.0)hexane; D014443:Tyrosine; D010649:Phenylalanine; D013937:Thymidine Kinase; D013936:Thymidine