RESUMO
The ternary complex crystal structures of Cryptococcus neoformans and Escherichia coli thymidylate synthase (TS) suggest mechanisms of species-specific inhibition of a highly conserved protein. The 2.1 Angstrom structure of C. neoformans TS cocrystallized with substrate and the cofactor analog CB3717 shows that the binding sites for substrate and cofactor are highly conserved with respect to human TS, but that the structure of the cofactor-binding site of C. neoformans TS is less constrained by surrounding residues. This feature might allow C. neoformans TS to form TS-dUMP-inhibitor complexes with a greater range of antifolates than human TS. 3',3''-Dibromophenol-4-chloro-1,8-naphthalein (GA9) selectively inhibits both E. coli TS and C. neoformans TS (K(i) = 4 microM) over human TS (K(i) >> 245 microM). The E. coli TS-dUMP-GA9 complex is in an open conformation, similar to that of the apoenzyme crystal structure. The GA9-binding site overlaps the binding site of the pABA-glutamyl moiety of the cofactor. The fact that human apoTS can adopt an unusual fold in which the GA9-binding site is disordered may explain the poor affinity of GA9 for the human enzyme. These observations highlight the critical need to incorporate multiple target conformations in any computational attempt to facilitate drug discovery.
Assuntos
Cryptococcus neoformans/enzimologia , Timidilato Sintase/química , Algoritmos , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Dimerização , Desenho de Fármacos , Escherichia coli/metabolismo , Deleção de Genes , Humanos , Cinética , Ligantes , Modelos Químicos , Modelos Moleculares , Conformação Molecular , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos , Software , Especificidade da Espécie , TemperaturaAssuntos
Complexos Multienzimáticos/genética , Mutação , Tetra-Hidrofolato Desidrogenase/genética , Timidilato Sintase/genética , Toxoplasma/genética , Sequência de Aminoácidos , Animais , Antiprotozoários/farmacologia , Western Blotting , Resistência a Medicamentos/genética , Humanos , Modelos Genéticos , Dados de Sequência Molecular , Complexos Multienzimáticos/química , Mutagênese Insercional , Mutação Puntual , Reação em Cadeia da Polimerase , Pirimetamina/farmacologia , Mapeamento por Restrição , Alinhamento de Sequência , Tetra-Hidrofolato Desidrogenase/química , Timidilato Sintase/química , Toxoplasma/efeitos dos fármacos , Toxoplasma/enzimologia , TransfecçãoRESUMO
We have determined the crystal structure of dihydrofolate reductase-thymidylate synthase (DHFR-TS) from Cryptosporidium hominis, revealing a unique linker domain containing an 11-residue alpha-helix that has extensive interactions with the opposite DHFR-TS monomer of the homodimeric enzyme. Analysis of the structure of DHFR-TS from C. hominis and of previously solved structures of DHFR-TS from Plasmodium falciparum and Leishmania major reveals that the linker domain primarily controls the relative orientation of the DHFR and TS domains. Using the tertiary structure of the linker domains, we have been able to place a number of protozoa in two distinct and dissimilar structural families corresponding to two evolutionary families and provide the first structural evidence validating the use of DHFR-TS as a tool of phylogenetic classification. Furthermore, the structure of C. hominis DHFR-TS calls into question surface electrostatic channeling as the universal means of dihydrofolate transport between TS and DHFR in the bifunctional enzyme.
