RESUMO
The glycolytic enzymes of trypanosomes are attractive drug targets, since the blood-stream form of Trypanosoma brucei lacks a functional citric acid cycle and is dependent solely on glycolysis for its energy requirements. Glyceraldehyde-3-phosphate dehydrogenases (GAPDH) from the pathogenic trypanosomatids T. brucei, Trypanosoma cruzi and Leishmania mexicana are quite similar to each other, and yet have sufficient structural differences compared to the human enzyme to enable the structure-based design of compounds that selectively inhibit all three trypanosomatid enzymes but not the human homologue. Adenosine analogs with substitutions on N-6 of the adenine ring and on the 2' position of the ribose moiety were designed, synthesized and tested for inhibition. Two crystal structures of L. mexicana glyceraldehyde-3-phosphate dehydrogenase in complex with high-affinity inhibitors that also block parasite growth were solved at a resolution of 2.6 A and 3.0 A. The complexes crystallized in the same crystal form, with one and a half tetramers in the crystallographic asymmetric unit. There is clear electron density for the inhibitor in all six copies of the binding site in each of the two structures. The L. mexicana GAPDH subunit exhibits substantial structural plasticity upon binding the inhibitor. Movements of the protein backbone, in response to inhibitor binding, enlarge a cavity at the binding site to accommodate the inhibitor in a classic example of induced fit. The extensive hydrophobic interactions between the protein and the two substituents on the adenine scaffold of the inhibitor provide a plausible explanation for the high affinity of these inhibitors for trypanosomatid GAPDHs.
Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Gliceraldeído-3-Fosfato Desidrogenases/antagonistas & inibidores , Gliceraldeído-3-Fosfato Desidrogenases/química , Leishmania mexicana/enzimologia , Adenosina/análogos & derivados , Adenosina/química , Adenosina/metabolismo , Adenosina/farmacologia , Sítio Alostérico , Animais , Cristalografia por Raios X , Inibidores Enzimáticos/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Humanos , Leishmania mexicana/efeitos dos fármacos , Leishmania mexicana/crescimento & desenvolvimento , Modelos Moleculares , Naftalenos/química , Naftalenos/metabolismo , Naftalenos/farmacologia , Ligação Proteica , Conformação Proteica/efeitos dos fármacos , Subunidades Proteicas , Especificidade da Espécie , Especificidade por SubstratoRESUMO
BACKGROUND: NAD-dependent glycerol-3-phosphate dehydrogenase (GPDH) catalyzes the interconversion of dihydroxyacetone phosphate and L-glycerol-3-phosphate. Although the enzyme has been characterized and cloned from a number of sources, until now no three-dimensional structure has been determined for this enzyme. Although the utility of this enzyme as a drug target against Leishmania mexicana is yet to be established, the critical role played by GPDH in the long slender bloodstream form of the related kinetoplastid Trypanosoma brucei makes it a viable drug target against sleeping sickness. RESULTS: The 1.75 A crystal structure of apo GPDH from L. mexicana was determined by multiwavelength anomalous diffraction (MAD) techniques, and used to solve the 2.8 A holo structure in complex with NADH. Each 39 kDa subunit of the dimeric enzyme contains a 189-residue N-terminal NAD-binding domain and a 156-residue C-terminal substrate-binding domain. Significant parts of both domains share structural similarity with plant acetohydroxyacid isomeroreductase. The discovery of extra, fatty-acid like, density buried inside the C-terminal domain indicates a possible post-translational modification with an associated biological function. CONCLUSIONS: The crystal structure of GPDH from L. mexicana is the first structure of this enzyme from any source and, in view of the sequence identity of 63%, serves as a valid model for the T. brucei enzyme. The differences between the human and trypanosomal enzymes are extensive, with only 29% sequence identity between the parasite and host enzyme, and support the feasibility of exploiting the NADH-binding site to develop selective inhibitors against trypanosomal GPDH. The structure also offers a plausible explanation for the observed inhibition of the T. brucei enzyme by melarsen oxide, the active form of the trypanocidal drugs melarsoprol and cymelarsan.
Assuntos
Glicerolfosfato Desidrogenase/química , Leishmania mexicana/enzimologia , Modelos Moleculares , Proteínas de Protozoários/química , Animais , Sítios de Ligação , Cristalografia por Raios X , Dimerização , Desenho de Fármacos , Evolução Molecular , Glicerol-3-Fosfato Desidrogenase (NAD+) , Glicerolfosfato Desidrogenase/genética , Glicerolfosfato Desidrogenase/metabolismo , Dados de Sequência Molecular , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Homologia de Sequência de Aminoácidos , Tripanossomicidas/química , Tripanossomicidas/metabolismoRESUMO
The structure of glycosomal glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the trypanosomatid parasite Leishmania mexicana in a new crystal form has been determined by X-ray crystallography. The protein crystallizes in space group P21 with one 156 kDa tetramer per asymmetric unit. The model of the protein with bound NAD+s and phosphates has been refined against 81% complete data from 10.0 to 2. 8 A to a crystallographic Rfactor of 0.217. The present structure confirms two key aspects of the previously reported orthorhombic crystal structure of L. mexicana GAPDH (LmGAPDH): the unusual conformation of a loop in the active site, and the repositioning of the inorganic phosphate binding site compared with crystal structures of GAPDHs from other organisms. As the monoclinic crystals of LmGAPDH were grown at a phosphate concentration and pH that were even closer to physiological conditions than were the orthorhombic LmGAPDH crystals, the present structure reinforces the physiological relevance of the active site structure seen in the previous orthorhombic crystal of LmGAPDH.
Assuntos
Gliceraldeído-3-Fosfato Desidrogenases/química , Leishmania mexicana/enzimologia , Animais , Sítios de Ligação , Cristalografia por Raios X , Humanos , Modelos Moleculares , Fosfatos/metabolismo , Conformação ProteicaRESUMO
The structure of glycosomal glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the trypanosomatid parasite Leishmania mexicana has been determined by X-ray crystallography. The protein crystallizes in space group P2(1)2(1)2(1) with unit cell parameters a = 99.0 A, b = 126.5 A, and c = 138.9 A. There is one 156,000 Da protein tetramer per asymmetric unit. The model of the protein with bound NAD+s and phosphates has been refined against 86% complete data from 10.0 to 2.8 A to a crystallographic Rfactor of 0.198. Density modification by noncrystallographic symmetry averaging was used during model building. The final model of the L. mexicana GAPDH tetramer shows small deviations of less than 0.5 degrees from ideal 222 molecular symmetry. The structure of L. mexicana GAPDH is very similar to that of glycosomal GAPDH from the related trypanosomatid Trypanosoma brucei. A significant structural difference between L. mexicana GAPDH and most previously determined GAPDH structures occurs in a loop region located at the active site. This unusual loop conformation in L. mexicana GAPDH occludes the inorganic phosphate binding site which has been seen in previous GAPDH structures. A new inorganic phosphate position is observed in the L. mexicana GAPDH structure. Model building studies indicate that this new anion binding site is well situated for nucleophilic attack of the inorganic phosphate on the thioester intermediate in the GAPDH-catalyzed reaction. Since crystals of L. mexicana GAPDH can be grown reproducibly and diffract much better than those of T. brucei GAPDH, L. mexicana GAPDH will be used as a basis for structure-based drug design targeted against trypanosomatid GAPDHs.