Báo cáo khoa học: Deciphering the key residues in Plasmodium falciparum b-ketoacyl acyl carrier protein reductase responsible for interactions with Plasmodium falciparum acyl carrier protein

Deciphering The Key Residues In Plasmodium Falciparum ẞ-Ketoacyl Acyl Carrier Protein Reductase Responsible For Interactions With Plasmodium Falciparum Acyl Carrier Protein

Tác giả: Krishanpal Karmodiya, Rahul Modak, Nirakar Sahoo, Syed Sajad and Namita Surolia
Lĩnh vực: Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bangalore, India
Nội dung tài liệu:
Nghiên cứu này tập trung vào việc làm sáng tỏ các gốc axit amin quan trọng trong enzyme β-ketoacyl acyl carrier protein reductase (PfFabG) của ký sinh trùng sốt rét Plasmodium falciparum, vốn chịu trách nhiệm cho sự tương tác với acyl carrier protein (PfACP) của cùng loại ký sinh trùng này. Pathway axit béo loại II (FAS) của P. falciparum là một mục tiêu đầy hứa hẹn cho việc phát triển các loại thuốc chống sốt rét mới do sự khác biệt của nó so với pathway loại I ở người. Nghiên cứu đã sử dụng các phương pháp chuyên sâu, bao gồm đo huỳnh quang nội tại và cộng hưởng plasmon bề mặt (SPR), để điều tra sự tương tác trực tiếp giữa PfFabG và PfACP. Các nhà nghiên cứu đã tạo ra các dạng đột biến của PfFabG, cụ thể là các gốc Arg187, Arg190 và Arg230, để xác định vai trò của chúng trong việc liên kết mạnh mẽ và đặc hiệu. Kết quả cho thấy các gốc Arg187 và Arg230, nằm trong một vùng kỵ nước gần cửa vào vị trí hoạt động của PfFabG, là những gốc quan trọng cho tương tác PfFabG-PfACP. Các phát hiện này nhấn mạnh tầm quan trọng của vùng mang điện tích dương/kỵ nước liền kề với các khoang vị trí hoạt động của PfFabG đối với sự tương tác với PfACP.

Mục lục chi tiết:
– Keywords: fatty acid synthase; fluorescence; malaria; protein-protein interactions; surface plasmon resonance
– Correspondence
– (Received 14 June 2008, revised 23 July 2008, accepted 25 July 2008)
– doi:10.1111/j.1742-4658.2008.06608.x
– The human malaria-causing parasite Plasmodium falciparum harbors the type II fatty acid synthase (FAS) [1,2], which is essential for its sustenance and survival.
– In contrast to the multifunctional FAS enzyme in the type I pathway operating in humans [3], the type II FAS system has discrete enzymes for each step of the pathway.
– Type II FAS in P. falciparum is one of the pathways specific to its ‘plastid’ and has been validated as a unique target for developing new antimalarials [4-8].
– During the elongation cycle of type II FAS, the growing acyl chain, i.e. butyryl-acyl carrier protein (ACP), is elongated successively in each round by two carbon units by the action of four enzymes acting consecutively.
– First, ẞ-ketoacyl ACP synthase (either FabB or FabF) elongates the acyl-ACP of the Cn acyl chain to a Cn + 2, ẞ-ketoacyl form.
– The ẞ-ketoacyl-ACP thus formed is reduced to ẞ-hydroxyacyl-ACP by an NADPH-dependent ẞ-ketoacyl ACP reductase (FabG).
– The ẞ-hydroxyacyl group is then dehydrated to an enoyl-ACP by a ẞ-hydroxyacyl ACP dehydratase (FabZ or FabA).
– Reduction of the enoyl group by an enoyl ACP reductase (FabI, FabK or FabL) finally produces Cn + 2 acyl-ACP, which can either re-enter the elongation cycle, or be hydrolyzed to ACO and the acyl moiety for the synthesis of phospholipids or sphingolipids, or become diverted for other modifications [9].
– All of the enzymes participating in type II FAS interact, but not much is known about the residues involved in interactions.
– Plasmodium falciparum ACP (PfACP) is a small protein, with a flexible conformation, which shuttles the substrates between the enzymes of the pathway.
– PfACP is a nuclear-encoded and plastid-targeted protein of 137 amino acids that includes leader and transit sequences.
– Mature PfACP consists of 79 amino acids (residues 58-137) with a preponderance of acidic residues [10].
– Two-dimensional NMR [11] has revealed that PfACP has a defined but flexible tertiary structure dominated by four a-helices located at residues 4-15 (helix I), 37-51 (helix II), 57-61 (helix II’) and 66-74 (helix III), all connected by loops with a long structured turn between helix I and helix II.
– The unusually mobile structure of ACP can be best represented as a dynamic equilibrium between two conformers.
– Highly mobile portions of PfACP include the loop regions and helix II.
– FabG is highly conserved across species, and is the only known isoform that functions as a ketoacyl reductase in the type II FAS system.
– Recently, the crystal structure of P. falciparum FabG (PfFabG) has been solved [12], and suggests that the interactions of PfFabG with the 4′-phosphopantetheine moiety of PfACP are hydrophobic in nature.
– Plasmodium FabG with a lone tryptophan provides an ideal system with which to study ligand-induced conformational changes by monitoring the change in its intrinsic fluorescence.
– In these studies, we have investigated the interactions between PfFabG and PfACP using a combination of computational, biochemical and biophysical methods.
– We have been able to identify specific surface features on PfFabG that are critical for these interactions.
– In PfFabG, Arg187 and Arg230 are located in a hydrophobic patch adjacent to the active site entrance of PfFabG, whereas Arg190 is located away from the active site.
– Hence, to characterize the role played by these residues in the interactions of PfFabG and PfACP, we generated the following mutants: R187E, R230E, R187A/R230A, R187E/R230E, R190E, R190A and R230K.
– We also generated R187K, as this is conservatively substituted throughout the apicomplexan group (present as Lys187 in other species of Plasmodium).
– In the type II FAS pathway, the growing acyl intermediates are attached to the terminal sulfhydryl of the 4′-phosphopentatheine prosthetic group [13], which is attached via a phosphodiester linkage to the Ser37 located at the beginning of helix II.
– The primary gene product is an apoprotein that is converted to holo-ACP (ACP) by the transfer of the 4′-phosphopentatheine moiety of CoA to Ser37 by holo-ACP synthase.
– ACP performs two functions: first, it sequesters the growing acyl chain from the aqueous environment; and second, upon binding to one of the type II FAS proteins, it releases its grip on the fatty acid, which is inserted into the active site of the enzyme.
– ACPs from various natural sources share significant primary sequence similarity, particularly at the prosthetic group attachment site, extending to helix II.
– However, the individual ACP-binding partners do not share any common ACP-binding motif.
– The molecular details that govern the specific interactions between Plasmodium ACP and type II FAS enzymes are poorly understood.
– Here, we report subtle aspects of the interactions between PfFabG and PfACP, with emphasis on association constants and number of binding sites with reference to the cofactor NADPH.
– The site-directed mutagenesis studies reveal that both electrostatic and hydrophobic interactions play important roles in PfFabG-PfACP complex formation.
– Results
– Identification of residues putatively involved in the PfFabG-PfACP interaction
– Multiple sequence alignment indicates that the FabG sequences from different species, including plants and bacteria, share a high degree of sequence identity (Fig. 1A).
– The crystal structures of FabG enzymes from Escherichia coli [14], Brassica napus [15] and P. falciparum [12] are also homologous, and show FabG to be a tetramer consisting of two homodimers of monomers arranged in a head-to-tail configuration.
– The crystal structure of FabG from E. coli shows that there is a conserved positively charged patch on its surface [14,16].
– This positively charged patch is positioned at the entrance of the active site and is involved in recognition of the highly conserved and negatively charged a2 helix of ACP.
– This patch is identical in E. coli FabG, Plasmodium FabG and counterparts from plants.
– Mutagenesis of E. coli FabG showed that two arginine residues (Arg29 and Arg172) present in this patch are central to the binding of ACP [16].
– Multiple sequence alignment of FabG sequences from different species shows that these residues are conserved in PfFabG too (Arg187 and Arg230, respectively) (Fig. 1A).
– Three residues of PfFabG selected for the mutagenesis studies, namely Arg187, Agr190 and Arg230, were highly conserved in all species of Plasmodium.
– Analysis of the Plasmodium FabG crystal structure shows that the conserved residues Arg187 and Arg230 are located at the surface, near its active site entrance (Fig. 1C).
– We replaced the positively charged arginines with glutamates to introduce electrostatic repulsion between PfFabG and PfACP and to test whether PfACP associates with PfFabG over the entire predicted surface.
– We also changed these positively charged residues to alanines to determine which of the electrostatic interactions are important for promoting the binding to PfACP.
– Interestingly, within the Plasmodium genus, Arg187 is substituted (Fig. 1B) with a lysine.
– Expression and purification of PfFabG, PfFabG mutants and PfACP
– The recombinant wild-type PfFabG, its mutants and PfACP were purified to homogeneity using an Ni2+-nitrilotriacetic acid affinity column as previously described [17,18].
– Figure 2 shows the apparent electrophoretic homogeneity of the purified proteins.
– The purified proteins on SDS/PAGE yielded a monomeric Mr of 31 000 ± 1000 for PfFabG as well as for PfFabG mutants.
– Gel filtration and CD analyses of the mutants
– Changes in the overall shape or the quaternary structure of the molecule, potentially introduced by mutagenesis, were first probed using size exclusion chromatography.
– Wild-type PfFabG was eluted as a single peak at a volume of 13.67 mL on a Superdex-200 gel filtration column [17].
– The PfFabG mutants were also eluted at the retention volume of their wild-type counterpart.
– The elution positions of the wild-type and mutants of PfFabG corresponded to a relative molecular mass of 110 kDa (± 10 kDa), indicating that the enzymes are homotetramers and that the mutations did not alter the overall shape or the quaternary structure of PfFabG.
– CD spectroscopy was used to investigate potential perturbations in the secondary and tertiary structure of PfFabG mutants.
– CD spectra of wild-type PfFabG and the PfFabG mutants were superimposable (Fig. S1), suggesting that the relative contents of a-helical and ẞ-sheet secondary structure in the PfFabG mutants are not changed as a result of the individual point mutations.
– Kinetic analyses of the PfFabG mutants
– In order to evaluate the effects of the mutations on the specific activity of PfFabG, we used an ACP-indepen-dent spectrophotometric assay, where acetoacetyl-CoA was used as a substrate in place of acetoacetyl-ACP, and the disappearance of NADPH was monitored at 340 nm.
– As can be seen in Table 1, the kinetic constants (Km and ACP-independent specific activities) of the R187A, R187E, R230A, R230E, R187A/R230A and R187E/R230E mutants remained largely unchanged with respect to wild-type PfFabG.
– Wild-type PfFabG shows less activity with acetoacetyl-CoA than with acetoacetyl-ACP.
– All the mutants exhibited very poor activity in the ACP-dependent spectroscopic assay, but not in the ACP-independent spectroscopic assay, which shows that PfFabG mutants are selec-tively compromised for utilization of the acyl-ACP substrate (acetoacetyl-ACP).
– The R230E and R187E/R230E mutants had higher Km values of 0.49 mm and 0.57 mm, respectively, than wild-type PfFabG for acetoacetyl-CoA (0.43 mm) [17].
– Moreover, the point mutation R187K gives similar results to those for wild-type PfFabG.
– The ACP-dependent activity assay clearly showed the involvement of the two surface arginine residues of PfFabG in the interaction with PfACP.
– In order to determine the ability of PfACP to function as the inhibitor, we used a spectrophotometric assay, utilizing acetoacetyl-CoA, with the indicated concentrations of PfACP.
– As can be seen in Fig. 3, wild-type PfFabG showed inhibition with increasing concentrations of PfACP, whereas no inhibition with the R187E, R230E, R187A and R230A mutants was observed.
– The effect was more deleterious when arginine was changed to glutamate than when it was changed to the neutral residue alanine (Table 2).
– Interaction of PfFabG mutants with wild-type PfACP monitored by measuring intrinsic PfFabG fluorescence
– The intrinsic fluorescence of PfFabG decreased when it was titrated with increasing concentrations of PfACP.
– As reported earlier [17], binding of PfACP to PfFabG, as analyzed by quenching of its fluorescence at 334 nm, gave an association constant of 400 nm-1 with n = 1.
– The value of Ka was determined for other mutants using nonlinear least squares fit of the data, using the Adair equation with one to four equivalent and independent, as well as equivalent and interdependent, binding sites (n).
– The Ka values for binding of the R187A and R230A mutants were, respectively, 150 and 82 nm-1.
– Thus, mutation of Arg187 and Arg230 to alanine decreased the binding affinities by three-fold and five-fold respectively.
– The Ka values for the binding decreased even more dra-matically to 93 and 9 nm-1, respectively, in the R187E and R230E mutants.
– Apparently, mutation of Arg187 and Arg230 to an acidic residue, glutamate, diminishes the strength of the PfACP-PfFabG inter-action in a relatively more significant manner than their replacement by a neutral alanine residue.
– The effect was more drastic when both the residues were converted to glutamate, there being an 80-fold reduc-tion in association constant (Table 2).
– The data shown here are in close agreement with those from the ACP-dependent assay.
– The affinity of wild-type PfACP increased three-fold (Ka = 1.10 μμ-1) in the presence of NADPH, and the number of binding sites increased from one to two, whereas in all PfFabG mutants examined except R187K, the number of binding sites remained the same, with decreased binding of the PfFabG mutants to PfACP in the presence of NADPH (Table 2).
– The maximum effect was observed on binding of the R230E mutant and the double mutant R187E/R230E, with association constants of 110.4 and 92 nm-1, respectively.
– Interaction of the PfFabG mutants with wild-type PfACP monitored by surface plasmon resonance (SPR)
– The PfFabG-PfACP interaction data were further ver-ified by direct measurement of binding of these two proteins by SPR (BIAcore).
– Wild-type PfFabG and its mutants were immobilized on the nitrilotriacetic acid sensor chip surface, following the manufacturer’s pro-tocol [19].
– Approximately 330–350 resonance units (RU) of His-tagged PfFabG was immobilized in each channel of the sensor chip.
– A continuous flow of buf-fer was maintained on the chip surface until a stable baseline was reached.
– All of the binding studies were conducted at 20 °C.
– Various concentrations of His-tag cleaved PfACP (ranging from 1 to 200 µm) were passed over the surface of the chip.
– The PfFabG immobilized surface devoid of the PfACP from a previous reaction could be regenerated by passing the buffer alone.
– A typical sensorgram for the binding of varying concentrations of PfACP to immobilized PfFabG is shown in Fig. 4A.
– The occurrence of an enhancement in the RUs is indic-ative of the increase in mass on the chip surface, which indicates binding.
– As shown in Fig. 4B, wild-type PfFabG showed strong binding, whereas the R230E and R187A/R230A mutants did not show any signifi-cant binding at similar PfACP concentrations.
– The apparent binding and association constants are shown in Table 2.
– There was a three-fold enhancement in PfACP binding to wild-type PfFabG in the presence of NADPH.
– PfACP binding to the mutant PfFabGs was also enhanced in the presence of NADPH.
– There was a three-fold to 100-fold decrease in the affinity of bind-ing of mutant PfFabGs to PfACP as compared to wild-type PfFabG (Table 2).
– The apparent binding constants determined by fluorescence quenching and SPR experiments for PfFabG and PfACP are essen-tially similar.
– Fluorescence quenching corroborated with SPR experiments shows the involvement of these residues in the PfFabG-PfACP interaction.
– Allosteric binding of NADPH to PfFabG mutants in the presence of PfACP
– The Ka value for the binding of NADPH to PfFabG, with n = 4, was found to be 40.90 µm-1,