Role of tryptophan-1 in hemolytic and phospholipase C activities of Clostridium perfringens alpha-toxin

Replacement of the Trp-1 in Clostridium perfringens alpha-toxin with tyrosine caused no effect on hemolytic and phospholipase C (PLC) activities or on binding to the zinc ion, but that of the residue with alanine, glycine and histidine led to drastic decreases in these activities and a significant reduction in binding to the zinc ion. The hemolytic and PLC activities of W1H and W1A were significantly increased by the preincubation of these variant toxins with zinc ions, but the preincubation of W1G with the metal ion caused little effect on these activities. Gly-Ile-alpha-toxin, which contained an additional Gly-Ile linked to the N-terminal amino acid of alpha-toxin, did not show hemolytic activity, but showed about 6% PLC activity of the wild-type toxin. A mutant toxin, which contained an additional Gly-Ile linked to the N-terminus of a protein lacking 4 N-terminal residues of alpha-toxin, showed about 1 and 6% hemolytic and PLC activities of the wild-type toxin, respectively. Incubation of the mutant toxin with zinc ions caused a significant increase in PLC activity. These observations suggested that Trp-1 is not essential for toxin activity, but plays a role in binding to zinc ions.     

Engineering GST M2-2 for high activity with indene 1,2-oxide and indication of an H-site residue sustaining catalytic promiscuity

The substrate-binding H-site of human glutathione transferase (GST) M2-2 was subjected to iterative saturation mutagenesis in order to obtain an efficient enzyme with the novel epoxide substrate indene 1,2-oxide. Residues 10, 116, and 210 were targeted, and the activities with the alternative substrates, benzyl isothiocyanate and the prodrug azathioprine, undergoing divergent chemical reactions were monitored for comparison. In general, increased activities were found when the smaller residues Gly, Ser, and Ala replaced the original Thr210. The most active mutant T210G was further mutated at position 116, but no mutant showed enhanced catalytic activity. However, saturation mutagenesis of position 10 identified one double mutant T210G/I10C with 100-fold higher specific activity with indene 1,2-oxide than wild-type GST M2-2. This enhanced epoxide activity of 50 μmol min^− 1 mg^− 1 resulted primarily from an increased k_cat value (70 s^− 1). The specific activity is 24-fold higher than that of wild-type GST M1-1, which is otherwise the most proficient GST enzyme with epoxide substrates. A second double mutant T210G/I10W displayed 30-fold increased activity with azathioprine, 0.56 μmol min^− 1 mg^− 1. In both double mutants, the replacement of Ile10 led to narrowed acceptance of alternative substrates. Ile10 is evolutionarily conserved in related class Mu GSTs. Conservation usually indicates preservation of a particular function, and in the Mu class, it would appear that the conserved Ile10 is not necessary to maintain catalytic functions but to prevent loss of broad substrate acceptance. In summary, our data underscore the facile transition between alternative substrate selectivity profiles in GSTs by a few mutations.     

Molecular Dynamics/Free Energy Perturbation Studies of the Thermostable V74I Mutant of Ribonuclease HI

Abstract

Recent site-directed mutagenesis and thermodynamic studies have shown that the V74I mutant of Escherichia coli ribonuclease HI (RNase HI) is more stable than the wild type protein [Ishikawa et al., Biochemistry 32, 6171 (1993)]. In order to clarify the stabilization mechanism of this mutant, we calculated the free energy change due to the mutation Val 74→Ile in both the native and denatured states by free energy perturbations based on molecular dynamics (MD) simulations. We carried out inclusive MD simulations for the protein in water; i.e., fully solvated, no artificial constraints applied, and all long-range Coulomb interactions included. We found that the free energy of the mutant increased slightly relative to the wild type, in the native state by 1.60 kcal/mol, and in the denatured state by 2.25 kcal/mol. The unfolding free energy increment of the mutant (0.66 ± 0.19 kcal/mol) was in good agreement with the experimental value (0.6 kcal/mol). The hysteresis error in the free energy calculations, i.e., forward and reverse perturbations, was only ±0.19 kcal/mol. These results show that the V74I mutant is stabilized relative to the wild type by the increased free energy of the denatured state and not by a decrease in the free energy of the native state as had been proposed earlier based on the mutant X-ray structure. It was found that the stabilization was caused by a loss of solvation energy in the mutant denatured state and not by improved packing interactions inside the native protein.     

Isolation and characteristics of staphylococcal alpha-toxin

A method for isolation of staphylococcal alpha-toxin preparations has been elaborated. Characteristics of the toxin isolated by the method are as follows: mol. mass = 35 Kd; HU = 0.1 microgram; DnD= 0.1 microgram; LD50 = 2 micrograms. It is for the first time that alpha-toxin was fragmented by papain and digested by alpha, gamma-chemotrypsin. The papain fragments (18.5 and 15 Kd) retained lethal activity but lost hemolytic and dermonecrotic activities. Alpha, gamma-chemotryptic digested fragments (18 and 15 Kd) retained hemolytic and lethal effects, but lost their dermonecrotic activity.     

Use of an EZ-Tn5-Based Random Mutagenesis System to Identify a Novel Toxin Regulatory Locus in Clostridium perfringens Strain 13

Background

Although useful for probing bacterial pathogenesis and physiology, current random mutagenesis systems suffer limitations for studying the toxin-producing bacterium Clostridium perfringens.

Methodology/Principal Findings

An EZ-Tn5-based random mutagenesis approach was developed for use in C. perfringens. This mutagenesis system identified a new regulatory locus controlling toxin production by strain 13, a C. perfringens type A strain. The novel locus, encoding proteins with homology to the AgrB and AgrD components of the Agr quorum sensing system of Staphylococcus aureus and two hypothetical proteins, was found to regulate early production of both alpha toxin and perfringolysin O (PFO) by strain 13. PFO production by the strain 13 ΔagrB mutant could be restored by genetic complementation or by physical complementation, i.e. by co-culture of the strain 13 ΔagrB mutant with a pfoA mutant of either strain 13 or C. perfringens type C CN3685. A similar AgrB- and AgrD-encoding locus is identifiable in all sequenced C. perfringens strains, including type B, C, D, and E isolates, suggesting this regulatory locus contributes to toxin regulation by most C. perfringens strains. In strain 13, the agrB and agrD genes were found to be co-transcribed in an operon with two upstream genes encoding hypothetical proteins.

Conclusions/Significance

The new Tn5-based random mutagenesis system developed in this study is more efficient and random than previously reported C. perfringens random mutagenesis approaches. It allowed identification of a novel C. perfringens toxin regulatory locus with homology to the Agr system of S. aureus and which functions as expected of an Agr-like quorum sensing system. Since previous studies have shown that alpha toxin and perfringolysin O are responsible for strain 13-induced clostridial myonecrosis in the mouse model, the new agr regulatory locus may have importance for strain 13 virulence.     

Identification and molecular analysis of a locus that regulates extracellular toxin production in Clostridium perfringens

The anaerobic bacterium Clostridium perfringens mediates clostridial myonecrosis, or gas gangrene, by producing a number of extracellular toxins and enzymes. Transposon mutagenesis with Tn916 was used to isolate a pleiotropic mutant of C. perfringens that produced reduced levels of phospholipase C, protease and sialidase, and did not produce any detectable perfringolysin O activity. Southern hybridization revealed that a single copy of Tn916 had inserted into a 2.7 kb Hindlll fragment in the C. perfringens chromosome. A 4.3 kb Pstl fragment, which spanned the Tn916 insertion site, was cloned from the wild-type strain. When subcloned into a shuttle vector and introduced into C. perfringens this fragment was able to complement the Tn916-derived mutation. Transformation of the mutant with plasmids containing the 2.7 kb Hindlll fragment, or the 4.3 kb Pstl fragment resulted in toxin and enzyme levels greater than or equal to those of the wild-type strain. The Pstl fragment was sequenced and found to potentially encode seven open reading frames, two of which appeared to be arranged in an operon and shared sequence similarity with members of two-component signal transduction systems. The putative virR gene encoded a protein with a deduced molecular weight of 30140, and with sequence similarity to activators in the response regulator family of proteins. The next gene, virS, into which Tn916 had inserted, was predicted to encode a membrane-spanning protein with a deduced molecular weight of 51 274. The putative VirS protein had sequence similarity to sensor proteins and also contained a histidine residue highly conserved in the histidine protein kinase family of sensor proteins. Virulence studies carried out using a mouse model implicated the virS gene in the pathogenesis of histotoxic C. perfringens infections. It was concluded that a two-component sensor regulator system that activated the expression of a number of extracellular toxins and enzymes involved In virulence had been cloned and sequenced. A model that described the regulation of extracellular toxin production in C. perfringens was constructed.     

Improvement in the alkaline stability of subtilisin using an efficient random mutagenesis and screening procedure

An efficient random mutagenesis procedure coupled to a replica plate screen facilitated the isolation of mutant subtilisins from Bacillus amyloliquefaciens that had altered autolytic stability under alkaline conditions. Out of about 4000 clones screened, approximately 70 produced subtilisins with reduced stability (negatives). Two clones produced a more stable subtilisin (positives) and were identified as having a single mutation, either Ile107Val or Lys213Arg (the wild-type amino acid is followed by the codon position and the mutant amino acid). One of the negative mutants, Met50Val, was at a site where other homologous subtilisins contained a Phe. When the Met50Phe mutation was introduced into the B. amyloliquefaciens gene, the mutant subtilisin was more alkaline stable. The double mutant (Ile107Val/Lys213Arg) was more stable than the isolated single mutant parents. The triple mutant (Met50Phe/Ile107Val/Lys213Arg) was even more stable than Ile107Val/Lys213Arg (up to two times the autolytic half-time of wild-type at pH 12). These studies demonstrate the feasibility for improving the alkaline stability of proteins by random mutagenesis and identifying potential sites where substitutions from homologous proteins can improve alkaline stability.     

Leucine 332 influences the CO2/O2 specificity factor of ribulose-1,5-bisphosphate carboxylase/oxygenase from Anacystis nidulans.

The role of Leu 332 in ribulose-1,5-bisphosphate carboxylase/oxygenase from the cyanobacterium Anacystis nidulans was investigated by site-directed mutagenesis. Substitutions of this residue with Met, Ile, Val, Thr, or Ala decreased the CO2/O2 specificity factor by as much as 67% and 96% for the Ile mutant in the presence of Mg2+ and Mn2+, respectively. For the Met, Ile, and Ala mutants in the presence of Mg2+, no loss of oxygenase activity was observed despite the loss of greater than 65% of the carboxylase activity relative to the wild-type enzyme. In the presence of Mn2+, carboxylase activities for mutant enzymes were reduced to approximately the same degree as was observed in the presence of Mg2+, although oxygenase activities were also reduced to similar extents as carboxylase activities. Only minor changes in Km(RuBP) were observed for all mutants in the presence of Mg2+ relative to the wild-type enzyme, indicating that Leu 332 does not function in RuBP binding. These results suggest that in the presence of Mg2+, Leu 332 contributes to the stabilization of the transition state for the carboxylase reaction, and demonstrate that it is possible to affect only one of the activities of this bifunctional enzyme.     

Protein stability and mutations in the axial methionine loop of a minimal cytochrome<Emphasis Type="Italic"> c</Emphasis>

The minimal mono-heme ferricytochrome c from Bacillus pasteurii, containing 71 amino acids, has been further investigated through mutagenesis of different positions in the loop containing the iron ligand Met71. These mutations have been designed to sample different aspects of the loop structure, in order to obtain insights into the determinants of the stability of the iron(III) environment. In particular, positions 68, 72 and 75 have been essayed. Gln68 has been mutated to Lys to provide a suitable alternate ligand that can displace Met71 under denaturing conditions. Pro72 has been mutated to Gly and Ala to modify the range of allowed backbone conformations. Ile75, which is in van der Waals contact with Met71 and partly shields a long-lived water molecule in a protein cavity, has been substituted by Val and Ala to affect the network of inter-residue interactions around the metal site. The different contributions of the above amino acids to protein parameters such as structure, redox potential and the overall stability against unfolding with guanidinium hydrochloride are analyzed. While the structure remains essentially the same, the stability decreases with mutations. The comparison with mitochondrial c-type cytochromes is instructive.Abbreviations Bpcytc soluble fragment of cytochrome c₅₅₃ from Bacillus pasteurii - GdmCl guanidinium chloride - I75A Ile75 to Ala mutant - I75V Ile75 to Val mutant - P72A Pro72 to Ala mutant - P72G Pro72 to Gly mutant - Q68K Gln75 to Lys mutant - WT wild type     

Identification,characterization and functional expression of a tyrosine ammonia-lyase and its mutants from the photosynthetic bacterium <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

A tyrosine ammonia-lyase (TAL) enzyme from the photosynthetic bacterium Rhodobacter sphaeroides (RsTAL) was identified, cloned and functionally expressed in Escherichia coli, where conversion of tyrosine to p-hydroxycinnamic acid (pHCA) was demonstrated. The RsTAL enzyme is implicated in production of pHCA, which serves as the cofactor for synthesis of the photoactive yellow protein (PYP) in photosynthetic bacteria. The wild type RsTAL enzyme, while accepting both tyrosine and phenylalanine as substrate, prefers tyrosine, but a serendipitous RsTAL mutant identified during PCR amplification of the RsTAL gene, demonstrates much higher preference for phenylalanine as substrate and deaminates it to produces cinnamic acid. Sequence analysis showed the presence of three mutations: Met4 → Ile, Ile325 → Val and Val409 → Met in this mutant. Sequence comparison with Rhodobacter capsulatus TAL (RcTAL) shows that Val409 is conserved between RcTAL and RsTAL. Two single mutants of RsTAL, Val409 → Met and Val 409 → Ile, generated by site-directed mutagenesis, demonstrate greater preference for phenylalanine compared to the wild type enzyme. Our studies illustrate that relatively minor changes in the primary structure of an ammonia-lyase enzyme can significantly affect its substrate specificity.     

An N-terminal Region of LukF of Staphylococcal Leukocidin/γ-Hemolysin Crucial for the Biological Activity of the Toxin

The two staphylococcal bi-component toxins, leukocidin and γ-hemolysin share LukF [Kamio et al, FEBS Lett., 321, 15-18 (1993)]. This report identifies the pivotal amino acid residues in the N-terminal region of LukF for the leukocytolytic and hemolytic activities in the presence of LukS and Hlg2, respectively, measuring the toxin activiy of a series of LukF mutants with truncated N-terminals. The data obtained showed that the LukF mutant TF21, lacking 20 amino acid residues at the N-terminus of LukF, failed to have any hemolytic activity and had less 10% leukocytolytic activity than that of the intact LukF, while 16-residue truncations retained both toxin activities without loss. The LukF mutants lacking 18- through 19-residue segments from the N-terminus showed low toxin activity on both target cells. All mutants having no toxin activity were also not capable of binding to the human erythrocytes. It can thus be concluded that the 3-residue segment, L¹⁸Y¹⁹K²⁰ of LukF is crucial for the biological activity of the toxin.     

Influence of membrane fluidity on the assembly of Staphylococcus aureus alpha-toxin, a channel-forming protein, in liposome membrane.

By use of multilamellar phosphatidylcholine (PC) liposomes of different acyl composition and cholesterol content as model membranes, we studied whether or not membrane fluidity affects the assembly process of Staphylococcus aureus alpha-toxin. Under conditions using fluid and solid membranes, we assayed accessibility (or hemolytic activity) of liposome-bound alpha-toxin to rabbit erythrocytes added, hexamerization of membrane-bound toxin using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nondenaturating conditions, and susceptibility of liposome-bound toxin to trypsin digestion. Our data indicated 1) that alpha-toxin bound to PC membrane as a hemolytically active monomer (or reversibly bound state); 2) that when the membrane was fluidized either by phase transition of PC or by inclusion of cholesterol over 20 mol %, the hemolytically active monomer of the toxin was irreversibly converted to nonhemolytic monomer (and/or unstable oligomer) in a first-order kinetics with a t1/2 of about 1 min, and thereafter hexamerization of the toxin gradually proceeded in the following 60-90 min; 3) that alpha-toxin might have different topology and/or conformation in PC membrane, depending on the presence or absence of cholesterol in the PC membrane; and 4) that coexistence of unsaturated acyl chain-carrying PC and cholesterol was a prerequisite for efficient hexamerization of alpha-toxin in membrane. Thus, increase in membrane fluidity promoted the assembly process of S. aureus alpha-toxin.     

Perturbations of the T1 copper site in the CotA laccase from Bacillus subtilis: structural, biochemical, enzymatic and stability studies

Site-directed mutagenesis has been used to replace Met502 in CotA laccase by the residues leucine and phenylalanine. X-ray structural comparison of M502L and M502F mutants with the wild-type CotA shows that the geometry of the T1 copper site is maintained as well as the overall fold of the proteins. The replacement of the weak so-called axial ligand of the T1 site leads to an increase in the redox potential by approximately 100 mV relative to that of the wild-type enzyme (E ⁰=455 mV). However the M502L mutant exhibits a twofold to fourfold decrease in the k _cat values for the all substrates tested and the catalytic activity in M502F is even more severely compromised; 10% activity and 0.15–0.05% for the non-phenolic substrates and for the phenolic substrates tested when compared with the wild-type enzyme. T1 copper depletion is a key event in the inactivation and thus it is a determinant of the thermodynamic stability of wild-type and mutant proteins. Whilst the unfolding of the tertiary structure in the wild-type enzyme is a two-state process displaying a midpoint at a guanidinium hydrochloride concentration of 4.6 M and a free-energy exchange in water of 10 kcal/mol, the unfolding for both mutant enzymes is clearly not a two-state process. At 1.9 M guanidinium hydrochloride, half of the molecules are in an intermediate conformation, only slightly less stable than the native state (approximately 1.4 kcal/mol). The T1 copper centre clearly plays a key role, from the structural, catalytic and stability viewpoints, in the regulation of CotA laccase activity.     

A recombinant carboxy‐terminal domain of alpha‐toxin protects mice against Clostridium perfringens

Clostridium perfringens alpha‐toxin (CP, 370 residues) is one of the main agents involved in the development of gas gangrene. In this study, the immunogenicity and protective efficacy of the C‐terminal domain (CP251‐370) of the toxin and phospholipase C (PLC; CB, 372 residues) of Clostridum bifermentans isolated from cases of clostridium necrosis were examined. The recombinant proteins were expressed as glutathione S‐transferase (GST) fusion proteins. Antibodies that cross‐reacted with alpha‐toxin were produced after immunization with recombinant proteins including GST‐CP251‐370, GST‐CP281‐370, GST‐CP311‐370, CB1‐372 and GST‐CB251‐372. Anti‐GST‐CP251‐370, anti‐GST‐CP281‐370 and anti‐GST‐CP311‐370 sera neutralized both the PLC and hemolytic activities of alpha‐toxin, whereas anti‐CB1‐372 and anti‐GST‐CB251‐372 weakly neutralized these activities. Immunization with GST‐CP251‐370 and GST‐CP281‐370 provided protection against the lethal effects of the toxin and C. perfringens type A NCTC8237. Partial protection from the toxin and C. perfringens was elicited by immunization with GST‐CP311‐370 and CB1‐372. GST‐CP251‐370 and GST‐CP281‐370 are promising candidates for vaccines for clostridial‐induced gas gangrene.     

Studies on the factors affecting the growth and hemolytic activity of <Emphasis Type="Italic">Anabaena variabilis</Emphasis>

The hemolytic activity of the cell-free culture supernatant of Anabaena variabilis OL S1 was investigated using the hemolysis of rabbit erythrocytes as an assay. The culture medium of A. variabilis started to exhibit hemolytic activity at the late exponential growth phase, and maximized at the stationary phase. The hemolytic toxin is heat-stable and can be extracted in dichloromethane. The hemolytic activities under different temperature, light intensity and pH showed a high correlation with the cell densities (r=0.965, 0.951, 0.865, respectively), and the optimum condition is 28~30°C, pH 7.5~8.0, light intensity 120 μmol photons m⁻²s⁻¹. The addition of 10~20 μg mL⁻¹ chloramphenicol, an inhibitor of protein synthesis, exhibited no marked suppression on the hemolytic activity. The supplement of 1~20 μg mL⁻¹ glycerol increased the hemolytic activity significantly, suggesting that synthesis of hemolysin was dependent on carbohydrate and lipid metabolism. The spectrum of erythrocyte sensitivity to the hemolysin indicated that rabbit erythrocytes were more sensitive to the hemolysin than were rat and human erythrocytes. Goldfish and cat erythrocytes were, however, insensitive to the hemolytic toxin of A. variabilis.     

Construction of an Alpha Toxin Gene Knockout Mutant of Clostridium perfringens Type A by Use of a Mobile Group II Intron

In developing Clostridium perfringens as a safe vaccine vector, the alpha toxin gene (plc) in the bacterial chromosome must be permanently inactivated. Disrupting genes in C. perfringens by traditional mutagenesis methods is very difficult. Therefore, we developed a new strategy using group II intron-based Target-Tron technology to inactivate the plc gene in C. perfringens ATCC 3624. Western blot analysis showed no production of alpha toxin protein in the culture supernatant of the plc mutant. Advantages of this technology, such as site specificity, relatively high frequency of insertion, and introduction of no antibiotic resistance genes into the chromosome, could facilitate construction of other C. perfringens mutants.     

Identification of residues critical for toxicity in Clostridium perfringens phospholipase C, the key toxin in gas gangrene.

Clostridium perfringens phospholipase C (PLC), also called alpha-toxin, is the major virulence factor in the pathogenesis of gas gangrene. The toxic activities of genetically engineered alpha-toxin variants harboring single amino-acid substitutions in three loops of its C-terminal domain were studied. The substitutions were made in aspartic acid residues which bind calcium, and tyrosine residues of the putative membrane-interacting region. The variants D269N and D336N had less than 20% of the hemolytic activity and displayed a cytotoxic potency 103-fold lower than that of the wild-type toxin. The variants in which Tyr275, Tyr307, and Tyr331 were substituted by Asn, Phe, or Leu had 11-73% of the hemolytic activity and exhibited a cytotoxic potency 102- to 105-fold lower than that of the wild-type toxin. The results demonstrated that the sphingomyelinase activity and the C-terminal domain are required for myotoxicity in vivo and that the variants D269N, D336N, Y275N, Y307F, and Y331L had less than 12% of the myotoxic activity displayed by the wild-type toxin. This work therefore identifies residues critical for the toxic activities of C. perfringens PLC and provides new insights toward understanding the mechanism of action of this toxin at a molecular level.     

Comparison of the metabolic activities of four wild-type <Emphasis Type="Italic">Clostridium perfringens</Emphasis> strains with their gatifloxacin-selected resistant mutants

The production of short-chain fatty acids, reductive enzymes, and hydrolytic enzymes by four gatifloxacin-selected, fluoroquinolone-resistant, mutant strains of C. perfringens, with stable mutations either in DNA gyrase or in both DNA gyrase and topoisomerase IV, was compared with that produced by the wild-type parent strains to investigate the effect of mutations associated with the selection of gatifloxacin resistance on bacterial metabolic activities. The mutants differed from their respective wild-type parent strains in the enzymatic activities of azoreductase, nitroreductase, and β-glucosidase and in the ratio of butyric acid to acetic acid production. Microarray analysis of one wild type and the corresponding mutant revealed different levels of mRNA expression for the enzymes involved in short-chain fatty acid (SCFA) synthesis and for β-glucosidase and oxidoreductases. In addition to mutations in the target genes, selection of resistance to gatifloxacin resulted in strain-specific physiological changes in the resistant mutants of C. perfringens that affected their metabolic activities.     

Origins of the specificity of inhibitor P218 toward wild-type and mutant PfDHFR: a molecular dynamics analysis

Molecular dynamics simulations were performed to evaluate the origin of the antimalarial effect of the lead compound P218. The simulations of the ligand in the cavities of wild-type, mutant Plasmodium falciparum Dihydrofolate Reductase (PfDHFR) and the human DHFR revealed the differences in the atomic-level interactions and also provided explanation for the specificity of this ligand toward PfDHFR. The binding free energy estimation using Molecular Mechanics Poisson-Boltzmann Surface Area method revealed that P218 has higher binding affinity (~ ?30 to ?35 kcal/mol) toward PfDHFR (both in wild-type and mutant forms) than human DHFR (~ ?22 kcal/mol), corroborating the experimental observations. Intermolecular hydrogen bonding analysis of the trajectories showed that P218 formed two stable hydrogen bonds with human DHFR (Ile7 and Glu30), wild-type and double-mutant PfDHFR’s (Asp54 and Arg122), while it formed three stable hydrogen bonds with quadruple-mutant PfDHFR (Asp54, Arg59, and Arg122). Additionally, P218 binding in PfDHFR is stabilized by hydrogen bonds with residues Ile14 and Ile164. It was found that mutant residues do not reduce the binding affinity of P218 to PfDHFR, in contrast, Cys59Arg mutation strongly favors inhibitor binding to quadruple-mutant PfDHFR. The atomistic-level details explored in this work will be highly useful for the design of non-resistant novel PfDHFR inhibitors as antimalarial agents.