Structural insights into <i>Plasmodium falciparum</i> nicotinamide mononucleotide adenylyltransferase: oligomeric assembly

The biochemical pathways involved in nicotinamide adenine dinucleotide (NAD) biosynthesis converge at the enzymatic step catalysed by nicotinamide mononucleotide adenylyltransferase (NMNAT, EC: 2.7.7.1). The majority of NMNATs are assembled into homo-oligomeric states that comprise 2-6 subunits. Recently, the NMNAT of Plasmodium falciparum (PfNMNAT) has been identified as a pharmacological target. The enzymatic characterisation, cellular location, and tertiary structure of the PfNMNAT protein have been reported. Nonetheless, its quaternary structure remains to be explored. The present study describes the oligomeric assembly of the 6 x His-PfNMNAT recombinant protein using immobilised metal affinity chromatography coupled with size exclusion chromatography (SEC) and native protein electrophoresis combined with Ferguson plot graphing. These chromatographic approaches resulted in the elution of an active monomer from the SEC column, whereas the Ferguson plot indicated a dimeric assembly of the 6 x His-PfNMNAT protein.

Key words:
NMNAT; oligomers; Plasmodium falciparum

Authorship

Luis Ernesto Contreras-Rodríguez

Universidad Nacional de Colombia, Facultad de Ciencias, Laboratorio de Investigaciones Básicas en Bioquímica, Bogotá, ColombiaUniversidad Nacional de ColombiaColombiaBogotá, ColombiaUniversidad Nacional de Colombia, Facultad de Ciencias, Laboratorio de Investigaciones Básicas en Bioquímica, Bogotá, Colombia

Catherin Yizet Marin-Mogollon

Lina Marcela Sánchez-Mejía

María Helena Ramírez-Hernández ⁺+ Corresponding author: mhramirezh@unal.edu.co

+ Corresponding author: mhramirezh@unal.edu.co

LE, CY and LM performed experiments; LE and MH designed the study, analysed the data and wrote the paper.

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Figures | Tables

Figures (6)
Tables (1)

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Fig. 1:
immobilised metal affinity chromatography coupled with size exclusion chromatography (IMAC-SEC) was used to purify an active 6 x His-Plasmodium falciparum nicotinamide mononucleotide adenylyltransferase (PfNMNAT) recombinant protein. (A) IMAC eluate 1 was subjected to SEC. The corresponding chromatogram shows two main peaks (A-B). (B) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) 12%T. Coomassie R-250 staining. Lanes: 1. Original soluble fraction; 2. IMAC eluate 1 injected in the SEC column; 3. Peaks B fraction; 4. Peaks A fraction; M. MW (kDa). (C) The enzymatic activity of the 6 x His-PfNMNAT recombinant protein from peak B was verified via coupled enzymatic assays. Negative control: elution buffer. Positive control: 6 x His-Leishmania braziliensis nicotinamide mononucleotide adenylyltransferase (LbNMNAT) recombinant protein.¹⁶16. Contreras LE, Neme R, Ramírez MH. Identification and functional evaluation of Leishmania braziliensis nicotinamide mononucleotide adenylyltransferase. Protein Expr Purif. 2015; 115: 26-33.

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Fig. 2:
the purified 6 x His-Plasmodium falciparum nicotinamide mononucleotide adenylyltransferase (PfNMNAT) recombinant protein was catalytically active. The enzymatic activity of the purified protein was corroborated by direct enzymatic assays and reverse phase high-performance liquid chromatography (RP-HPLC). (A) Standard analytes. (B) Analytes from the 6 x His-PfNMNAT reaction. (C) Negative control reaction (without enzyme).

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Fig. 3:
molecular weight (MW) analysis of protein standards by size exclusion chromatography (SEC). The standardised SEC conditions separated the protein standards referred to in Table.

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Fig. 4:
the 6 x His-Plasmodium falciparum nicotinamide mononucleotide adenylyltransferase (PfNMNAT) recombinant protein eluted as a monomer from the size exclusion chromatography (SEC) column. Molecular weight (MW) calibration curve. The equation of the curve and the K _av value were used to calculate the MW of the 6 x His-PfNMNAT recombinant protein.

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Fig. 5:
native protein electrophoresis (PAGE) and Ferguson plot analysis of the protein standards and the 6 x His-Plasmodium falciparum nicotinamide mononucleotide adenylyltransferase (PfNMNAT) recombinant protein. (A) native protein electrophoresis (PAGE) 10%T. Coomassie R-250 staining. Lanes: 1. Lactalbumin (14 kDa); 2. Trypsin inhibitor (20 kDa); 3. Carbonic anhydrase (29 kDa); 4. Ovalbumin (45 kDa); 5. BSA (66 kDa); 6. 6 x His-PfNMNAT. (B) %T effect on the relative mobility (R _f ) of the standards proteins. (C) Ferguson plot. The -Log of the slopes from figure in panel B (retardation coefficients, K _r ) is plotted as a function of the Log molecular weight (MW) of the corresponding standards.

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Fig. 6:
the Plasmodium falciparum nicotinamide mononucleotide adenylyltransferase (PfNMNAT) protein possesses the structural elements involved in oligomeric assembly. The quaternary structures of NMNATs exhibit a common structural element (red) between the Rossmann fold (blue) and the C-terminal domain (green). (A-D) NMNAT structures solved by X-ray crystallography. MjNMNAT, SaNaMNAT, HsNMNAT3, PfNMNAT: NMNATs from Methanococcus jannaschii, Staphylococcus aureus, Homo sapiens and Plasmodium falciparum, respectively. The image design was adapted from.⁴4. Zhai RG, Rizzi M, Garavaglia S. Nicotinamide/nicotinic acid mononucleotide adenylyltransferase, new insights into an ancient enzyme. Cell Mol Life Sci. 2009; 66(17): 2805-18. Protein Data Bank (PDB) codes are indicated in parentheses. The image was generated with UCSF Chimera.²⁹29. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, et al. UCSF Chimera-a visualization system for exploratory research and analysis. J Comput Chem. 2004; 25(13): 1605-12.

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TABLE Partition
coefficients (K _av ) of the protein standards analysed by size exclusion chromatography (SEC)

Fig. 1: immobilised metal affinity chromatography coupled with size exclusion chromatography (IMAC-SEC) was used to purify an active 6 x His-Plasmodium falciparum nicotinamide mononucleotide adenylyltransferase (PfNMNAT) recombinant protein. (A) IMAC eluate 1 was subjected to SEC. The corresponding chromatogram shows two main peaks (A-B). (B) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) 12%T. Coomassie R-250 staining. Lanes: 1. Original soluble fraction; 2. IMAC eluate 1 injected in the SEC column; 3. Peaks B fraction; 4. Peaks A fraction; M. MW (kDa). (C) The enzymatic activity of the 6 x His-PfNMNAT recombinant protein from peak B was verified via coupled enzymatic assays. Negative control: elution buffer. Positive control: 6 x His-Leishmania braziliensis nicotinamide mononucleotide adenylyltransferase (LbNMNAT) recombinant protein.¹⁶16. Contreras LE, Neme R, Ramírez MH. Identification and functional evaluation of Leishmania braziliensis nicotinamide mononucleotide adenylyltransferase. Protein Expr Purif. 2015; 115: 26-33.

Fig. 2: the purified 6 x His-Plasmodium falciparum nicotinamide mononucleotide adenylyltransferase (PfNMNAT) recombinant protein was catalytically active. The enzymatic activity of the purified protein was corroborated by direct enzymatic assays and reverse phase high-performance liquid chromatography (RP-HPLC). (A) Standard analytes. (B) Analytes from the 6 x His-PfNMNAT reaction. (C) Negative control reaction (without enzyme).

Fig. 3: molecular weight (MW) analysis of protein standards by size exclusion chromatography (SEC). The standardised SEC conditions separated the protein standards referred to in Table.

Fig. 4: the 6 x His-Plasmodium falciparum nicotinamide mononucleotide adenylyltransferase (PfNMNAT) recombinant protein eluted as a monomer from the size exclusion chromatography (SEC) column. Molecular weight (MW) calibration curve. The equation of the curve and the K _av value were used to calculate the MW of the 6 x His-PfNMNAT recombinant protein.

Fig. 5: native protein electrophoresis (PAGE) and Ferguson plot analysis of the protein standards and the 6 x His-Plasmodium falciparum nicotinamide mononucleotide adenylyltransferase (PfNMNAT) recombinant protein. (A) native protein electrophoresis (PAGE) 10%T. Coomassie R-250 staining. Lanes: 1. Lactalbumin (14 kDa); 2. Trypsin inhibitor (20 kDa); 3. Carbonic anhydrase (29 kDa); 4. Ovalbumin (45 kDa); 5. BSA (66 kDa); 6. 6 x His-PfNMNAT. (B) %T effect on the relative mobility (R _f ) of the standards proteins. (C) Ferguson plot. The -Log of the slopes from figure in panel B (retardation coefficients, K _r ) is plotted as a function of the Log molecular weight (MW) of the corresponding standards.

Fig. 6: the Plasmodium falciparum nicotinamide mononucleotide adenylyltransferase (PfNMNAT) protein possesses the structural elements involved in oligomeric assembly. The quaternary structures of NMNATs exhibit a common structural element (red) between the Rossmann fold (blue) and the C-terminal domain (green). (A-D) NMNAT structures solved by X-ray crystallography. MjNMNAT, SaNaMNAT, HsNMNAT3, PfNMNAT: NMNATs from Methanococcus jannaschii, Staphylococcus aureus, Homo sapiens and Plasmodium falciparum, respectively. The image design was adapted from.⁴4. Zhai RG, Rizzi M, Garavaglia S. Nicotinamide/nicotinic acid mononucleotide adenylyltransferase, new insights into an ancient enzyme. Cell Mol Life Sci. 2009; 66(17): 2805-18. Protein Data Bank (PDB) codes are indicated in parentheses. The image was generated with UCSF Chimera.²⁹29. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, et al. UCSF Chimera-a visualization system for exploratory research and analysis. J Comput Chem. 2004; 25(13): 1605-12.

TABLE Partition coefficients (K _av ) of the protein standards analysed by size exclusion chromatography (SEC)

Standard	Molecular weight (MW) (Da)	Log MW	Elution volume (V_e) (mL)	K_av = V_e-V_o/V_c-V_o
1: 6xHis-HsNMNAT1 (4-mer)	132000	5,12	12,27	0,26
2: BSA	66463	4,82	14,75	0,41
3: Carbonic anhydrase	29000	4,46	16,85	0,55
4: Lysozyme	14307	4,16	19,76	0,73
Additional data				0,73
Dextran blue				Void volume (V_o) = 8,22 mL
Column: Superdex 200 10/300 GL				Column volume (V_c) = 24 mL

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10.1590/0074-02760180073ER

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Structural insights into <i>Plasmodium falciparum</i> nicotinamide mononucleotide adenylyltransferase: oligomeric assembly

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[1] + Corresponding author: mhramirezh@unal.edu.co