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Structural and mechanistic investigations on Salmonella typhimurium acetate kinase (AckA): identification of a putative ligand binding pocket at the dimeric interface.

Chittori S, Savithri HS, Murthy MR - BMC Struct. Biol. (2012)

Bottom Line: These domains adopt an intermediate conformation compared to that of open and closed forms of ligand-bound Methanosarcina thermophila AckA (MtAckA).Unexpectedly, Form-II StAckA structure showed a drastic change in the conformation of residues 230-300 compared to that of Form-I.Dramatic conformational differences observed between unliganded and citrate-bound forms of StAckA led to identification of a putative ligand-binding pocket at the dimeric interface of StAckA with implications for enzymatic function.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Biophysics Unit, Indian Institute of Science, Bangalore, Karnataka 560012, India.

ABSTRACT

Background: Bacteria such as Escherichia coli and Salmonella typhimurium can utilize acetate as the sole source of carbon and energy. Acetate kinase (AckA) and phosphotransacetylase (Pta), key enzymes of acetate utilization pathway, regulate flux of metabolites in glycolysis, gluconeogenesis, TCA cycle, glyoxylate bypass and fatty acid metabolism.

Results: Here we report kinetic characterization of S. typhimurium AckA (StAckA) and structures of its unliganded (Form-I, 2.70 Å resolution) and citrate-bound (Form-II, 1.90 Å resolution) forms. The enzyme showed broad substrate specificity with k(cat)/K(m) in the order of acetate > propionate > formate. Further, the Km for acetyl-phosphate was significantly lower than for acetate and the enzyme could catalyze the reverse reaction (i.e. ATP synthesis) more efficiently. ATP and Mg(2+) could be substituted by other nucleoside 5'-triphosphates (GTP, UTP and CTP) and divalent cations (Mn(2+) and Co(2+)), respectively. Form-I StAckA represents the first structural report of an unliganded AckA. StAckA protomer consists of two domains with characteristic βββαβαβα topology of ASKHA superfamily of proteins. These domains adopt an intermediate conformation compared to that of open and closed forms of ligand-bound Methanosarcina thermophila AckA (MtAckA). Spectroscopic and structural analyses of StAckA further suggested occurrence of inter-domain motion upon ligand-binding. Unexpectedly, Form-II StAckA structure showed a drastic change in the conformation of residues 230-300 compared to that of Form-I. Further investigation revealed electron density corresponding to a citrate molecule in a pocket located at the dimeric interface of Form-II StAckA. Interestingly, a similar dimeric interface pocket lined with largely conserved residues could be identified in Form-I StAckA as well as in other enzymes homologous to AckA suggesting that ligand binding at this pocket may influence the function of these enzymes.

Conclusions: The biochemical and structural characterization of StAckA reported here provides insights into the biochemical specificity, overall fold, thermal stability, molecular basis of ligand binding and inter-domain motion in AckA family of enzymes. Dramatic conformational differences observed between unliganded and citrate-bound forms of StAckA led to identification of a putative ligand-binding pocket at the dimeric interface of StAckA with implications for enzymatic function.

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Comparison of Form-I and -II St AckA. (A) Plot of residual distance between sequentially equivalent Cα atoms against residue number obtained after pairwise superposition of A-subunit of Form-I and A- and B-subunits of Form-II StAckA. Labeling scheme – e.g. IIA-IIB corresponds to distances between equivalent Cα atoms after superposition of Form-II B-subunit on Form-II A-subunit. The insets show the variable segment (residues 230–300) Cα atom distances highlighting large conformational differences (corresponding to ~18% of the total length of the enzyme). Region corresponding to residues 251–260 (refer Additional file 1: Figure S1 for fit of the electron density) are marked by vertical dotted lines. (B) Structural superposition of the A-subunit of Form-I (salmon-red) with A- (yellow) and B- (cyan) subunits of the Form-II StAckA highlighting the structural differences of the variable segment. Secondary structures corresponding to the core helices and variable segments (labels corresponding to I-A, II-A and II-B subunits are enclosed using ○, □ and Δ shapes, respectively) are labeled. Met230 and Lys300 occur at the ends of the variable segment. (C) Form-II StAckA dimer highlighting regions of variable segment (A-subunit, yellow except for the variable segment shown in bright-orange; B-subunit, cyan with variable segment highlighted in blue). Citrate (pink, CIT-501) bound at the dimeric interface is shown in ball and stick representation. Secondary structures corresponding to the variable segments of each subunit are labeled.
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Figure 6: Comparison of Form-I and -II St AckA. (A) Plot of residual distance between sequentially equivalent Cα atoms against residue number obtained after pairwise superposition of A-subunit of Form-I and A- and B-subunits of Form-II StAckA. Labeling scheme – e.g. IIA-IIB corresponds to distances between equivalent Cα atoms after superposition of Form-II B-subunit on Form-II A-subunit. The insets show the variable segment (residues 230–300) Cα atom distances highlighting large conformational differences (corresponding to ~18% of the total length of the enzyme). Region corresponding to residues 251–260 (refer Additional file 1: Figure S1 for fit of the electron density) are marked by vertical dotted lines. (B) Structural superposition of the A-subunit of Form-I (salmon-red) with A- (yellow) and B- (cyan) subunits of the Form-II StAckA highlighting the structural differences of the variable segment. Secondary structures corresponding to the core helices and variable segments (labels corresponding to I-A, II-A and II-B subunits are enclosed using ○, □ and Δ shapes, respectively) are labeled. Met230 and Lys300 occur at the ends of the variable segment. (C) Form-II StAckA dimer highlighting regions of variable segment (A-subunit, yellow except for the variable segment shown in bright-orange; B-subunit, cyan with variable segment highlighted in blue). Citrate (pink, CIT-501) bound at the dimeric interface is shown in ball and stick representation. Secondary structures corresponding to the variable segments of each subunit are labeled.

Mentions: Another crystal form of StAckA (Form-II) was obtained in the presence of citrate [17]. The structure of this form was determined at 1.90 Å resolution using a polyalanine model of the Form-I StAckA monomer. Although the overall polypeptide fold in these two forms is similar, residues 230–300 (variable segment) were in significantly different conformations in the two subunits of Form-II dimer and these conformations in turn were different from that observed for this segment in Form-I (Figure 6A). Superposition of A- and B-subunits of Form-II StAckA with the A-subunit of Form-I StAckA excluding the variable segment results in rmsds of 1.03 Å and 1.53 Å for 326 and 313 pairs of Cα atoms, respectively (Figure 6B). The variable segment is located between strand βII3 and helix αII1 (Figures 3A and B). In the A-subunit of Form-II, residues 230–237 and 247–255 are at a location close to the corresponding region of Form-I StAckA (Figure 6B). However, position and conformation of residues 258–270 and 277–294 are completely different from those of Form-I. The segment consisting of residues 230–263 of the B-subunit of Form-II is initially close to the corresponding segment of Form-I but progressively departs from the structure observed in Form-I towards the C-terminal end. Residues 277–287 of B-subunit are close to the active site cleft present between the domains and occupy the space where ligands are expected to bind.


Structural and mechanistic investigations on Salmonella typhimurium acetate kinase (AckA): identification of a putative ligand binding pocket at the dimeric interface.

Chittori S, Savithri HS, Murthy MR - BMC Struct. Biol. (2012)

Comparison of Form-I and -II St AckA. (A) Plot of residual distance between sequentially equivalent Cα atoms against residue number obtained after pairwise superposition of A-subunit of Form-I and A- and B-subunits of Form-II StAckA. Labeling scheme – e.g. IIA-IIB corresponds to distances between equivalent Cα atoms after superposition of Form-II B-subunit on Form-II A-subunit. The insets show the variable segment (residues 230–300) Cα atom distances highlighting large conformational differences (corresponding to ~18% of the total length of the enzyme). Region corresponding to residues 251–260 (refer Additional file 1: Figure S1 for fit of the electron density) are marked by vertical dotted lines. (B) Structural superposition of the A-subunit of Form-I (salmon-red) with A- (yellow) and B- (cyan) subunits of the Form-II StAckA highlighting the structural differences of the variable segment. Secondary structures corresponding to the core helices and variable segments (labels corresponding to I-A, II-A and II-B subunits are enclosed using ○, □ and Δ shapes, respectively) are labeled. Met230 and Lys300 occur at the ends of the variable segment. (C) Form-II StAckA dimer highlighting regions of variable segment (A-subunit, yellow except for the variable segment shown in bright-orange; B-subunit, cyan with variable segment highlighted in blue). Citrate (pink, CIT-501) bound at the dimeric interface is shown in ball and stick representation. Secondary structures corresponding to the variable segments of each subunit are labeled.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 6: Comparison of Form-I and -II St AckA. (A) Plot of residual distance between sequentially equivalent Cα atoms against residue number obtained after pairwise superposition of A-subunit of Form-I and A- and B-subunits of Form-II StAckA. Labeling scheme – e.g. IIA-IIB corresponds to distances between equivalent Cα atoms after superposition of Form-II B-subunit on Form-II A-subunit. The insets show the variable segment (residues 230–300) Cα atom distances highlighting large conformational differences (corresponding to ~18% of the total length of the enzyme). Region corresponding to residues 251–260 (refer Additional file 1: Figure S1 for fit of the electron density) are marked by vertical dotted lines. (B) Structural superposition of the A-subunit of Form-I (salmon-red) with A- (yellow) and B- (cyan) subunits of the Form-II StAckA highlighting the structural differences of the variable segment. Secondary structures corresponding to the core helices and variable segments (labels corresponding to I-A, II-A and II-B subunits are enclosed using ○, □ and Δ shapes, respectively) are labeled. Met230 and Lys300 occur at the ends of the variable segment. (C) Form-II StAckA dimer highlighting regions of variable segment (A-subunit, yellow except for the variable segment shown in bright-orange; B-subunit, cyan with variable segment highlighted in blue). Citrate (pink, CIT-501) bound at the dimeric interface is shown in ball and stick representation. Secondary structures corresponding to the variable segments of each subunit are labeled.
Mentions: Another crystal form of StAckA (Form-II) was obtained in the presence of citrate [17]. The structure of this form was determined at 1.90 Å resolution using a polyalanine model of the Form-I StAckA monomer. Although the overall polypeptide fold in these two forms is similar, residues 230–300 (variable segment) were in significantly different conformations in the two subunits of Form-II dimer and these conformations in turn were different from that observed for this segment in Form-I (Figure 6A). Superposition of A- and B-subunits of Form-II StAckA with the A-subunit of Form-I StAckA excluding the variable segment results in rmsds of 1.03 Å and 1.53 Å for 326 and 313 pairs of Cα atoms, respectively (Figure 6B). The variable segment is located between strand βII3 and helix αII1 (Figures 3A and B). In the A-subunit of Form-II, residues 230–237 and 247–255 are at a location close to the corresponding region of Form-I StAckA (Figure 6B). However, position and conformation of residues 258–270 and 277–294 are completely different from those of Form-I. The segment consisting of residues 230–263 of the B-subunit of Form-II is initially close to the corresponding segment of Form-I but progressively departs from the structure observed in Form-I towards the C-terminal end. Residues 277–287 of B-subunit are close to the active site cleft present between the domains and occupy the space where ligands are expected to bind.

Bottom Line: These domains adopt an intermediate conformation compared to that of open and closed forms of ligand-bound Methanosarcina thermophila AckA (MtAckA).Unexpectedly, Form-II StAckA structure showed a drastic change in the conformation of residues 230-300 compared to that of Form-I.Dramatic conformational differences observed between unliganded and citrate-bound forms of StAckA led to identification of a putative ligand-binding pocket at the dimeric interface of StAckA with implications for enzymatic function.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Biophysics Unit, Indian Institute of Science, Bangalore, Karnataka 560012, India.

ABSTRACT

Background: Bacteria such as Escherichia coli and Salmonella typhimurium can utilize acetate as the sole source of carbon and energy. Acetate kinase (AckA) and phosphotransacetylase (Pta), key enzymes of acetate utilization pathway, regulate flux of metabolites in glycolysis, gluconeogenesis, TCA cycle, glyoxylate bypass and fatty acid metabolism.

Results: Here we report kinetic characterization of S. typhimurium AckA (StAckA) and structures of its unliganded (Form-I, 2.70 Å resolution) and citrate-bound (Form-II, 1.90 Å resolution) forms. The enzyme showed broad substrate specificity with k(cat)/K(m) in the order of acetate > propionate > formate. Further, the Km for acetyl-phosphate was significantly lower than for acetate and the enzyme could catalyze the reverse reaction (i.e. ATP synthesis) more efficiently. ATP and Mg(2+) could be substituted by other nucleoside 5'-triphosphates (GTP, UTP and CTP) and divalent cations (Mn(2+) and Co(2+)), respectively. Form-I StAckA represents the first structural report of an unliganded AckA. StAckA protomer consists of two domains with characteristic βββαβαβα topology of ASKHA superfamily of proteins. These domains adopt an intermediate conformation compared to that of open and closed forms of ligand-bound Methanosarcina thermophila AckA (MtAckA). Spectroscopic and structural analyses of StAckA further suggested occurrence of inter-domain motion upon ligand-binding. Unexpectedly, Form-II StAckA structure showed a drastic change in the conformation of residues 230-300 compared to that of Form-I. Further investigation revealed electron density corresponding to a citrate molecule in a pocket located at the dimeric interface of Form-II StAckA. Interestingly, a similar dimeric interface pocket lined with largely conserved residues could be identified in Form-I StAckA as well as in other enzymes homologous to AckA suggesting that ligand binding at this pocket may influence the function of these enzymes.

Conclusions: The biochemical and structural characterization of StAckA reported here provides insights into the biochemical specificity, overall fold, thermal stability, molecular basis of ligand binding and inter-domain motion in AckA family of enzymes. Dramatic conformational differences observed between unliganded and citrate-bound forms of StAckA led to identification of a putative ligand-binding pocket at the dimeric interface of StAckA with implications for enzymatic function.

Show MeSH
Related in: MedlinePlus