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Structure of an engineered β-lactamase maltose binding protein fusion protein: insights into heterotropic allosteric regulation.

Ke W, Laurent AH, Armstrong MD, Chen Y, Smith WE, Liang J, Wright CM, Ostermeier M, van den Akker F - PLoS ONE (2012)

Bottom Line: Combined with previous mutagenesis results we therefore hypothesize the presence of two or more inter-domain mutually exclusive inhibitory Zn(2+) sites.Structural analysis indicated that the linker attachment sites on MBP are at a site that, upon maltose binding, harbors both the largest local Cα distance changes and displays surface curvature changes, from concave to relatively flat becoming thus less sterically intrusive.Maltose activation and zinc inhibition of RG13 are hypothesized to have opposite effects on productive relaxation of the TEM-1 β3 linker region via steric and/or linker juxtapositioning mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Departments of Biochemistry, Case Western Reserve University, Cleveland, Ohio, United States of America.

ABSTRACT
Engineering novel allostery into existing proteins is a challenging endeavor to obtain novel sensors, therapeutic proteins, or modulate metabolic and cellular processes. The RG13 protein achieves such allostery by inserting a circularly permuted TEM-1 β-lactamase gene into the maltose binding protein (MBP). RG13 is positively regulated by maltose yet is, serendipitously, inhibited by Zn(2+) at low µM concentration. To probe the structure and allostery of RG13, we crystallized RG13 in the presence of mM Zn(2+) concentration and determined its structure. The structure reveals that the MBP and TEM-1 domains are in close proximity connected via two linkers and a zinc ion bridging both domains. By bridging both TEM-1 and MBP, Zn(2+) acts to "twist tie" the linkers thereby partially dislodging a linker between the two domains from its original catalytically productive position in TEM-1. This linker 1 contains residues normally part of the TEM-1 active site including the critical β3 and β4 strands important for activity. Mutagenesis of residues comprising the crystallographically observed Zn(2+) site only slightly affected Zn(2+) inhibition 2- to 4-fold. Combined with previous mutagenesis results we therefore hypothesize the presence of two or more inter-domain mutually exclusive inhibitory Zn(2+) sites. Mutagenesis and molecular modeling of an intact TEM-1 domain near MBP within the RG13 framework indicated a close surface proximity of the two domains with maltose switching being critically dependent on MBP linker anchoring residues and linker length. Structural analysis indicated that the linker attachment sites on MBP are at a site that, upon maltose binding, harbors both the largest local Cα distance changes and displays surface curvature changes, from concave to relatively flat becoming thus less sterically intrusive. Maltose activation and zinc inhibition of RG13 are hypothesized to have opposite effects on productive relaxation of the TEM-1 β3 linker region via steric and/or linker juxtapositioning mechanisms.

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Active site changes in TEM-1 domain of RG13 compared to wt TEM-1 structure.Superpositioning of wt TEM-1 structure onto the TEM domain in RG13 reveals displacement of β3 strand and part of β4 strand (inset is a zoomed in view with secondary structure elements in transparent representation). The TEM-1 β4 strand is mostly intact yet starts diverging at position R244. The α10 TEM-1 helix which is observed to be more extended in RG13. Also labeled and underlined are the MBP helices α14 and α15 that form part of the linker anchor points in the RG13 fusion protein. TEM-1 β3 strand residue positions K234, S235, A237, and β4 strand residue K244 are indicated as well as the new position of A237 with the RG13 sequence A325. The zinc ion is indicated via a grey sphere.
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pone-0039168-g003: Active site changes in TEM-1 domain of RG13 compared to wt TEM-1 structure.Superpositioning of wt TEM-1 structure onto the TEM domain in RG13 reveals displacement of β3 strand and part of β4 strand (inset is a zoomed in view with secondary structure elements in transparent representation). The TEM-1 β4 strand is mostly intact yet starts diverging at position R244. The α10 TEM-1 helix which is observed to be more extended in RG13. Also labeled and underlined are the MBP helices α14 and α15 that form part of the linker anchor points in the RG13 fusion protein. TEM-1 β3 strand residue positions K234, S235, A237, and β4 strand residue K244 are indicated as well as the new position of A237 with the RG13 sequence A325. The zinc ion is indicated via a grey sphere.

Mentions: TEM-1 is a β-lactamase that hydrolyses bicyclic β-lactam compounds that contain a carboxyl moiety and a carbonyl moiety. Key active site elements for TEM-1 are the catalytic S70, the carboxyl binding pocket, the oxy-anion hole for carbonyl oxygen, and the deacylation-water priming E166 residue. The TEM-1 domain of RG13 has an overall fold similar to the wild-type TEM-1 structure (PDB ID: 1ZG4) with r.m.s.d. values of 1.02Å (P1 molA), 1.02 Å (P1 molB), and 1.1 Å (C2) for ∼240 Cα as calculated using COOT [10]. The most significant structural change in the TEM-1 domain of RG13, compared to the wt TEM-1 structure, is the displacement of TEM-1 residues 229–244 which comprises the critical active site β3 strand, part of the β4 strand, and the connecting loop between these strands. This TEM-1 active site section in RG13 is now part of the linker 1 connecting the MBP and TEM domains (Figures 1B and 3). In addition, the preceding residues 214–228 are in a somewhat different conformation as the TEM-1 α10 helix extends in RG13 forming a longer helix (Figure 3). Despite these substantial active site differences of missing active site β-strands, the positions of most other active site TEM-1 residues including S70, Y105, S130, N132, E166, and N170 (in TEM-1 residue numbering) are relatively unchanged in RG13. The β3-strand plays an important role for TEM-1 activity because it forms one of the active site walls providing part of both the carboxyl binding pocket (via S235) and oxy-anion hole for the carbonyl oxygen (via the backbone nitrogen of residue A237) which are thus no longer in the correct position in the Zn2+ bound structure of RG13. β3-strand residue K234 is also critical for β-lactamase functioning [11] and is no longer in the native TEM-1 position (Figure 3). Furthermore, β4-strand residue R244, which is also involved in electrostatic interactions with the β-lactam carboxyl moiety [12], has also shifted. This β3-β4-strand section, comprising TEM-1 residues 229–244 is in close proximity to one of the fusion sites, i.e. residue 229, between TEM-1 and MBP (Figures 1 and 3) [5]. In addition to lacking some key active site features, the entry to the TEM-1 active site in RG13 is partially blocked by this displaced region that now forms a short helix (Figure 3). Taken together, loss of β3-β4 strand section adopting a new conformation, now sterically blocking the active site, likely explains the lack of activity of the Zn2+-bound RG13 structure at mM concentration of Zn2+. Further away from the active site, residues V216 and A217 have also shifted somewhat and these shifts could also affect enzyme activity as those residues have been shown to not tolerate changes [13]. However, this disturbance is likely not a main contributor to the loss of activity.


Structure of an engineered β-lactamase maltose binding protein fusion protein: insights into heterotropic allosteric regulation.

Ke W, Laurent AH, Armstrong MD, Chen Y, Smith WE, Liang J, Wright CM, Ostermeier M, van den Akker F - PLoS ONE (2012)

Active site changes in TEM-1 domain of RG13 compared to wt TEM-1 structure.Superpositioning of wt TEM-1 structure onto the TEM domain in RG13 reveals displacement of β3 strand and part of β4 strand (inset is a zoomed in view with secondary structure elements in transparent representation). The TEM-1 β4 strand is mostly intact yet starts diverging at position R244. The α10 TEM-1 helix which is observed to be more extended in RG13. Also labeled and underlined are the MBP helices α14 and α15 that form part of the linker anchor points in the RG13 fusion protein. TEM-1 β3 strand residue positions K234, S235, A237, and β4 strand residue K244 are indicated as well as the new position of A237 with the RG13 sequence A325. The zinc ion is indicated via a grey sphere.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3375305&req=5

pone-0039168-g003: Active site changes in TEM-1 domain of RG13 compared to wt TEM-1 structure.Superpositioning of wt TEM-1 structure onto the TEM domain in RG13 reveals displacement of β3 strand and part of β4 strand (inset is a zoomed in view with secondary structure elements in transparent representation). The TEM-1 β4 strand is mostly intact yet starts diverging at position R244. The α10 TEM-1 helix which is observed to be more extended in RG13. Also labeled and underlined are the MBP helices α14 and α15 that form part of the linker anchor points in the RG13 fusion protein. TEM-1 β3 strand residue positions K234, S235, A237, and β4 strand residue K244 are indicated as well as the new position of A237 with the RG13 sequence A325. The zinc ion is indicated via a grey sphere.
Mentions: TEM-1 is a β-lactamase that hydrolyses bicyclic β-lactam compounds that contain a carboxyl moiety and a carbonyl moiety. Key active site elements for TEM-1 are the catalytic S70, the carboxyl binding pocket, the oxy-anion hole for carbonyl oxygen, and the deacylation-water priming E166 residue. The TEM-1 domain of RG13 has an overall fold similar to the wild-type TEM-1 structure (PDB ID: 1ZG4) with r.m.s.d. values of 1.02Å (P1 molA), 1.02 Å (P1 molB), and 1.1 Å (C2) for ∼240 Cα as calculated using COOT [10]. The most significant structural change in the TEM-1 domain of RG13, compared to the wt TEM-1 structure, is the displacement of TEM-1 residues 229–244 which comprises the critical active site β3 strand, part of the β4 strand, and the connecting loop between these strands. This TEM-1 active site section in RG13 is now part of the linker 1 connecting the MBP and TEM domains (Figures 1B and 3). In addition, the preceding residues 214–228 are in a somewhat different conformation as the TEM-1 α10 helix extends in RG13 forming a longer helix (Figure 3). Despite these substantial active site differences of missing active site β-strands, the positions of most other active site TEM-1 residues including S70, Y105, S130, N132, E166, and N170 (in TEM-1 residue numbering) are relatively unchanged in RG13. The β3-strand plays an important role for TEM-1 activity because it forms one of the active site walls providing part of both the carboxyl binding pocket (via S235) and oxy-anion hole for the carbonyl oxygen (via the backbone nitrogen of residue A237) which are thus no longer in the correct position in the Zn2+ bound structure of RG13. β3-strand residue K234 is also critical for β-lactamase functioning [11] and is no longer in the native TEM-1 position (Figure 3). Furthermore, β4-strand residue R244, which is also involved in electrostatic interactions with the β-lactam carboxyl moiety [12], has also shifted. This β3-β4-strand section, comprising TEM-1 residues 229–244 is in close proximity to one of the fusion sites, i.e. residue 229, between TEM-1 and MBP (Figures 1 and 3) [5]. In addition to lacking some key active site features, the entry to the TEM-1 active site in RG13 is partially blocked by this displaced region that now forms a short helix (Figure 3). Taken together, loss of β3-β4 strand section adopting a new conformation, now sterically blocking the active site, likely explains the lack of activity of the Zn2+-bound RG13 structure at mM concentration of Zn2+. Further away from the active site, residues V216 and A217 have also shifted somewhat and these shifts could also affect enzyme activity as those residues have been shown to not tolerate changes [13]. However, this disturbance is likely not a main contributor to the loss of activity.

Bottom Line: Combined with previous mutagenesis results we therefore hypothesize the presence of two or more inter-domain mutually exclusive inhibitory Zn(2+) sites.Structural analysis indicated that the linker attachment sites on MBP are at a site that, upon maltose binding, harbors both the largest local Cα distance changes and displays surface curvature changes, from concave to relatively flat becoming thus less sterically intrusive.Maltose activation and zinc inhibition of RG13 are hypothesized to have opposite effects on productive relaxation of the TEM-1 β3 linker region via steric and/or linker juxtapositioning mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Departments of Biochemistry, Case Western Reserve University, Cleveland, Ohio, United States of America.

ABSTRACT
Engineering novel allostery into existing proteins is a challenging endeavor to obtain novel sensors, therapeutic proteins, or modulate metabolic and cellular processes. The RG13 protein achieves such allostery by inserting a circularly permuted TEM-1 β-lactamase gene into the maltose binding protein (MBP). RG13 is positively regulated by maltose yet is, serendipitously, inhibited by Zn(2+) at low µM concentration. To probe the structure and allostery of RG13, we crystallized RG13 in the presence of mM Zn(2+) concentration and determined its structure. The structure reveals that the MBP and TEM-1 domains are in close proximity connected via two linkers and a zinc ion bridging both domains. By bridging both TEM-1 and MBP, Zn(2+) acts to "twist tie" the linkers thereby partially dislodging a linker between the two domains from its original catalytically productive position in TEM-1. This linker 1 contains residues normally part of the TEM-1 active site including the critical β3 and β4 strands important for activity. Mutagenesis of residues comprising the crystallographically observed Zn(2+) site only slightly affected Zn(2+) inhibition 2- to 4-fold. Combined with previous mutagenesis results we therefore hypothesize the presence of two or more inter-domain mutually exclusive inhibitory Zn(2+) sites. Mutagenesis and molecular modeling of an intact TEM-1 domain near MBP within the RG13 framework indicated a close surface proximity of the two domains with maltose switching being critically dependent on MBP linker anchoring residues and linker length. Structural analysis indicated that the linker attachment sites on MBP are at a site that, upon maltose binding, harbors both the largest local Cα distance changes and displays surface curvature changes, from concave to relatively flat becoming thus less sterically intrusive. Maltose activation and zinc inhibition of RG13 are hypothesized to have opposite effects on productive relaxation of the TEM-1 β3 linker region via steric and/or linker juxtapositioning mechanisms.

Show MeSH