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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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Structural analysis of the MBP subdomain angle variations.(A) Superposition of subdomain 1 of MBP reveals different domain closure angles of subdomain 2. The superposed structures include uncomplexed MBP structure (PDB ID: 1OMP, grey), RG13 structure of P1 space group (yellow), RG13 structure of C2 space group (wheat), and maltotriose complexed MBP structure (PDB ID: 3MBP, green). Subdomain 1 of MBP with RG13 is defined by residues 1–109 and 261–316 and subdomain 2 is defined as 110–260 & 586–637. (B) Hypothesized surface change of MBP upon maltose binding in the vicinity the fusion sites R316 and A586 (A319). The surface of the maltose-bound MBP (green) and uncomplexed MBP domains (grey) are shown within the RG13 framework with TEM-1 depicted in a magenta Cα trace. The surface change from concave (grey curved interrupted line) to flat (green straight interrupted line) going from uncomplexed to maltose-bound is indicated. (C) Close-up view of the fusion site between MBP and TEM-1 showing the RG13 linker anchor residues R316 and S585 and their interactions with the rest of the MBP domain.
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pone-0039168-g002: Structural analysis of the MBP subdomain angle variations.(A) Superposition of subdomain 1 of MBP reveals different domain closure angles of subdomain 2. The superposed structures include uncomplexed MBP structure (PDB ID: 1OMP, grey), RG13 structure of P1 space group (yellow), RG13 structure of C2 space group (wheat), and maltotriose complexed MBP structure (PDB ID: 3MBP, green). Subdomain 1 of MBP with RG13 is defined by residues 1–109 and 261–316 and subdomain 2 is defined as 110–260 & 586–637. (B) Hypothesized surface change of MBP upon maltose binding in the vicinity the fusion sites R316 and A586 (A319). The surface of the maltose-bound MBP (green) and uncomplexed MBP domains (grey) are shown within the RG13 framework with TEM-1 depicted in a magenta Cα trace. The surface change from concave (grey curved interrupted line) to flat (green straight interrupted line) going from uncomplexed to maltose-bound is indicated. (C) Close-up view of the fusion site between MBP and TEM-1 showing the RG13 linker anchor residues R316 and S585 and their interactions with the rest of the MBP domain.

Mentions: MBP adopts a periplasmic binding fold and is capable of undergoing a ligand-induced subdomain closure in which the ligand maltose binds to both subdomain halves of MBP leading to a domain closure of ∼35° [9]. Superpositioning shows that the MBP domain of RG13 conformation is more similar to the uncomplexed MBP structure (PDB ID: 1OMP). The latter superpositioning yielded relative low r.m.s.d. values of 1.25Å monomer A in P1, 1.39Å for monomer B in P1, and 1.81Å in space group C2 compared to superpositioning with the maltotriose complexed MBP structure (PDB ID: 3MBP) which yielded larger r.m.s.d.s. of 2.76 (P1 monA), 2.80 (P1 monB), and 2.0 Å (C2) for about 360 Cα atoms (Figure 2). This result is in agreement with a subdomain rotation analysis indicating that RG13 is in a more unliganded open conformation in both P1 and C2 spacegroups since its domain closure angle is about 10° and 17°, respectively, compared to that of the uncomplexed MBP structure (Figure 2). The linker regions, where TEM-1 and MBP are fused, extend from the backside of the MBP domain opposite of where maltose binds. Therefore, the TEM-1 domain in RG13 will see a changing surface upon maltose binding going from concave to more flat (Figure 2B). This will have likely steric consequences for the linker tension between the MBP and TEM-1 domains as will be discussed later. The residues of the linkers closest to the MBP domain that appear to be anchoring the linker to MBP are residues S585 and R316 as the mainchain and sidechain residues of these residues make numerous stabilizing interactions in the P1 space group (Figure 2C). Furthermore, S585 and R316 are also the last residues visible in the C2 RG13 structure whereas the rest of the linker regions are too disordered to be modeled in this C2 spacegroup.


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)

Structural analysis of the MBP subdomain angle variations.(A) Superposition of subdomain 1 of MBP reveals different domain closure angles of subdomain 2. The superposed structures include uncomplexed MBP structure (PDB ID: 1OMP, grey), RG13 structure of P1 space group (yellow), RG13 structure of C2 space group (wheat), and maltotriose complexed MBP structure (PDB ID: 3MBP, green). Subdomain 1 of MBP with RG13 is defined by residues 1–109 and 261–316 and subdomain 2 is defined as 110–260 & 586–637. (B) Hypothesized surface change of MBP upon maltose binding in the vicinity the fusion sites R316 and A586 (A319). The surface of the maltose-bound MBP (green) and uncomplexed MBP domains (grey) are shown within the RG13 framework with TEM-1 depicted in a magenta Cα trace. The surface change from concave (grey curved interrupted line) to flat (green straight interrupted line) going from uncomplexed to maltose-bound is indicated. (C) Close-up view of the fusion site between MBP and TEM-1 showing the RG13 linker anchor residues R316 and S585 and their interactions with the rest of the MBP domain.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3375305&req=5

pone-0039168-g002: Structural analysis of the MBP subdomain angle variations.(A) Superposition of subdomain 1 of MBP reveals different domain closure angles of subdomain 2. The superposed structures include uncomplexed MBP structure (PDB ID: 1OMP, grey), RG13 structure of P1 space group (yellow), RG13 structure of C2 space group (wheat), and maltotriose complexed MBP structure (PDB ID: 3MBP, green). Subdomain 1 of MBP with RG13 is defined by residues 1–109 and 261–316 and subdomain 2 is defined as 110–260 & 586–637. (B) Hypothesized surface change of MBP upon maltose binding in the vicinity the fusion sites R316 and A586 (A319). The surface of the maltose-bound MBP (green) and uncomplexed MBP domains (grey) are shown within the RG13 framework with TEM-1 depicted in a magenta Cα trace. The surface change from concave (grey curved interrupted line) to flat (green straight interrupted line) going from uncomplexed to maltose-bound is indicated. (C) Close-up view of the fusion site between MBP and TEM-1 showing the RG13 linker anchor residues R316 and S585 and their interactions with the rest of the MBP domain.
Mentions: MBP adopts a periplasmic binding fold and is capable of undergoing a ligand-induced subdomain closure in which the ligand maltose binds to both subdomain halves of MBP leading to a domain closure of ∼35° [9]. Superpositioning shows that the MBP domain of RG13 conformation is more similar to the uncomplexed MBP structure (PDB ID: 1OMP). The latter superpositioning yielded relative low r.m.s.d. values of 1.25Å monomer A in P1, 1.39Å for monomer B in P1, and 1.81Å in space group C2 compared to superpositioning with the maltotriose complexed MBP structure (PDB ID: 3MBP) which yielded larger r.m.s.d.s. of 2.76 (P1 monA), 2.80 (P1 monB), and 2.0 Å (C2) for about 360 Cα atoms (Figure 2). This result is in agreement with a subdomain rotation analysis indicating that RG13 is in a more unliganded open conformation in both P1 and C2 spacegroups since its domain closure angle is about 10° and 17°, respectively, compared to that of the uncomplexed MBP structure (Figure 2). The linker regions, where TEM-1 and MBP are fused, extend from the backside of the MBP domain opposite of where maltose binds. Therefore, the TEM-1 domain in RG13 will see a changing surface upon maltose binding going from concave to more flat (Figure 2B). This will have likely steric consequences for the linker tension between the MBP and TEM-1 domains as will be discussed later. The residues of the linkers closest to the MBP domain that appear to be anchoring the linker to MBP are residues S585 and R316 as the mainchain and sidechain residues of these residues make numerous stabilizing interactions in the P1 space group (Figure 2C). Furthermore, S585 and R316 are also the last residues visible in the C2 RG13 structure whereas the rest of the linker regions are too disordered to be modeled in this C2 spacegroup.

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