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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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Structure of uncomplexed MBP (1OMP) showing maltose-induced local Cα-Cα distance shifts.Maltose-dependent Cα-Cα distance differences for residues i = i+3 and i = i+6 are averaged and mapped onto the structure with residues color ramped from blue to red for averaged shifts ranging from 0 to 1.4Å, respectively. Residue 312 yielded the largest maltose-dependent local shifts (1.4Å). Labeled also are residues 315 (i+3) and 318 (i+6) with arrows to residue 312 (i) indicating the local distances that were maximally shifted by maltose. Helices α14 and α15 are labeled as well as residue 316 (black), the site of the RG13’s TEM-1 insertion in MBP, to show that the insertion occurred within the maltose-dependent locally shifting region of 312–318 situated between the two helices. Labeled residues are also shown as spheres. The orientation of MBP is similar to that of Figure 5.
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pone-0039168-g007: Structure of uncomplexed MBP (1OMP) showing maltose-induced local Cα-Cα distance shifts.Maltose-dependent Cα-Cα distance differences for residues i = i+3 and i = i+6 are averaged and mapped onto the structure with residues color ramped from blue to red for averaged shifts ranging from 0 to 1.4Å, respectively. Residue 312 yielded the largest maltose-dependent local shifts (1.4Å). Labeled also are residues 315 (i+3) and 318 (i+6) with arrows to residue 312 (i) indicating the local distances that were maximally shifted by maltose. Helices α14 and α15 are labeled as well as residue 316 (black), the site of the RG13’s TEM-1 insertion in MBP, to show that the insertion occurred within the maltose-dependent locally shifting region of 312–318 situated between the two helices. Labeled residues are also shown as spheres. The orientation of MBP is similar to that of Figure 5.

Mentions: It is interesting as to why MBP residue 316 is selected for during the generation of two different maltose-regulated TEM-1 fusion constructs as residue 316 is not near where the largest conformational differences are observed upon maltose binding that include a 35° domain closure conformational change. Residue 316 is actually situated on the backside of the hinge region of the bi-lobal MBP structure. However, one has to consider as to how these allosteric fusion proteins are generated as the TEM-1 domain is inserted into a single site of MBP (with possibly some residues deleted as in RG13, Figure 1). Although the linker anchor points of MBP are not where there are the largest differences in distance between an uncomplexed and complexed MBP are found, the anchor points are near where the largest differences are when one only considers Cα positions of a few residues downstream from each other in the continuous polypeptide chain to allow for insertion of the non-homologous TEM-1 gene at a single site. By calculating Cα-Cα Δdistance measurement of residues i and i+x via substracting the distances from the maltose-bound from the maltose-free MBP structure, the MBP region at residue 312 lights up as to having the largest Cα-Cα difference for i and i+3 and i and i+6 (Figure 7). These calculations suggest that the MBP region from 312 to 312+i (i.e. 312–318) has the largest shifts thereby likely explaining why insertions near residue 316, in particular since R316 has adopted the linker anchor role in RG13 (Figure 2C), are capable of transmitting maltose signals to TEM-1 (as is also evident mechanistically in Figure 5A). An additional potential benefit of this region, being on the backside of MBP, is that this region could exert a lever/torque effect as the fusion points are close to the hinge region. As such, this region is thus perhaps able to exert a larger force on the movements near its rotation point, which might be needed as the β3 TEM-1 strand is normally well lodged into the active site. Furthermore, the backside of MBP, near the hinge region where TEM-1 is attached, changes from concave to more flat upon maltose binding (Figure 2B) and could therefore decrease steric contacts between TEM-1 and MBP and thus provide additional slack for the β3 linker regions such that they can adopt the wt catalytically competent conformation. It is remarkable that the forced evolutionary pressure resulted in this successful RG13 of having indeed found the MBP 312–318 region and the 230 β3-strand region of TEM-1 indicating that this approach works to find this narrow signaling window region being almost a ‘needle in a hay stack’.


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)

Structure of uncomplexed MBP (1OMP) showing maltose-induced local Cα-Cα distance shifts.Maltose-dependent Cα-Cα distance differences for residues i = i+3 and i = i+6 are averaged and mapped onto the structure with residues color ramped from blue to red for averaged shifts ranging from 0 to 1.4Å, respectively. Residue 312 yielded the largest maltose-dependent local shifts (1.4Å). Labeled also are residues 315 (i+3) and 318 (i+6) with arrows to residue 312 (i) indicating the local distances that were maximally shifted by maltose. Helices α14 and α15 are labeled as well as residue 316 (black), the site of the RG13’s TEM-1 insertion in MBP, to show that the insertion occurred within the maltose-dependent locally shifting region of 312–318 situated between the two helices. Labeled residues are also shown as spheres. The orientation of MBP is similar to that of Figure 5.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3375305&req=5

pone-0039168-g007: Structure of uncomplexed MBP (1OMP) showing maltose-induced local Cα-Cα distance shifts.Maltose-dependent Cα-Cα distance differences for residues i = i+3 and i = i+6 are averaged and mapped onto the structure with residues color ramped from blue to red for averaged shifts ranging from 0 to 1.4Å, respectively. Residue 312 yielded the largest maltose-dependent local shifts (1.4Å). Labeled also are residues 315 (i+3) and 318 (i+6) with arrows to residue 312 (i) indicating the local distances that were maximally shifted by maltose. Helices α14 and α15 are labeled as well as residue 316 (black), the site of the RG13’s TEM-1 insertion in MBP, to show that the insertion occurred within the maltose-dependent locally shifting region of 312–318 situated between the two helices. Labeled residues are also shown as spheres. The orientation of MBP is similar to that of Figure 5.
Mentions: It is interesting as to why MBP residue 316 is selected for during the generation of two different maltose-regulated TEM-1 fusion constructs as residue 316 is not near where the largest conformational differences are observed upon maltose binding that include a 35° domain closure conformational change. Residue 316 is actually situated on the backside of the hinge region of the bi-lobal MBP structure. However, one has to consider as to how these allosteric fusion proteins are generated as the TEM-1 domain is inserted into a single site of MBP (with possibly some residues deleted as in RG13, Figure 1). Although the linker anchor points of MBP are not where there are the largest differences in distance between an uncomplexed and complexed MBP are found, the anchor points are near where the largest differences are when one only considers Cα positions of a few residues downstream from each other in the continuous polypeptide chain to allow for insertion of the non-homologous TEM-1 gene at a single site. By calculating Cα-Cα Δdistance measurement of residues i and i+x via substracting the distances from the maltose-bound from the maltose-free MBP structure, the MBP region at residue 312 lights up as to having the largest Cα-Cα difference for i and i+3 and i and i+6 (Figure 7). These calculations suggest that the MBP region from 312 to 312+i (i.e. 312–318) has the largest shifts thereby likely explaining why insertions near residue 316, in particular since R316 has adopted the linker anchor role in RG13 (Figure 2C), are capable of transmitting maltose signals to TEM-1 (as is also evident mechanistically in Figure 5A). An additional potential benefit of this region, being on the backside of MBP, is that this region could exert a lever/torque effect as the fusion points are close to the hinge region. As such, this region is thus perhaps able to exert a larger force on the movements near its rotation point, which might be needed as the β3 TEM-1 strand is normally well lodged into the active site. Furthermore, the backside of MBP, near the hinge region where TEM-1 is attached, changes from concave to more flat upon maltose binding (Figure 2B) and could therefore decrease steric contacts between TEM-1 and MBP and thus provide additional slack for the β3 linker regions such that they can adopt the wt catalytically competent conformation. It is remarkable that the forced evolutionary pressure resulted in this successful RG13 of having indeed found the MBP 312–318 region and the 230 β3-strand region of TEM-1 indicating that this approach works to find this narrow signaling window region being almost a ‘needle in a hay stack’.

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
Related in: MedlinePlus