Conversion of a maltose receptor into a zinc biosensor by computational design

Marvin, Jonathan S.; Hellinga, Homme W.

doi:10.1073/pnas.091083898

Cited by 150 publications

(117 citation statements)

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“…The creation of ligand-binding proteins has applications in the areas of biosensors, diagnostics, chiral separations, therapeutic proteins, and basic biological studies. Periplasmic binding proteins, like MBP, have been shown to be a versatile template for the computational design of ligand binding functionalities (27,28). We hypothesized that the mutations necessary to convert the maltose switch into one for sucrose could be used to convert MBP into a sucrose-binding protein.…”

Section: Application Of Switches To the Creation Of Ligand-binding Prmentioning

confidence: 99%

Directed evolution of protein switches and their application to the creation of ligand-binding proteins

Guntas

Mansell

Kim

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

199

246

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We describe an iterative approach for creating protein switches involving the in vitro recombination of two nonhomologous genes. We demonstrate this approach by recombining the genes coding for TEM1 ␤-lactamase (BLA) and the Escherichia coli maltose binding protein (MBP) to create a family of MBP-BLA hybrids in which maltose is a positive or negative effector of ␤-lactam hydrolysis. Some of these MBP-BLA switches were effectively ''on-off'' in nature, with maltose altering catalytic activity by as much as 600-fold. The ability of these switches to confer an effector-dependent growth͞no growth phenotype to E. coli cells was exploited to rapidly identify, from a library of 4 ؋ 10 6 variants, MBP-BLA switch variants that respond to sucrose as the effector. The transplantation of these mutations into wild-type MBP converted MBP into a ''sucrose-binding protein,'' illustrating the switches potential as a tool to rapidly identify ligand-binding proteins.allostery ͉ ␤-lactamase ͉ maltose binding protein R egulation of protein activity is fundamental to cellular function.One of the mechanisms a cell uses to modulate the level of protein activity is regulation of the amount of a protein present in a cell. Accordingly, many strategies for engineering control of cellular protein activity have focused on modulation of protein production and degradation, often through small-moleculedependent switches that regulate transcription, translation, localization, degradation, or protein splicing (1) or through the engineering of artificial gene-regulatory networks (2). The key strength of many of these approaches is that they are easily generalized. For example, a switch that regulates transcription of one gene can easily be adapted to regulate transcription of an arbitrary gene. However, the significant limitation of these approaches is the slow dynamics stemming from the indirect nature of the regulation (i.e., protein activity is regulated by controlling the amount of protein and not by regulating the protein's specific activity directly). In addition, modulation is only feasible in the context of the cell and cannot be easily transferred to an in vitro setting.A more satisfying approach is to modulate the protein activity directly, but this presents a considerable design problem. Inhibitors, if they can be found, can only can be used to down-regulate activity, and such a strategy suffers from the fact that one is not free to choose the signal that modulates the activity: The signal must be an inhibitor of the protein. One clever way around this limitation is to engineer the inhibitor such that a third molecule can regulate the inhibitor's affinity for the regulated protein, as was demonstrated for RNA aptamer inhibitors that could be regulated by an organic small molecule (3). Natural allosteric proteins solve this problem by having spatially distinct regulatory and active sites. Ligand binding or covalent modifications at one site affects the output function at a distant site through a conformational change. This mechanism has ...

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Section: Application Of Switches To the Creation Of Ligand-binding Prmentioning

confidence: 99%

Directed evolution of protein switches and their application to the creation of ligand-binding proteins

Guntas

Mansell

Kim

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

199

246

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“…The increase in affinity is due primarily to a decrease in the rate of dissociation between MBP and its ligand. The introduction of larger, bulky groups into the balancing interface has been used previously to engineer MBP (12,30). For example, mutation of Ile-329 to cysteine followed by modification with thionitropyridine, produced an MBP molecule with a K D for maltose of 7.4 nM.…”

Section: High Resolution Analysis Of Structural Changes In Mbp-del Anmentioning

confidence: 99%

Insights into the Conformational Equilibria of Maltose-binding Protein by Analysis of High Affinity Mutants

Telmer

Shilton

2003

Journal of Biological Chemistry

109

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ATP binding cassette transporters couple ATP hydrolysis to the transmembrane transport of a diverse range of compounds. Members of the ATP binding cassette transporter superfamily are characterized by two membrane-integral domains that each contain 6 or more membrane spanning helices, but are otherwise poorly conserved, and two peripheral ATP binding cassette domains that display sequence conservation across the entire superfamily (1). In addition to the membrane complex, ATP binding cassette systems that catalyze nutrient uptake have primary receptors (binding proteins) that serve two functions: they provide a high affinity binding site for the transported molecule and they regulate the ATPase activity of the integral membrane complex.We are interested in the function of the primary receptors in the transport process. As a group, these proteins have been intensively studied by x-ray crystallography and other biophysical techniques (for a review, see Ref.2). They typically contain two domains separated by a hinge region; the substrate binds in the cleft between the two domains, and the protein undergoes a large conformational change, leading to closure of the cleft. With respect to the maltose transport system, domain closure in the binding protein is thought to be the first step toward molecular shape recognition by the membrane complex (3, 4), although it has been shown that both substrate-loaded and substrate-free binding proteins have a role in the transport cycle (5-8). The association and dissociation of the substrate, and attendant conformational changes in the binding protein (MBP), 1 may have direct effects on transport kinetics and regulation of ATP hydrolysis by MalFGK 2 . To investigate the role of binding protein affinity on the transport process, our goal was to engineer MBP molecules with greater affinity for maltose, without changing residues in either the maltose binding site or in regions thought to interact with MalFGK 2 .Crystal structures of MBP in both the closed and open conformations have been solved (9, 10), and they show that binding of maltose results in a large conformational change of the protein, bringing the two domains together such that the substrate is buried inside the cleft. In solution, unliganded MBP is in the open conformation (11); however, there is no obvious energetic barrier to closure of the ligand binding cleft, either in the hinge or in the interface surrounding the ligand binding site. Rather, an interface on the opposite side of the hinge from the ligand binding site appears to maintain the protein in an * This work was supported by Natural Sciences and Engineering Research Council Grant 21749-1999 (to B. H. S.); the macromolecular x-ray facility at the University of Western Ontario was financed with grants from the Canada Foundation for Innovation, Ontario Challenge Fund, and Western's Academic Development Fund; fluorescence and SPR studies were carried out at the University of Western Ontario Biomolecular Interactions and Conformations Facility, supported by a Mu...

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“…Whereas the sites for introduction of histidyl residues in these cases were identified by inspection of their structures, Hellinga and colleagues (3)(4)(5)(6)(7)(8) developed computational methods for identifying sites in structurally defined proteins that are particularly amenable to introduction of metal ion binding sites (3)(4)(5) that they have applied successfully to several proteins (6)(7)(8).…”

mentioning

confidence: 99%

Introduction and characterization of a functionally linked metal ion binding site at the exposed heme edge of myoglobin

Hunter

Maurus²,

Mauk³

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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A binding site for metal ions has been created on the surface of horse heart myoglobin (Mb) near the heme 6-propionate group by replacing K45 and K63 with glutamyl residues. One-dimensional 1 H NMR spectroscopy indicates that Mn 2؉ binds in the vicinity of the heme 6-propionate as anticipated, and potentiometric titrations establish that the affinity of the new site for Mn 2؉ is 1.28(4) ؋ 10 4 M ؊1 (pH 6.96, ionic strength I ‫؍‬ 17.2 M, 25°C). In addition, these substitutions lower the reduction potential of the protein and increase the pK a for the water molecule coordinated to the heme iron of metmyoglobin. The peroxidase [2,2 -azinobis(3-ethylbenzthiazoline-6-sulfonic acid), ABTS, as substrate] and the Mn 2؉ -peroxidase activity of the variant are both increased Ϸ3-fold. In contrast to wild-type Mb, both the affinity for azide and the midpoint potential of the variant are significantly influenced by the addition of Mn 2؉ . The structure of the variant has been determined by x-ray crystallography to define the coordination environment of bound Mn 2؉ and Cd 2؉ . Although slight differences are observed between the geometry of the binding of the two metal ions, both are hexacoordinate, and neither involves coordination by E63.

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Conversion of a maltose receptor into a zinc biosensor by computational design

Cited by 150 publications

References 50 publications

Directed evolution of protein switches and their application to the creation of ligand-binding proteins

Directed evolution of protein switches and their application to the creation of ligand-binding proteins

Insights into the Conformational Equilibria of Maltose-binding Protein by Analysis of High Affinity Mutants

Introduction and characterization of a functionally linked metal ion binding site at the exposed heme edge of myoglobin

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