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Trapping conformational states along ligand-binding dynamics of peptide deformylase: the impact of induced fit on enzyme catalysis.

Fieulaine S, Boularot A, Artaud I, Desmadril M, Dardel F, Meinnel T, Giglione C - PLoS Biol. (2011)

Bottom Line: Ligand-induced reshaping of a hydrophobic pocket drives closure of the active site, which is finally "zipped up" by additional binding interactions.Together with biochemical analyses, these data allow a coherent reconstruction of the sequence of events leading from the encounter complex to the key-lock binding state of the enzyme.A "movie" that reconstructs this entire process can be further extrapolated to catalysis.

View Article: PubMed Central - PubMed

Affiliation: CNRS, ISV, UPR2355, Gif-sur-Yvette, France.

ABSTRACT
For several decades, molecular recognition has been considered one of the most fundamental processes in biochemistry. For enzymes, substrate binding is often coupled to conformational changes that alter the local environment of the active site to align the reactive groups for efficient catalysis and to reach the transition state. Adaptive substrate recognition is a well-known concept; however, it has been poorly characterized at a structural level because of its dynamic nature. Here, we provide a detailed mechanism for an induced-fit process at atomic resolution. We take advantage of a slow, tight binding inhibitor-enzyme system, actinonin-peptide deformylase. Crystal structures of the initial open state and final closed state were solved, as well as those of several intermediate mimics captured during the process. Ligand-induced reshaping of a hydrophobic pocket drives closure of the active site, which is finally "zipped up" by additional binding interactions. Together with biochemical analyses, these data allow a coherent reconstruction of the sequence of events leading from the encounter complex to the key-lock binding state of the enzyme. A "movie" that reconstructs this entire process can be further extrapolated to catalysis.

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Related in: MedlinePlus

Inhibition and enzymatic reactions progress through an induced fit pathway.(A) The catalytic parameters Km and kcat, for all AtPDF variants are provided as a percentage of the wild-type values (WT). Detailed values are presented in Table S2. (B) Schematic model for actinonin binding to AtPDF in favor of an induced-fit pathway. PDF might exist in at least two conformational states, open (O) or closed (C). The relative abundance of each conformation would vary, depending on the enzyme type. With AtPDF, it is likely that the most abundant form is the O one, which is the only form leading to a productive complex. The superclosed form (S) is likely to show reduced affinity for the ligand because of steric occlusion of the active site. At the initial stage, the inhibitor (shown in red) binds to AtPDF (indicated in brown) in the O conformation. To reach the final key-lock state (productive closed conformation, C), two major and extreme pathways can be used. According to the conformational selection pathway, the inhibitor selects the C conformation. This pathway, which is represented by the dashed arrow, does not occur within the crystal. In contrast, the G41Q and G41M mutants, by providing the structure of the enzyme in intermediate conformations (I), prove the existence of the so-called encounter complex and confirm that the inhibitor binds to the enzyme when it is in the O conformation. The ligand-binding site is then reorganized to yield the C enzyme conformation, that is, the key-lock state. Indeed, the inhibitor binds to the enzyme through the induced-fit pathway. Each timescale was calculated using the data available in the text and corresponds to t1/2 values deduced from the calculation of 0.693/(kinetic constant of interest). The kcat value (k2) was used to assess the timescale of catalysis in panel C, whereas, in (B), k4 assesses the first step of inhibition, and k6 is used in the case of the slow step. For the SO conversion (left, B), the lifetime of the minor form of EcPDF was used to assess the order of magnitude (see text and [38]). (C) Schematic model for the deformylation reaction catalyzed by PDF. Since actinonin is a pseudo-peptidic inhibitor, it is likely that a peptidic substrate can bind to the PDF enzyme through an induced-fit pathway, as described in (B). The key-lock state represents a transition state in which the N-formylated substrate is deformylated to yield the final reaction product.
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pbio-1001066-g005: Inhibition and enzymatic reactions progress through an induced fit pathway.(A) The catalytic parameters Km and kcat, for all AtPDF variants are provided as a percentage of the wild-type values (WT). Detailed values are presented in Table S2. (B) Schematic model for actinonin binding to AtPDF in favor of an induced-fit pathway. PDF might exist in at least two conformational states, open (O) or closed (C). The relative abundance of each conformation would vary, depending on the enzyme type. With AtPDF, it is likely that the most abundant form is the O one, which is the only form leading to a productive complex. The superclosed form (S) is likely to show reduced affinity for the ligand because of steric occlusion of the active site. At the initial stage, the inhibitor (shown in red) binds to AtPDF (indicated in brown) in the O conformation. To reach the final key-lock state (productive closed conformation, C), two major and extreme pathways can be used. According to the conformational selection pathway, the inhibitor selects the C conformation. This pathway, which is represented by the dashed arrow, does not occur within the crystal. In contrast, the G41Q and G41M mutants, by providing the structure of the enzyme in intermediate conformations (I), prove the existence of the so-called encounter complex and confirm that the inhibitor binds to the enzyme when it is in the O conformation. The ligand-binding site is then reorganized to yield the C enzyme conformation, that is, the key-lock state. Indeed, the inhibitor binds to the enzyme through the induced-fit pathway. Each timescale was calculated using the data available in the text and corresponds to t1/2 values deduced from the calculation of 0.693/(kinetic constant of interest). The kcat value (k2) was used to assess the timescale of catalysis in panel C, whereas, in (B), k4 assesses the first step of inhibition, and k6 is used in the case of the slow step. For the SO conversion (left, B), the lifetime of the minor form of EcPDF was used to assess the order of magnitude (see text and [38]). (C) Schematic model for the deformylation reaction catalyzed by PDF. Since actinonin is a pseudo-peptidic inhibitor, it is likely that a peptidic substrate can bind to the PDF enzyme through an induced-fit pathway, as described in (B). The key-lock state represents a transition state in which the N-formylated substrate is deformylated to yield the final reaction product.

Mentions: To unravel the dynamics of the recognition process, we surmised that it should be possible to freeze the conformational change along the pathway by introducing selected, minor variations within the above-mentioned crucial residues involved in the collective motion. In this respect, site-directed mutagenesis of AtPDF was performed on Gly41, Ile42, and Ile130. Single substitutions were made at Gly41 (G41A/Q/M), Ile42 (I42A/F/N/W), and Ile130 (I130A/F), and the variants were purified and characterized. These mutant proteins showed no change in overall stability, as evidenced by DSC experiments (unpublished data). However, two variants of G41, G41Q and G41M, showed dramatic effects; the kcat/Km values were reduced by three orders of magnitude due to large decreases in the kcat values compared to the WT enzyme (Figure 5A and Table S1). The reduced kcat/Km values suggest an altered ability of these variants to attain the final enzyme-transition state complex and, as a result, to give rise to possible states different from the final E:I* complex. Substitutions at positions 42 and 130 only caused small reductions in the kcat values (Figure 5A, Figure S2C, and Table S1). The actinonin-binding potency of both G41 variants was also greatly reduced (Table S1 and Figure S2B). The time-dependent inhibition by actinonin of the most active variants was then studied (Table S3). The half-lives of the final complexes—as assessed by comparison of the 1/k6 values—were always significantly smaller (Table S3), suggesting that the conformational change induced by actinonin binding still occurred, but the C state is destabilized relative to the O state in the mutants compared to the WT. Accordingly, actinonin strongly stabilized almost all of the variants; Tm was increased by more than 20°C. This differs from the G41M and G41Q variants, which both showed increases in the Tm of only 12°C, consistent with reduced binding potency (Table S1).


Trapping conformational states along ligand-binding dynamics of peptide deformylase: the impact of induced fit on enzyme catalysis.

Fieulaine S, Boularot A, Artaud I, Desmadril M, Dardel F, Meinnel T, Giglione C - PLoS Biol. (2011)

Inhibition and enzymatic reactions progress through an induced fit pathway.(A) The catalytic parameters Km and kcat, for all AtPDF variants are provided as a percentage of the wild-type values (WT). Detailed values are presented in Table S2. (B) Schematic model for actinonin binding to AtPDF in favor of an induced-fit pathway. PDF might exist in at least two conformational states, open (O) or closed (C). The relative abundance of each conformation would vary, depending on the enzyme type. With AtPDF, it is likely that the most abundant form is the O one, which is the only form leading to a productive complex. The superclosed form (S) is likely to show reduced affinity for the ligand because of steric occlusion of the active site. At the initial stage, the inhibitor (shown in red) binds to AtPDF (indicated in brown) in the O conformation. To reach the final key-lock state (productive closed conformation, C), two major and extreme pathways can be used. According to the conformational selection pathway, the inhibitor selects the C conformation. This pathway, which is represented by the dashed arrow, does not occur within the crystal. In contrast, the G41Q and G41M mutants, by providing the structure of the enzyme in intermediate conformations (I), prove the existence of the so-called encounter complex and confirm that the inhibitor binds to the enzyme when it is in the O conformation. The ligand-binding site is then reorganized to yield the C enzyme conformation, that is, the key-lock state. Indeed, the inhibitor binds to the enzyme through the induced-fit pathway. Each timescale was calculated using the data available in the text and corresponds to t1/2 values deduced from the calculation of 0.693/(kinetic constant of interest). The kcat value (k2) was used to assess the timescale of catalysis in panel C, whereas, in (B), k4 assesses the first step of inhibition, and k6 is used in the case of the slow step. For the SO conversion (left, B), the lifetime of the minor form of EcPDF was used to assess the order of magnitude (see text and [38]). (C) Schematic model for the deformylation reaction catalyzed by PDF. Since actinonin is a pseudo-peptidic inhibitor, it is likely that a peptidic substrate can bind to the PDF enzyme through an induced-fit pathway, as described in (B). The key-lock state represents a transition state in which the N-formylated substrate is deformylated to yield the final reaction product.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3101196&req=5

pbio-1001066-g005: Inhibition and enzymatic reactions progress through an induced fit pathway.(A) The catalytic parameters Km and kcat, for all AtPDF variants are provided as a percentage of the wild-type values (WT). Detailed values are presented in Table S2. (B) Schematic model for actinonin binding to AtPDF in favor of an induced-fit pathway. PDF might exist in at least two conformational states, open (O) or closed (C). The relative abundance of each conformation would vary, depending on the enzyme type. With AtPDF, it is likely that the most abundant form is the O one, which is the only form leading to a productive complex. The superclosed form (S) is likely to show reduced affinity for the ligand because of steric occlusion of the active site. At the initial stage, the inhibitor (shown in red) binds to AtPDF (indicated in brown) in the O conformation. To reach the final key-lock state (productive closed conformation, C), two major and extreme pathways can be used. According to the conformational selection pathway, the inhibitor selects the C conformation. This pathway, which is represented by the dashed arrow, does not occur within the crystal. In contrast, the G41Q and G41M mutants, by providing the structure of the enzyme in intermediate conformations (I), prove the existence of the so-called encounter complex and confirm that the inhibitor binds to the enzyme when it is in the O conformation. The ligand-binding site is then reorganized to yield the C enzyme conformation, that is, the key-lock state. Indeed, the inhibitor binds to the enzyme through the induced-fit pathway. Each timescale was calculated using the data available in the text and corresponds to t1/2 values deduced from the calculation of 0.693/(kinetic constant of interest). The kcat value (k2) was used to assess the timescale of catalysis in panel C, whereas, in (B), k4 assesses the first step of inhibition, and k6 is used in the case of the slow step. For the SO conversion (left, B), the lifetime of the minor form of EcPDF was used to assess the order of magnitude (see text and [38]). (C) Schematic model for the deformylation reaction catalyzed by PDF. Since actinonin is a pseudo-peptidic inhibitor, it is likely that a peptidic substrate can bind to the PDF enzyme through an induced-fit pathway, as described in (B). The key-lock state represents a transition state in which the N-formylated substrate is deformylated to yield the final reaction product.
Mentions: To unravel the dynamics of the recognition process, we surmised that it should be possible to freeze the conformational change along the pathway by introducing selected, minor variations within the above-mentioned crucial residues involved in the collective motion. In this respect, site-directed mutagenesis of AtPDF was performed on Gly41, Ile42, and Ile130. Single substitutions were made at Gly41 (G41A/Q/M), Ile42 (I42A/F/N/W), and Ile130 (I130A/F), and the variants were purified and characterized. These mutant proteins showed no change in overall stability, as evidenced by DSC experiments (unpublished data). However, two variants of G41, G41Q and G41M, showed dramatic effects; the kcat/Km values were reduced by three orders of magnitude due to large decreases in the kcat values compared to the WT enzyme (Figure 5A and Table S1). The reduced kcat/Km values suggest an altered ability of these variants to attain the final enzyme-transition state complex and, as a result, to give rise to possible states different from the final E:I* complex. Substitutions at positions 42 and 130 only caused small reductions in the kcat values (Figure 5A, Figure S2C, and Table S1). The actinonin-binding potency of both G41 variants was also greatly reduced (Table S1 and Figure S2B). The time-dependent inhibition by actinonin of the most active variants was then studied (Table S3). The half-lives of the final complexes—as assessed by comparison of the 1/k6 values—were always significantly smaller (Table S3), suggesting that the conformational change induced by actinonin binding still occurred, but the C state is destabilized relative to the O state in the mutants compared to the WT. Accordingly, actinonin strongly stabilized almost all of the variants; Tm was increased by more than 20°C. This differs from the G41M and G41Q variants, which both showed increases in the Tm of only 12°C, consistent with reduced binding potency (Table S1).

Bottom Line: Ligand-induced reshaping of a hydrophobic pocket drives closure of the active site, which is finally "zipped up" by additional binding interactions.Together with biochemical analyses, these data allow a coherent reconstruction of the sequence of events leading from the encounter complex to the key-lock binding state of the enzyme.A "movie" that reconstructs this entire process can be further extrapolated to catalysis.

View Article: PubMed Central - PubMed

Affiliation: CNRS, ISV, UPR2355, Gif-sur-Yvette, France.

ABSTRACT
For several decades, molecular recognition has been considered one of the most fundamental processes in biochemistry. For enzymes, substrate binding is often coupled to conformational changes that alter the local environment of the active site to align the reactive groups for efficient catalysis and to reach the transition state. Adaptive substrate recognition is a well-known concept; however, it has been poorly characterized at a structural level because of its dynamic nature. Here, we provide a detailed mechanism for an induced-fit process at atomic resolution. We take advantage of a slow, tight binding inhibitor-enzyme system, actinonin-peptide deformylase. Crystal structures of the initial open state and final closed state were solved, as well as those of several intermediate mimics captured during the process. Ligand-induced reshaping of a hydrophobic pocket drives closure of the active site, which is finally "zipped up" by additional binding interactions. Together with biochemical analyses, these data allow a coherent reconstruction of the sequence of events leading from the encounter complex to the key-lock binding state of the enzyme. A "movie" that reconstructs this entire process can be further extrapolated to catalysis.

Show MeSH
Related in: MedlinePlus