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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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Effect of 6b binding on the conformation of key residues of PDF.Superimposition of free, 6b-, and actinonin-bound AtPDF indicated in brown, red, and yellow, respectively. (A) Molecule A in the three models was superimposed, resulting in an r.m.s.d. of 0.9 Å for 100% of the Cα. Actinonin is shown in yellow and 6b in red. (B) Conformation of key residues Ile42, Phe58, and Ile130 in the different complexes and in unbound WT AtPDF. Actinonin is shown in yellow and 6b in red. (C) A detailed view of the AtPDF ligand-binding site for both actinonin and 6b complexes, which are indicated by sticks and are superimposed. The two ligands are colored in pale and dark grey, respectively. The hydrogen bond made by actinonin only is shown.
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pbio-1001066-g006: Effect of 6b binding on the conformation of key residues of PDF.Superimposition of free, 6b-, and actinonin-bound AtPDF indicated in brown, red, and yellow, respectively. (A) Molecule A in the three models was superimposed, resulting in an r.m.s.d. of 0.9 Å for 100% of the Cα. Actinonin is shown in yellow and 6b in red. (B) Conformation of key residues Ile42, Phe58, and Ile130 in the different complexes and in unbound WT AtPDF. Actinonin is shown in yellow and 6b in red. (C) A detailed view of the AtPDF ligand-binding site for both actinonin and 6b complexes, which are indicated by sticks and are superimposed. The two ligands are colored in pale and dark grey, respectively. The hydrogen bond made by actinonin only is shown.

Mentions: Given the similarity between actinonin and natural substrate binding, the very slow kinetics of inhibitor binding (10-s time-scale) remains puzzling compared to the 10 ms required for catalysis (deduced from the kcat). This finding could be explained as a conformational effect during the formation of the hydrogen bond, aligning the substrate as an additional beta-sheet and eventually stabilizing the entire enzyme-ligand complex. The significantly longer time needed to reach the most stable state compared to the substrate would most likely be due to the presence of the flexible and one carbon longer metal-binding group in actinonin (i.e., hydroxamate versus formyl, Figure 1B). This suggestion is in line with the overall data obtained when we investigated more deeply the role of the first carbonyl group of the ligand. This group is well known to exert a crucial effect in both productive and unproductive ligand binding (i.e., substrate and inhibitor) [54]. In this respect, we studied the binding of compound 6b (Figure S5B), a PDF ligand that does not exhibit a reactive group at this position [49]. We observed that this compound binds strongly to both EcPDF (KI*  = 63±6 nM) and AtPDF (KI*  = 400±35 nM) but, unlike actinonin, does not display slow, tight binding as KI*  =  KI. This impact on binding is consistent with the absence of the hydrogen bond involving the first carbonyl group of the ligand. The 3D structure of AtPDF was determined after soaking the compound in crystals of the free, open AtPDF form. Upon binding, 6b induced a complete conformational change, identical to that observed with actinonin (Figures 2B and 6A; “O” state). This result further suggests that the conformational change is not induced initially by the formation of this hydrogen bond and that the encounter complex is primarily driven by the fit within the S1' pocket. This also reveals that the timescale of the large conformational change is several orders of magnitude faster than the kinetics of slow binding and fully compatible with both the first step of actinonin binding (k4  = 140 s−1; see Table 1) and the catalytic rate of the substrate (kcat  = 37 s−1; see Table 1 and Table S3). The 3D structure also revealed that both the P1' and the hydroxamate groups are bound similarly to the corresponding groups of actinonin (Figure 6B). As expected, no additional bonding occurs, especially around the backbone nitrogen of Ile42 (Figure 6C).


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)

Effect of 6b binding on the conformation of key residues of PDF.Superimposition of free, 6b-, and actinonin-bound AtPDF indicated in brown, red, and yellow, respectively. (A) Molecule A in the three models was superimposed, resulting in an r.m.s.d. of 0.9 Å for 100% of the Cα. Actinonin is shown in yellow and 6b in red. (B) Conformation of key residues Ile42, Phe58, and Ile130 in the different complexes and in unbound WT AtPDF. Actinonin is shown in yellow and 6b in red. (C) A detailed view of the AtPDF ligand-binding site for both actinonin and 6b complexes, which are indicated by sticks and are superimposed. The two ligands are colored in pale and dark grey, respectively. The hydrogen bond made by actinonin only is shown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3101196&req=5

pbio-1001066-g006: Effect of 6b binding on the conformation of key residues of PDF.Superimposition of free, 6b-, and actinonin-bound AtPDF indicated in brown, red, and yellow, respectively. (A) Molecule A in the three models was superimposed, resulting in an r.m.s.d. of 0.9 Å for 100% of the Cα. Actinonin is shown in yellow and 6b in red. (B) Conformation of key residues Ile42, Phe58, and Ile130 in the different complexes and in unbound WT AtPDF. Actinonin is shown in yellow and 6b in red. (C) A detailed view of the AtPDF ligand-binding site for both actinonin and 6b complexes, which are indicated by sticks and are superimposed. The two ligands are colored in pale and dark grey, respectively. The hydrogen bond made by actinonin only is shown.
Mentions: Given the similarity between actinonin and natural substrate binding, the very slow kinetics of inhibitor binding (10-s time-scale) remains puzzling compared to the 10 ms required for catalysis (deduced from the kcat). This finding could be explained as a conformational effect during the formation of the hydrogen bond, aligning the substrate as an additional beta-sheet and eventually stabilizing the entire enzyme-ligand complex. The significantly longer time needed to reach the most stable state compared to the substrate would most likely be due to the presence of the flexible and one carbon longer metal-binding group in actinonin (i.e., hydroxamate versus formyl, Figure 1B). This suggestion is in line with the overall data obtained when we investigated more deeply the role of the first carbonyl group of the ligand. This group is well known to exert a crucial effect in both productive and unproductive ligand binding (i.e., substrate and inhibitor) [54]. In this respect, we studied the binding of compound 6b (Figure S5B), a PDF ligand that does not exhibit a reactive group at this position [49]. We observed that this compound binds strongly to both EcPDF (KI*  = 63±6 nM) and AtPDF (KI*  = 400±35 nM) but, unlike actinonin, does not display slow, tight binding as KI*  =  KI. This impact on binding is consistent with the absence of the hydrogen bond involving the first carbonyl group of the ligand. The 3D structure of AtPDF was determined after soaking the compound in crystals of the free, open AtPDF form. Upon binding, 6b induced a complete conformational change, identical to that observed with actinonin (Figures 2B and 6A; “O” state). This result further suggests that the conformational change is not induced initially by the formation of this hydrogen bond and that the encounter complex is primarily driven by the fit within the S1' pocket. This also reveals that the timescale of the large conformational change is several orders of magnitude faster than the kinetics of slow binding and fully compatible with both the first step of actinonin binding (k4  = 140 s−1; see Table 1) and the catalytic rate of the substrate (kcat  = 37 s−1; see Table 1 and Table S3). The 3D structure also revealed that both the P1' and the hydroxamate groups are bound similarly to the corresponding groups of actinonin (Figure 6B). As expected, no additional bonding occurs, especially around the backbone nitrogen of Ile42 (Figure 6C).

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