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Conformational flexibility and changes underlying activation of the SUMO-specific protease SENP1 by remote substrate binding.

Chen CH, Namanja AT, Chen Y - Nat Commun (2014)

Bottom Line: SENP1 is a model for this protease family and responsible for processing SUMO.The β-grasp domain of SUMO1 alone induces structural changes at ~20 Å away in the active site of SENP1, revealing the importance of this domain in activating the enzyme.These findings likely represent general properties of the mechanism of substrate recognition and processing by SENPs and other Ubl-specific proteases, and illuminate how adaptive substrate binding can allosterically enhance enzyme activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine, Beckman Research Institute of the City of Hope, 1500 East Duarte Road, Duarte, California 91010, USA.

ABSTRACT
Ubiquitin-like (Ubl) modifications regulate nearly all cellular functions in eukaryotes with the largest superfamily of Ubl-specific proteases being Cys proteases. SENP1 is a model for this protease family and responsible for processing SUMO. Here using nuclear magnetic resonance relaxation measurements, chemical shift perturbation and enzyme kinetic analysis, we provide structural insights into the mechanism of substrate recognition coupled enzymatic activation within SENP1. We find that residues in the catalytic channel of SENP1, including the 'lid' residue Trp465, exhibit dynamics over a range of timescales, both in the presence and absence of bound substrates. The β-grasp domain of SUMO1 alone induces structural changes at ~20 Å away in the active site of SENP1, revealing the importance of this domain in activating the enzyme. These findings likely represent general properties of the mechanism of substrate recognition and processing by SENPs and other Ubl-specific proteases, and illuminate how adaptive substrate binding can allosterically enhance enzyme activity.

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Characterization of the binding of SUMO1 constructs to SENP1. (A) Representative region of the superimposed 1H-15N HSQC spectra for monitoring the titration of SENP1 with SUMO1-FL. Spectra are colored as a rainbow from red to violet, corresponding to increasing SENP1:SUMO1 molar ratios (1:0, 1:0.2, 1:0.4, 1:0.6, 1:0.8, 1:1.07, and 1:1.39). Arrows indicate the direction of CSP. (B) Correlation of CSP of SENP1 upon binding SUMO1-GG and SUMO1-FL at a SENP1:SUMO1 molar ratio of 1:1.39. (C) The same region as shown in (A) but of the superimposed 1H-15N HSQC spectra monitoring the titration of SENP1 with SUMO11-92. (D) Overlay of the 1H-15N HSQC spectra of SENP1, free (green) and in the presence of a 2.4-fold higher concentration of the S1-HSTV peptide (red). (E) ITC profiles of SENP1 titration with SUMO11-92 (left), or the S1-HSTV peptide (right, black) that is superimposed onto the profile of S1-HSTV titration into the SENP1-SUMO11-92 complex (right, red). (F) Overlay of a region of the 1H-15N HSQC spectra showing the resonances of Trp sidechains of SENP1 free (red) and in complex with SUMO1-FL (blue). (G) Overlay of the same region of the 1H-15N HSQC spectra as that in (D) and (F) of the SENP1-SUMO11-92 complex (green), and that in the presence of a 2.7-fold higher concentration of S1-HSTV (red).
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Figure 2: Characterization of the binding of SUMO1 constructs to SENP1. (A) Representative region of the superimposed 1H-15N HSQC spectra for monitoring the titration of SENP1 with SUMO1-FL. Spectra are colored as a rainbow from red to violet, corresponding to increasing SENP1:SUMO1 molar ratios (1:0, 1:0.2, 1:0.4, 1:0.6, 1:0.8, 1:1.07, and 1:1.39). Arrows indicate the direction of CSP. (B) Correlation of CSP of SENP1 upon binding SUMO1-GG and SUMO1-FL at a SENP1:SUMO1 molar ratio of 1:1.39. (C) The same region as shown in (A) but of the superimposed 1H-15N HSQC spectra monitoring the titration of SENP1 with SUMO11-92. (D) Overlay of the 1H-15N HSQC spectra of SENP1, free (green) and in the presence of a 2.4-fold higher concentration of the S1-HSTV peptide (red). (E) ITC profiles of SENP1 titration with SUMO11-92 (left), or the S1-HSTV peptide (right, black) that is superimposed onto the profile of S1-HSTV titration into the SENP1-SUMO11-92 complex (right, red). (F) Overlay of a region of the 1H-15N HSQC spectra showing the resonances of Trp sidechains of SENP1 free (red) and in complex with SUMO1-FL (blue). (G) Overlay of the same region of the 1H-15N HSQC spectra as that in (D) and (F) of the SENP1-SUMO11-92 complex (green), and that in the presence of a 2.7-fold higher concentration of S1-HSTV (red).

Mentions: The substrate SUMO1 consists of a β-grasp domain that encompasses residues 20-92 and a flexible C-terminus that begins with residue 93 in the SUMO1 precursor (referred to as SUMO1-FL, residues 1-101) or in mature SUMO1 (referred to as SUMO-GG, residues 1-97) that is the product of SENP1 cleavage. Both the β-grasp domain and the unstructured C-terminus interact with SENPs in the crystal structures of their complexes with SUMO precursors or conjugated substrates 10-14. We compared SENP1 interactions with SUMO1-FL, SUMO1-GG, SUMO11-92 (containing residues 1-92, with C-terminus truncated) or peptides corresponding to the C-terminus of SUMO1. Each of these SUMO1 constructs was titrated into 15N or 13C-methyl-labeled SENP1, and NMR chemical shift perturbation (CSP) was monitored in a series of two-dimensional 1H-15N HSQC or 1H-13C HMQC correlation spectra (Fig. 2A-D, and 2F-G, and Supplementary Fig. 4). The exchange rates between free SENP1 and SENP1 in complex with SUMO1-FL or SUMO1-GG were mostly slow to intermediate, relative to the NMR chemical shift timescale, and both SUMO-GG and SUMO-FL produced similar CSP (Fig. 2B). Upon titration with SUMO11-92, the resonances of some residues (i.e., R449, Fig. 2C and Supplementary Fig. 4) showed similarly slow exchange between free and bound states and similar CSP as that observed for binding of SUMO1-FL or SUMO1-GG. The resonances of some other residues (i.e., T451 and Q507, Fig. 2C and Supplementary Fig. 4) showed fast exchange between the free and bound states but with similar CSP trends. These data suggest that the β-grasp domain of SUMO1 interacts with SENP1 in a manner similar to that in the context of SUMO1-FL.


Conformational flexibility and changes underlying activation of the SUMO-specific protease SENP1 by remote substrate binding.

Chen CH, Namanja AT, Chen Y - Nat Commun (2014)

Characterization of the binding of SUMO1 constructs to SENP1. (A) Representative region of the superimposed 1H-15N HSQC spectra for monitoring the titration of SENP1 with SUMO1-FL. Spectra are colored as a rainbow from red to violet, corresponding to increasing SENP1:SUMO1 molar ratios (1:0, 1:0.2, 1:0.4, 1:0.6, 1:0.8, 1:1.07, and 1:1.39). Arrows indicate the direction of CSP. (B) Correlation of CSP of SENP1 upon binding SUMO1-GG and SUMO1-FL at a SENP1:SUMO1 molar ratio of 1:1.39. (C) The same region as shown in (A) but of the superimposed 1H-15N HSQC spectra monitoring the titration of SENP1 with SUMO11-92. (D) Overlay of the 1H-15N HSQC spectra of SENP1, free (green) and in the presence of a 2.4-fold higher concentration of the S1-HSTV peptide (red). (E) ITC profiles of SENP1 titration with SUMO11-92 (left), or the S1-HSTV peptide (right, black) that is superimposed onto the profile of S1-HSTV titration into the SENP1-SUMO11-92 complex (right, red). (F) Overlay of a region of the 1H-15N HSQC spectra showing the resonances of Trp sidechains of SENP1 free (red) and in complex with SUMO1-FL (blue). (G) Overlay of the same region of the 1H-15N HSQC spectra as that in (D) and (F) of the SENP1-SUMO11-92 complex (green), and that in the presence of a 2.7-fold higher concentration of S1-HSTV (red).
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Figure 2: Characterization of the binding of SUMO1 constructs to SENP1. (A) Representative region of the superimposed 1H-15N HSQC spectra for monitoring the titration of SENP1 with SUMO1-FL. Spectra are colored as a rainbow from red to violet, corresponding to increasing SENP1:SUMO1 molar ratios (1:0, 1:0.2, 1:0.4, 1:0.6, 1:0.8, 1:1.07, and 1:1.39). Arrows indicate the direction of CSP. (B) Correlation of CSP of SENP1 upon binding SUMO1-GG and SUMO1-FL at a SENP1:SUMO1 molar ratio of 1:1.39. (C) The same region as shown in (A) but of the superimposed 1H-15N HSQC spectra monitoring the titration of SENP1 with SUMO11-92. (D) Overlay of the 1H-15N HSQC spectra of SENP1, free (green) and in the presence of a 2.4-fold higher concentration of the S1-HSTV peptide (red). (E) ITC profiles of SENP1 titration with SUMO11-92 (left), or the S1-HSTV peptide (right, black) that is superimposed onto the profile of S1-HSTV titration into the SENP1-SUMO11-92 complex (right, red). (F) Overlay of a region of the 1H-15N HSQC spectra showing the resonances of Trp sidechains of SENP1 free (red) and in complex with SUMO1-FL (blue). (G) Overlay of the same region of the 1H-15N HSQC spectra as that in (D) and (F) of the SENP1-SUMO11-92 complex (green), and that in the presence of a 2.7-fold higher concentration of S1-HSTV (red).
Mentions: The substrate SUMO1 consists of a β-grasp domain that encompasses residues 20-92 and a flexible C-terminus that begins with residue 93 in the SUMO1 precursor (referred to as SUMO1-FL, residues 1-101) or in mature SUMO1 (referred to as SUMO-GG, residues 1-97) that is the product of SENP1 cleavage. Both the β-grasp domain and the unstructured C-terminus interact with SENPs in the crystal structures of their complexes with SUMO precursors or conjugated substrates 10-14. We compared SENP1 interactions with SUMO1-FL, SUMO1-GG, SUMO11-92 (containing residues 1-92, with C-terminus truncated) or peptides corresponding to the C-terminus of SUMO1. Each of these SUMO1 constructs was titrated into 15N or 13C-methyl-labeled SENP1, and NMR chemical shift perturbation (CSP) was monitored in a series of two-dimensional 1H-15N HSQC or 1H-13C HMQC correlation spectra (Fig. 2A-D, and 2F-G, and Supplementary Fig. 4). The exchange rates between free SENP1 and SENP1 in complex with SUMO1-FL or SUMO1-GG were mostly slow to intermediate, relative to the NMR chemical shift timescale, and both SUMO-GG and SUMO-FL produced similar CSP (Fig. 2B). Upon titration with SUMO11-92, the resonances of some residues (i.e., R449, Fig. 2C and Supplementary Fig. 4) showed similarly slow exchange between free and bound states and similar CSP as that observed for binding of SUMO1-FL or SUMO1-GG. The resonances of some other residues (i.e., T451 and Q507, Fig. 2C and Supplementary Fig. 4) showed fast exchange between the free and bound states but with similar CSP trends. These data suggest that the β-grasp domain of SUMO1 interacts with SENP1 in a manner similar to that in the context of SUMO1-FL.

Bottom Line: SENP1 is a model for this protease family and responsible for processing SUMO.The β-grasp domain of SUMO1 alone induces structural changes at ~20 Å away in the active site of SENP1, revealing the importance of this domain in activating the enzyme.These findings likely represent general properties of the mechanism of substrate recognition and processing by SENPs and other Ubl-specific proteases, and illuminate how adaptive substrate binding can allosterically enhance enzyme activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine, Beckman Research Institute of the City of Hope, 1500 East Duarte Road, Duarte, California 91010, USA.

ABSTRACT
Ubiquitin-like (Ubl) modifications regulate nearly all cellular functions in eukaryotes with the largest superfamily of Ubl-specific proteases being Cys proteases. SENP1 is a model for this protease family and responsible for processing SUMO. Here using nuclear magnetic resonance relaxation measurements, chemical shift perturbation and enzyme kinetic analysis, we provide structural insights into the mechanism of substrate recognition coupled enzymatic activation within SENP1. We find that residues in the catalytic channel of SENP1, including the 'lid' residue Trp465, exhibit dynamics over a range of timescales, both in the presence and absence of bound substrates. The β-grasp domain of SUMO1 alone induces structural changes at ~20 Å away in the active site of SENP1, revealing the importance of this domain in activating the enzyme. These findings likely represent general properties of the mechanism of substrate recognition and processing by SENPs and other Ubl-specific proteases, and illuminate how adaptive substrate binding can allosterically enhance enzyme activity.

Show MeSH
Related in: MedlinePlus