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Solution structure and Rpn1 interaction of the UBL domain of human RNA polymerase II C-terminal domain phosphatase.

Yun JH, Ko S, Lee CK, Cheong HK, Cheong C, Yoon JB, Lee W - PLoS ONE (2013)

Bottom Line: The UBL domain of hUBLCP1 has a unique β-strand (β3) and β3-α2 loop, instead of the canonical β4 observed in other UBL domains.The molecular topology and secondary structures are different from those of known UBL domains including that of fly UBLCP1.The positively charged residues of the β3-α2 loop are involved in interacting with the C-terminal leucine-rich repeat-like domain of Rpn1.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul, Korea.

ABSTRACT
The ubiquitin-like modifier (UBL) domain of ubiquitin-like domain proteins (UDPs) interacts specifically with subunits of the 26 S proteasome. A novel UDP, ubiquitin-like domain-containing C-terminal domain phosphatase (UBLCP1), has been identified as an interacting partner of the 26 S proteasome. We determined the high-resolution solution structure of the UBL domain of human UBLCP1 by nuclear magnetic resonance spectroscopy. The UBL domain of hUBLCP1 has a unique β-strand (β3) and β3-α2 loop, instead of the canonical β4 observed in other UBL domains. The molecular topology and secondary structures are different from those of known UBL domains including that of fly UBLCP1. Data from backbone dynamics shows that the β3-α2 loop is relatively rigid although it might have intrinsic dynamic profile. The positively charged residues of the β3-α2 loop are involved in interacting with the C-terminal leucine-rich repeat-like domain of Rpn1.

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Related in: MedlinePlus

Intermolecular interaction between hUBLCP1 and Rpn1.(A) Immunoprecipitation (IP) of both hUBLCP1 and Rpn1 was performed. Immunoblotting was conducted using HA- and FLAG-antibodies to distinguish the two proteins. IP was performed for the a-FLAG-RPN1 and co-precipitation was confirmed by IB. (B) In vitro GST pull-down assay using the GST-fused UBL domain of hUBLCP1 (UBLCP11–81), the TRX-His6-fused regulatory subunit 1 (Rpn1394–568), and subunit 2 (Rpn1640–772). Lane 1 (upper and lower), GST-fused UBL domain of UBLCP1; Lane 2 (upper), TRX-His6-fused Rpn1394–568 and (lower), TRX-His6-fused Rpn1640–772; Lane 3 (upper and lower), pull-down elution using GST elution buffer containing 10 mM reduced glutathione. (C) 15N-labeled UBL domain was titrated with Rpn1394–568. Superimposed 2D 1H-15N HSQC spectra of UBL domain without Rpn1394–568 (black) and with Rpn1394–568 (purple). To assess chemical shift perturbations of the UBL domain on Rpn1 binding, titration of 0–2 molar equivalents of Rpn1394–568 was performed. Residues K49 and A55 in the β3-α2 loop, K65 in the α2-β4 loop and W9 in the β1-β2 loop are displayed. (D) Chemical shift changes (△δ) of the UBL domain upon Rpn1394–568 binding are summarized. Chemical shift perturbations are shown by different colors: blue, 0 ppm<△δ ≤0.05 ppm; orange, 0.05 ppm<△δ ≤0.1 ppm; red, 0.1 ppm<△δ ≤ disappeared. Residues that "disappeared," due to peak broadening, are marked by asterisks and the secondary structures are also displayed. (E) Residues with significant chemical shift changes and less perturbed changes upon Rpn1 binding drawn by the Pymol program are shown by spheres and sticks, respectively. (F) Surface charge model of the UBL domain of the hUBLCP1. Electrostatic surfaces are shown for negative (red), positive (blue), and neutral (white) potential.
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pone-0062981-g003: Intermolecular interaction between hUBLCP1 and Rpn1.(A) Immunoprecipitation (IP) of both hUBLCP1 and Rpn1 was performed. Immunoblotting was conducted using HA- and FLAG-antibodies to distinguish the two proteins. IP was performed for the a-FLAG-RPN1 and co-precipitation was confirmed by IB. (B) In vitro GST pull-down assay using the GST-fused UBL domain of hUBLCP1 (UBLCP11–81), the TRX-His6-fused regulatory subunit 1 (Rpn1394–568), and subunit 2 (Rpn1640–772). Lane 1 (upper and lower), GST-fused UBL domain of UBLCP1; Lane 2 (upper), TRX-His6-fused Rpn1394–568 and (lower), TRX-His6-fused Rpn1640–772; Lane 3 (upper and lower), pull-down elution using GST elution buffer containing 10 mM reduced glutathione. (C) 15N-labeled UBL domain was titrated with Rpn1394–568. Superimposed 2D 1H-15N HSQC spectra of UBL domain without Rpn1394–568 (black) and with Rpn1394–568 (purple). To assess chemical shift perturbations of the UBL domain on Rpn1 binding, titration of 0–2 molar equivalents of Rpn1394–568 was performed. Residues K49 and A55 in the β3-α2 loop, K65 in the α2-β4 loop and W9 in the β1-β2 loop are displayed. (D) Chemical shift changes (△δ) of the UBL domain upon Rpn1394–568 binding are summarized. Chemical shift perturbations are shown by different colors: blue, 0 ppm<△δ ≤0.05 ppm; orange, 0.05 ppm<△δ ≤0.1 ppm; red, 0.1 ppm<△δ ≤ disappeared. Residues that "disappeared," due to peak broadening, are marked by asterisks and the secondary structures are also displayed. (E) Residues with significant chemical shift changes and less perturbed changes upon Rpn1 binding drawn by the Pymol program are shown by spheres and sticks, respectively. (F) Surface charge model of the UBL domain of the hUBLCP1. Electrostatic surfaces are shown for negative (red), positive (blue), and neutral (white) potential.

Mentions: Because the structure of the UBL domain of hUBLCP1 is unique, detailed analysis of Rpn1 binding is of the essence in understanding the mechanism underlying the interaction between the two molecules. Using an immunoprecipitation assay, we showed that hUBLCP1 directly interacts with the Rpn1 of the regulatory particle of the 26 S proteasome (Fig. 3A). A recent study suggests that leucine-rich repeat-like domains of Rpn1 recognize the ubiquitin motif [25]. Homology modeling and secondary structure predictions show that Rpn1 has two structural subunits, regulatory subunits 1 (Rpn1394–568) and 2 (Rpn1640–772) with leucine-rich repeat-like-rich sequences (data not shown). Data from the GST pull-down assay showed that regulatory subunit 1 (Rpn1394–568) directly interacts with UBLCP11–81, whereas regulatory subunit 2 (Rpn1640–772) of Rpn1 does not (Fig. 3B).


Solution structure and Rpn1 interaction of the UBL domain of human RNA polymerase II C-terminal domain phosphatase.

Yun JH, Ko S, Lee CK, Cheong HK, Cheong C, Yoon JB, Lee W - PLoS ONE (2013)

Intermolecular interaction between hUBLCP1 and Rpn1.(A) Immunoprecipitation (IP) of both hUBLCP1 and Rpn1 was performed. Immunoblotting was conducted using HA- and FLAG-antibodies to distinguish the two proteins. IP was performed for the a-FLAG-RPN1 and co-precipitation was confirmed by IB. (B) In vitro GST pull-down assay using the GST-fused UBL domain of hUBLCP1 (UBLCP11–81), the TRX-His6-fused regulatory subunit 1 (Rpn1394–568), and subunit 2 (Rpn1640–772). Lane 1 (upper and lower), GST-fused UBL domain of UBLCP1; Lane 2 (upper), TRX-His6-fused Rpn1394–568 and (lower), TRX-His6-fused Rpn1640–772; Lane 3 (upper and lower), pull-down elution using GST elution buffer containing 10 mM reduced glutathione. (C) 15N-labeled UBL domain was titrated with Rpn1394–568. Superimposed 2D 1H-15N HSQC spectra of UBL domain without Rpn1394–568 (black) and with Rpn1394–568 (purple). To assess chemical shift perturbations of the UBL domain on Rpn1 binding, titration of 0–2 molar equivalents of Rpn1394–568 was performed. Residues K49 and A55 in the β3-α2 loop, K65 in the α2-β4 loop and W9 in the β1-β2 loop are displayed. (D) Chemical shift changes (△δ) of the UBL domain upon Rpn1394–568 binding are summarized. Chemical shift perturbations are shown by different colors: blue, 0 ppm<△δ ≤0.05 ppm; orange, 0.05 ppm<△δ ≤0.1 ppm; red, 0.1 ppm<△δ ≤ disappeared. Residues that "disappeared," due to peak broadening, are marked by asterisks and the secondary structures are also displayed. (E) Residues with significant chemical shift changes and less perturbed changes upon Rpn1 binding drawn by the Pymol program are shown by spheres and sticks, respectively. (F) Surface charge model of the UBL domain of the hUBLCP1. Electrostatic surfaces are shown for negative (red), positive (blue), and neutral (white) potential.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3646893&req=5

pone-0062981-g003: Intermolecular interaction between hUBLCP1 and Rpn1.(A) Immunoprecipitation (IP) of both hUBLCP1 and Rpn1 was performed. Immunoblotting was conducted using HA- and FLAG-antibodies to distinguish the two proteins. IP was performed for the a-FLAG-RPN1 and co-precipitation was confirmed by IB. (B) In vitro GST pull-down assay using the GST-fused UBL domain of hUBLCP1 (UBLCP11–81), the TRX-His6-fused regulatory subunit 1 (Rpn1394–568), and subunit 2 (Rpn1640–772). Lane 1 (upper and lower), GST-fused UBL domain of UBLCP1; Lane 2 (upper), TRX-His6-fused Rpn1394–568 and (lower), TRX-His6-fused Rpn1640–772; Lane 3 (upper and lower), pull-down elution using GST elution buffer containing 10 mM reduced glutathione. (C) 15N-labeled UBL domain was titrated with Rpn1394–568. Superimposed 2D 1H-15N HSQC spectra of UBL domain without Rpn1394–568 (black) and with Rpn1394–568 (purple). To assess chemical shift perturbations of the UBL domain on Rpn1 binding, titration of 0–2 molar equivalents of Rpn1394–568 was performed. Residues K49 and A55 in the β3-α2 loop, K65 in the α2-β4 loop and W9 in the β1-β2 loop are displayed. (D) Chemical shift changes (△δ) of the UBL domain upon Rpn1394–568 binding are summarized. Chemical shift perturbations are shown by different colors: blue, 0 ppm<△δ ≤0.05 ppm; orange, 0.05 ppm<△δ ≤0.1 ppm; red, 0.1 ppm<△δ ≤ disappeared. Residues that "disappeared," due to peak broadening, are marked by asterisks and the secondary structures are also displayed. (E) Residues with significant chemical shift changes and less perturbed changes upon Rpn1 binding drawn by the Pymol program are shown by spheres and sticks, respectively. (F) Surface charge model of the UBL domain of the hUBLCP1. Electrostatic surfaces are shown for negative (red), positive (blue), and neutral (white) potential.
Mentions: Because the structure of the UBL domain of hUBLCP1 is unique, detailed analysis of Rpn1 binding is of the essence in understanding the mechanism underlying the interaction between the two molecules. Using an immunoprecipitation assay, we showed that hUBLCP1 directly interacts with the Rpn1 of the regulatory particle of the 26 S proteasome (Fig. 3A). A recent study suggests that leucine-rich repeat-like domains of Rpn1 recognize the ubiquitin motif [25]. Homology modeling and secondary structure predictions show that Rpn1 has two structural subunits, regulatory subunits 1 (Rpn1394–568) and 2 (Rpn1640–772) with leucine-rich repeat-like-rich sequences (data not shown). Data from the GST pull-down assay showed that regulatory subunit 1 (Rpn1394–568) directly interacts with UBLCP11–81, whereas regulatory subunit 2 (Rpn1640–772) of Rpn1 does not (Fig. 3B).

Bottom Line: The UBL domain of hUBLCP1 has a unique β-strand (β3) and β3-α2 loop, instead of the canonical β4 observed in other UBL domains.The molecular topology and secondary structures are different from those of known UBL domains including that of fly UBLCP1.The positively charged residues of the β3-α2 loop are involved in interacting with the C-terminal leucine-rich repeat-like domain of Rpn1.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul, Korea.

ABSTRACT
The ubiquitin-like modifier (UBL) domain of ubiquitin-like domain proteins (UDPs) interacts specifically with subunits of the 26 S proteasome. A novel UDP, ubiquitin-like domain-containing C-terminal domain phosphatase (UBLCP1), has been identified as an interacting partner of the 26 S proteasome. We determined the high-resolution solution structure of the UBL domain of human UBLCP1 by nuclear magnetic resonance spectroscopy. The UBL domain of hUBLCP1 has a unique β-strand (β3) and β3-α2 loop, instead of the canonical β4 observed in other UBL domains. The molecular topology and secondary structures are different from those of known UBL domains including that of fly UBLCP1. Data from backbone dynamics shows that the β3-α2 loop is relatively rigid although it might have intrinsic dynamic profile. The positively charged residues of the β3-α2 loop are involved in interacting with the C-terminal leucine-rich repeat-like domain of Rpn1.

Show MeSH
Related in: MedlinePlus