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Crystal Structure of Human ASB9-2 and Substrate-Recognition of CKB

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Abstract

Human ankyrin repeat and suppressor of cytokine signaling box protein 9 (hASB9) is a specific substrate-recognition subunit of an elongin C-cullin-SOCS box E3 ubiquitin ligase complex. It recognizes its substrate, brain type creatine kinase (CKB), using the ankyrin repeat domain; and facilitates the polyubiquitination of CKB to mediate proteasomal degradation through the SOCS box domain. HASB9-2 is an isoform of hASB9 that contains one ankyrin repeat domain. In this study, the crystal structure of hASB9-2 is shown at 2.2-Å resolution using molecular replacement. Overall, hASB9-2 forms a slightly curved arch with a characteristic L-shaped cross-section. Amino acid substitution analysis based on docking experiments revealed that His103 and Phe107 in hASB9-2 are essential for binding to CKB. Analysis of truncation mutants demonstrated that the first six ankyrin repeats along with the N-terminal region of hASB9-2 contribute to the interaction with CKB.

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Abbreviations

hASB9:

Human ankyrin repeat and suppressor of cytokine signaling box protein 9

ASBs:

Ankyrin repeat and suppressor of cytokine signaling box proteins

SOCS:

Suppressor of cytokine signaling

ECS:

Elongin C-cullin-SOCS box

CKB:

Brain type creatine kinase

CK:

Creatine kinase

E1:

Ubiquitin-activating enzyme

E2:

Ubiquitin-conjugating enzyme

PDB:

Protein Data Bank

GST:

Glutathione S-transferase

PCR:

Polymerase chain reaction

SDS-PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

PBS:

Phosphate buffered saline

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Acknowledgments

This work was supported by the National Basic Research Program of China (973 Program, 2007CB914304 & 2009CB825505), the National Natural Science Foundation of China (30770427), the New Century Excellent Talents in University (NCET-06-0356), the Shanghai Leading Academic Discipline Project (B111) and the National Talent Training Fund in Basic Research of China (No. J0630643).

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Authors and Affiliations

  1. State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, 200433, People’s Republic of China

    Xiangwei Fei, Xing Gu, Zhenxing Yang, Fan Li, Yumin Mao & Chaoneng Ji

  2. National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, People’s Republic of China

    Shilong Fan, Cheng Zhang & Weimin Gong

Authors
  1. Xiangwei Fei
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  2. Xing Gu
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  3. Shilong Fan
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  4. Zhenxing Yang
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  5. Fan Li
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  6. Cheng Zhang
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  7. Weimin Gong
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  8. Yumin Mao
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  9. Chaoneng Ji
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Corresponding author

Correspondence to Chaoneng Ji.

Additional information

The crystal structure reported in this paper has been submitted to the PDB with ID code 3D9H.

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Supplementary material 1 (DOC 346 kb)

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Fei, X., Gu, X., Fan, S. et al. Crystal Structure of Human ASB9-2 and Substrate-Recognition of CKB. Protein J 31, 275–284 (2012). https://doi.org/10.1007/s10930-012-9401-1

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