Structural study of MCPIP1 N-terminal conserved domain reveals a PIN-like RNase

MCP-1-induced protein 1 (MCPIP1) plays an important role in the downregulation of the LPS-induced immune response by acting as an RNase targeting IL-6 and IL-12b mRNAs. A conserved domain located in the N-terminal part of MCPIP1 is thought to be responsible for its RNase activity, but its catalytic mechanism is not well understood due to the lack of an atomic resolution structure. We determined the 3D crystal structure of this MCPIP1 N-terminal conserved RNase domain at a resolution of 2.0 Å. The overall structure of MCPIP1 N-terminal conserved domain shares high structural homology with PilT N-terminal domain. We show that the RNase catalytic center is composed of several acidic residues, verifying their importance by site-specific mutagenesis. A positively charged arm close to the catalytic center may act as an RNA substrate-binding site, since exchange of critical positively charged residues on this arm with alanine partially abolish the RNase activity of MCPIP1 in vivo. Our structure of the MCPIP1 N-terminal conserved domain reveals the details of the catalytic center and provides a greater understanding of the RNA degradation mechanism.

[1]  Mahua Choudhury,et al.  Monocyte Chemoattractant Protein-1 Induces a Novel Transcription Factor That Causes Cardiac Myocyte Apoptosis and Ventricular Dysfunction , 2006, Circulation research.

[2]  M. Fu,et al.  A Novel CCCH-Zinc Finger Protein Family Regulates Proinflammatory Activation of Macrophages* , 2008, Journal of Biological Chemistry.

[3]  A. Kasza,et al.  Interleukin‐1‐inducible MCPIP protein has structural and functional properties of RNase and participates in degradation of IL‐1β mRNA , 2009, The FEBS journal.

[4]  Liisa Holm,et al.  Dali server: conservation mapping in 3D , 2010, Nucleic Acids Res..

[5]  Haruki Nakamura,et al.  Zc3h12a is an RNase essential for controlling immune responses by regulating mRNA decay , 2009, Nature.

[6]  Randy J. Read,et al.  Phaser crystallographic software , 2007, Journal of applied crystallography.

[7]  A. Zernecke,et al.  MCP-1 induces a novel transcription factor with proapoptotic activity. , 2006, Circulation research.

[8]  F. Liu,et al.  Retinoic acid inducible gene-I, more than a virus sensor , 2011, Protein & Cell.

[9]  Patrice Gouet,et al.  ESPript: analysis of multiple sequence alignments in PostScript , 1999, Bioinform..

[10]  R. Méndez,et al.  Specificity of RNA Binding by CPEB: Requirement for RNA Recognition Motifs and a Novel Zinc Finger , 1998, Molecular and Cellular Biology.

[11]  L. Skalniak,et al.  Regulatory feedback loop between NF‐κB and MCP‐1‐induced protein 1 RNase , 2009, The FEBS journal.

[12]  Collaborative Computational,et al.  The CCP4 suite: programs for protein crystallography. , 1994, Acta crystallographica. Section D, Biological crystallography.

[13]  Martin Phillips,et al.  Toward the structural genomics of complexes: crystal structure of a PE/PPE protein complex from Mycobacterium tuberculosis. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[14]  C. M. Dupureur.  One is enough: insights into the two-metal ion nuclease mechanism from global analysis and computational studies. , 2010, Metallomics : integrated biometal science.

[15]  M. Harding,et al.  The geometry of metal-ligand interactions relevant to proteins. , 1999, Acta crystallographica. Section D, Biological crystallography.

[16]  George M Sheldrick,et al.  Substructure solution with SHELXD. , 2002, Acta crystallographica. Section D, Biological crystallography.

[17]  Thomas R. Schneider,et al.  HKL2MAP: a graphical user interface for macromolecular phasing with SHELX programs , 2004 .

[18]  Jing Wang,et al.  MCP-induced protein 1 deubiquitinates TRAF proteins and negatively regulates JNK and NF-κB signaling , 2010, The Journal of experimental medicine.

[19]  T. Ceska,et al.  A helical arch allowing single-stranded DNA to thread through T5 5'-exonuclease , 1996, Nature.

[20]  E. Conti,et al.  Structures of the PIN domains of SMG6 and SMG5 reveal a nuclease within the mRNA surveillance complex , 2006, The EMBO journal.

[21]  W. Delano.  The PyMOL Molecular Graphics System , 2002 .

[22]  E. Birney,et al.  Pfam: the protein families database , 2013, Nucleic Acids Res..

[23]  Wei Yang,et al.  An equivalent metal ion in one- and two-metal-ion catalysis , 2008, Nature Structural &Molecular Biology.

[24]  B. Matthews.  Solvent content of protein crystals. , 1968, Journal of molecular biology.

[25]  M. Fu,et al.  Genome-Wide Survey and Expression Profiling of CCCH-Zinc Finger Family Reveals a Functional Module in Macrophage Activation , 2008, PloS one.

[26]  C. Janeway,et al.  Innate immune recognition. , 2002, Annual review of immunology.

[27]  Z. Otwinowski,et al.  Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[28]  George M. Sheldrick,et al.  Macromolecular phasing with SHELXE , 2002 .

[29]  Wei Yang,et al.  Nucleases: diversity of structure, function and mechanism , 2010, Quarterly Reviews of Biophysics.

[30]  H. Dyson,et al.  Recognition of the mRNA AU-rich element by the zinc finger domain of TIS11d , 2004, Nature Structural &Molecular Biology.

[31]  Hiroshi I. Suzuki,et al.  MCPIP1 ribonuclease antagonizes dicer and terminates microRNA biogenesis through precursor microRNA degradation. , 2011, Molecular cell.

[32]  Rodrigo Lopez,et al.  Clustal W and Clustal X version 2.0 , 2007, Bioinform..

[33]  P. Emsley,et al.  Features and development of Coot , 2010, Acta crystallographica. Section D, Biological crystallography.

[34]  P. Blackshear,et al.  Interactions of CCCH Zinc Finger Proteins with mRNA , 2002, The Journal of Biological Chemistry.

[35]  Marcin Piechota,et al.  Identification of interleukin-1 and interleukin-6-responsive genes in human monocyte-derived macrophages using microarrays. , 2008, Biochimica et biophysica acta.

[36]  G. Gao,et al.  Analyses of SELEX-derived ZAP-binding RNA aptamers suggest that the binding specificity is determined by both structure and sequence of the RNA , 2010, Protein & Cell.