Structural and Functional Assays of AtTLP18.3 Identify Its Novel Acid Phosphatase Activity in Thylakoid Lumen 1[W][OA]

The membrane protein AtTLP18.3 of Arabidopsis ( Arabidopsis thaliana ) contains a domain of unknown function, DUF477; it forms a polysome with photosynthetic apparatuses in the thylakoid lumen. To explore the molecular function of AtTLP18.3, we resolved its crystal structures with residues 83 to 260, the DUF477 only, and performed a series of biochemical analyses to discover its function. The gene expression of AtTLP18.3 followed a circadian rhythm. X-ray crystallography revealed the folding of AtTLP18.3 as a three-layer sandwich with three a -helices in the upper layer, four b -sheets in the middle layer, and two a -helices in the lower layer, which resembles a Rossmann fold. Structural comparison suggested that AtTLP18.3 might be a phosphatase. The enzymatic activity of AtTLP18.3 was further confirmed by phosphatase assay with various substrates (e.g. p -nitrophenyl phosphate, 6,8-difluoro-4-methylumbelliferyl phosphate, O -phospho- L -serine, and several synthetic phospho-peptides). Furthermore, we obtained the structure of AtTLP18.3 in complex with O -phospho- L -serine to identify the binding site of AtTLP18.3. Our structural and biochemical studies revealed that AtTLP18.3 has the molecular function of a novel acid phosphatase in the thylakoid lumen. The PCR for site-directed mutagenesis denaturation at 95 (cid:4) followed 16 cycles of 30 s at 95 (cid:4) s at 55 (cid:4) 72 (cid:4) After amplification, the digested with Dpn I for 1 at 37 (cid:4) C to remove template plasmid The mutants containing a single point mutation were first made individually and expressed as for overexpression of the wild-type protein for checking protein solubility. The mutant with a double point mutation, L128M and I159M, was finally chosen and generated from the soluble proteins. All plasmids containing point mutations were confirmed for substitution of the target residues by DNA sequencing.

[1]  E. Weis,et al.  Regulatory Subunit B′γ of Protein Phosphatase 2A Prevents Unnecessary Defense Reactions under Low Light in Arabidopsis1[W][OA] , 2011, Plant Physiology.

[2]  Keisuke Kawakami,et al.  Crystal structure of oxygen-evolving photosystem II at a resolution of 1.9 Å , 2011, Nature.

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

[4]  M. Suorsa,et al.  Auxiliary proteins involved in the assembly and sustenance of photosystem II , 2008, Photosynthesis Research.

[5]  V. Paakkarinen,et al.  TLP18.3, a novel thylakoid lumen protein regulating photosystem II repair cycle. , 2007, The Biochemical journal.

[6]  Nathan Nelson,et al.  The structure of a plant photosystem I supercomplex at 3.4 Å resolution , 2007, Nature.

[7]  Frances M. G. Pearl,et al.  The CATH domain structure database: new protocols and classification levels give a more comprehensive resource for exploring evolution , 2006, Nucleic Acids Res..

[8]  T. Kieselbach,et al.  The chloroplast lumen and stromal proteomes of Arabidopsis thaliana show differential sensitivity to short- and long-term exposure to low temperature. , 2006, The Plant journal : for cell and molecular biology.

[9]  D. Leister,et al.  Abundantly and Rarely Expressed Lhc Protein Genes Exhibit Distinct Regulation Patterns in Plants1[W] , 2006, Plant Physiology.

[10]  Jan Kern,et al.  Towards complete cofactor arrangement in the 3.0 Å resolution structure of photosystem II , 2005, Nature.

[11]  Kevin Cowtan,et al.  research papers Acta Crystallographica Section D Biological , 2005 .

[12]  K. V. van Wijk,et al.  New Functions of the Thylakoid Membrane Proteome of Arabidopsis thaliana Revealed by a Simple, Fast, and Versatile Fractionation Strategy* , 2004, Journal of Biological Chemistry.

[13]  D. Kramer,et al.  How acidic is the lumen? , 1999, Photosynthesis Research.

[14]  D. Godde,et al.  Phosphorylation of PS II polypeptides inhibits D1 protein-degradation and increases PS II stability , 1996, Photosynthesis Research.

[15]  T. Higgins,et al.  A Sensitive, High-Volume, Colorimetric Assay for Protein Phosphatases , 1994, Pharmaceutical Research.

[16]  G. Friso,et al.  In-Depth Analysis of the Thylakoid Membrane Proteome of Arabidopsis thalianaChloroplasts:NewProteins,NewFunctions, and a Plastid Proteome Database , 2004 .

[17]  J. Popot,et al.  An atypical haem in the cytochrome b6f complex , 2003, Nature.

[18]  Takeshi Kawabata,et al.  MATRAS: a program for protein 3D structure comparison , 2003, Nucleic Acids Res..

[19]  C. Wilkerson,et al.  Proteomic study of the Arabidopsis thaliana chloroplastic envelope membrane utilizing alternatives to traditional two-dimensional electrophoresis. , 2003, Journal of proteome research.

[20]  J. Garin,et al.  Proteomics of the Chloroplast Envelope Membranes from Arabidopsis thaliana*S , 2003, Molecular & Cellular Proteomics.

[21]  G. Groth.  Structure of spinach chloroplast F1-ATPase complexed with the phytopathogenic inhibitor tentoxin , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[22]  B. Haas,et al.  Proteome Map of the Chloroplast Lumen of Arabidopsis thaliana * , 2002, The Journal of Biological Chemistry.

[23]  Peter Roepstorff,et al.  Central Functions of the Lumenal and Peripheral Thylakoid Proteome of Arabidopsis Determined by Experimentation and Genome-Wide Prediction Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.010304. , 2002, The Plant Cell Online.

[24]  Kathleen Marchal,et al.  PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences , 2002, Nucleic Acids Res..

[25]  Jae Young Lee,et al.  Crystal structure and functional analysis of the SurE protein identify a novel phosphatase family , 2001, Nature Structural Biology.

[26]  Petra Fromme,et al.  Three-dimensional structure of cyanobacterial photosystem I at 2.5 Å resolution , 2001, Nature.

[27]  T. Ueda,et al.  Tolerance of point substitution of methionine for isoleucine in hen egg white lysozyme. , 2001, Protein engineering.

[28]  M. Sussman,et al.  Mass Spectrometric Resolution of Reversible Protein Phosphorylation in Photosynthetic Membranes ofArabidopsis thaliana* , 2001, The Journal of Biological Chemistry.

[29]  Petra Fromme,et al.  Crystal structure of photosystem II from Synechococcus elongatus at 3.8 Å resolution , 2001, Nature.

[30]  Liam J. McGuffin,et al.  The PSIPRED protein structure prediction server , 2000, Bioinform..

[31]  B. Matthews,et al.  Use of differentially substituted selenomethionine proteins in X-ray structure determination. , 1999, Acta crystallographica. Section D, Biological crystallography.

[32]  D T Jones,et al.  Protein secondary structure prediction based on position-specific scoring matrices. , 1999, Journal of molecular biology.

[33]  Yunje Cho,et al.  Structure-based identification of a novel NTPase from Methanococcus jannaschii , 1999, Nature Structural Biology.

[34]  Thomas C. Terwilliger,et al.  Automated MAD and MIR structure solution , 1999, Acta crystallographica. Section D, Biological crystallography.

[35]  B. Andersson,et al.  A cyclophilin-regulated PP2A-like protein phosphatase in thylakoid membranes of plant chloroplasts. , 1999, Biochemistry.

[36]  Stephane Rombauts,et al.  PlantCARE, a plant cis-acting regulatory element database , 1999, Nucleic Acids Res..

[37]  Yoshihiro Ugawa,et al.  Plant cis-acting regulatory DNA elements (PLACE) database: 1999 , 1999, Nucleic Acids Res..

[38]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.

[39]  Kenichi Higo,et al.  PLACE: a database of plant cis-acting regulatory DNA elements , 1998, Nucleic Acids Res..

[40]  Chris Sander,et al.  Touring protein fold space with Dali/FSSP , 1998, Nucleic Acids Res..

[41]  Thomas L. Madden,et al.  Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. , 1997, Nucleic acids research.

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

[43]  G. Schönknecht,et al.  Ion Channel Permeable for Divalent and Monovalent Cations in Native Spinach Thylakoid Membranes , 1996, The Journal of Membrane Biology.

[44]  E. Aro,et al.  Degradation of the D1- and D2-proteins of photosystem II in higher plants is regulated by reversible phosphorylation. , 1995, Biochemistry.

[45]  M. Weiner,et al.  Site-directed mutagenesis of double-stranded DNA by the polymerase chain reaction. , 1994, Gene.

[46]  H. Michel,et al.  Tandem mass spectrometry identifies sites of three post-translational modifications of spinach light-harvesting chlorophyll protein II. Proteolytic cleavage, acetylation, and phosphorylation. , 1991, The Journal of biological chemistry.

[47]  A. McPherson,et al.  Current approaches to macromolecular crystallization. , 1990, European journal of biochemistry.

[48]  M. W. Jones,et al.  Chloroplast thylakoid protein phosphatase is a membrane surface-associated activity. , 1989, Plant physiology.

[49]  R. Selvam,et al.  Isolation and properties of an acid phosphatase from thylakoid membranes of Sorghum vulgare. , 1981, Archives of biochemistry and biophysics.