Wednesday, December 22, 2010

Detection and Functional Characterization of a 215 Amino Acid N-Terminal Extension in the Xanthomonas Type III Effector XopD

PLoS ONE, 2010, 5 (12), art. no. e15773
Joanne Canonne1Daniel Marino1Laurent D.Noël1Ignacio Arechaga3Carole Pichereaux2Michel Rossignol2,Dominique Roby1Susana Rivas1*

1 Laboratoire des Interactions Plantes Micro-organismes (LIPM), UMR CNRS-INRA 2594/441, Castanet Tolosan, France, 2 Institut Fédératif de Recherche (IFR40), Plateforme protéomique Génopole Toulouse Midi-Pyrénées, Institut de Pharmacologie et Biologie Structurale, Université de Toulouse, Toulouse, France, 3 Departamento de Biología Molecular, Universidad de Cantabria (UC) and Instituto de Biomedicina y Biotecnología de Cantabria, IBBTEC (CSIC-UC-IDICAN), Santander, Spain.

Abstract

During evolution, pathogens have developed a variety of strategies to suppress plant-triggered immunity and promote successful infection. In Gram-negative phytopathogenic bacteria, the so-called type III protein secretion system works as a molecular syringe to inject type III effectors (T3Es) into plant cells. The XopD T3E from the strain 85-10 of Xanthomonas campestris pathovar vesicatoria (Xcv) delays the onset of symptom development and alters basal defence responses to promote pathogen growth in infected tomato leaves. XopD was previously described as a modular protein that contains (i) an N-terminal DNA-binding domain (DBD), (ii) two tandemly repeated EAR (ERF-associated amphiphillic repression) motifs involved in transcriptional repression, and (iii) a C-terminal cysteine protease domain, involved in release of SUMO (small ubiquitin-like modifier) from SUMO-modified proteins. Here, we show that the XopD protein that is produced and secreted by Xcv presents an additional N-terminal extension of 215 amino acids. Closer analysis of this newly identified N-terminal domain shows a low complexity region rich in lysine, alanine and glutamic acid residues (KAE-rich) with high propensity to form coiled-coil structures that confers to XopD the ability to form dimers when expressed in E. coli. The full length XopD protein identified in this study (XopD1-760) displays stronger repression of the XopD plant target promoter PR1, as compared to the XopD version annotated in the public databases (XopD216-760). Furthermore, the N-terminal extension of XopD, which is absent in XopD216-760, is essential for XopD type III-dependent secretion and, therefore, for complementation of an Xcv mutant strain deleted from XopD in its ability to delay symptom development in tomato susceptible cultivars. The identification of the complete sequence of XopD opens new perspectives for future studies on the XopD protein and its virulence-associated functions in planta.


Tuesday, December 21, 2010

Electron microscopy analysis of mammalian phosphofructokinase reveals an unusual 3-dimensional structure with significant implications for enzyme function

Arechaga, I., Martínez-Costa, O.H. , Ferreras, C., Carrascosa, J.L., Aragón, J.J. FASEB Journal. 2010, 24, 12, 4960-4968.

Abstract
Phosphofructokinase is a sophisticated allosteric enzyme that is fundamental for the control of glycolysis. The structure of the bacterial enzyme is well characterized. However, little is known about the structural organization of the more complex enzyme from mammals. We have obtained the structure of human muscle phosphofructokinase in the presence of fructose 6-phosphate at a resolution of 1.8 nm by electron microscopy (EM). Particles of the tetrameric enzyme corresponded to an elongated molecule (14.5×9 nm) arranged into 2 dimeric subdomains. Image analysis and 3-dimensional reconstruction showed the presence of a prominent channel in one of the dimers but not in the opposite one, revealing that they are in greatly different conformations. Fitting of bacterial structures into the EM model suggested disruption of the fructose 6-phosphate catalytic and the fructose 2,6-bisphophate allosteric sites in the cavity-containing dimer. Therefore, the reported structure might have major implications for the function of mammalian phosphofructokinase.—Arechaga, I., Martínez-Costa, O. H., Ferreras, C., Carrascosa, J. L., Aragón, J. J. Electron microscopy analysis of mammalian phosphofructokinase reveals an unusual 3-dimensional structure with significant implications for enzyme function.

Structural characterization of the TCR complex by electron microscopy

Ignacio Arechaga, Mahima Swamy, David Abia, Wolfgang A. Schamel, Balbino Alarcón and José María Valpuesta. International Inmunology. 2010, 22, 11, 897-903

Abstract
Structural information on how the TCR transmits signals upon binding of its antigen peptide MHC molecule ligand is still lacking. The ectodomains of the TCRα/β, CD3εγ and CD3εδ dimers, as well as the transmembrane domain of CD3ζ, have been characterized by X-ray crystallography and nuclear magnetic resonance (NMR). However, no structural data have been obtained for the entire TCR complex. In this study, we have purified the TCR from T cells under native conditions and used electron microscopy to derive a three-dimensional structure. The TCR complex appears as a pear-shaped structure of 180 × 120 × 65 . Furthermore, the use of mAbs has allowed to determine the orientation of the TCRα/β and CD3 subunits and to suggest a model of interactions. Interestingly, the reconstructed TCR is larger than expected for a complex with a αβγεδεζζ stoichiometry. The accommodation of a second TCRαβ to fill in the extra volume is discussed.