Journal Article

The molecular basis of ubiquitin-like protein NEDD8 deamidation by the bacterial effector protein Cif

Institut National de la Santé et de la Recherche Médicale (INSERM), Centre de Physiopathologie de Toulouse-Purpan (INSERM U563 - CNRS UMR1037) ; Centre Hospitalier Universitaire de Toulouse (CHU Toulouse)-Institut Claudius Regaud (ICR)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre de lutte contre le cancer (CLCC)-Centre National de la Recherche Scientifique (CNRS), Oswald, Eric, Taieb, Frederic, Interactions hôtes-agents pathogènes [Toulouse] (IHAP) ; Institut National de la Recherche Agronomique (INRA)-Ecole Nationale Vétérinaire de Toulouse (ENVT) ; Institut National Polytechnique (Toulouse) (Toulouse INP) ; Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National Polytechnique (Toulouse) (Toulouse INP) ; Université de Toulouse (UT)-Université de Toulouse (UT), Biotechnology and Biological Sciences Research Council, United Kingdom [F008732]; Royal Society (United Kingdom) University Research Fellowship; Ligue Nationale Contre le Cancer, Banfield, Mark

2012

Overview

The cycle inhibiting factors (Cifs) are a family of translocated effector proteins, found in diverse pathogenic bacteria, that interfere with the host cell cycle by catalyzing the deamidation of a specific glutamine residue (Gln40) in NEDD8 and the related protein ubiquitin. This modification prevents recycling of neddylated cullin-RING ligases, leading to stabilization of various cullin-RING ligase targets, and also prevents polyubiquitin chain formation. Here, we report the crystal structures of two Cif/NEDD8 complexes, revealing a conserved molecular interface that defines enzyme/substrate recognition. Mutation of residues forming the interface suggests that shape complementarity, rather than specific individual interactions, is a critical feature for complex formation. We show that Cifs from diverse bacteria bind NEDD8 in vitro and conclude that they will all interact with their substrates in the same way. The “occluding loop” in Cif gates access to Gln40 by forcing a conformational change in the C terminus of NEDD8. We used native PAGE to follow the activity of Cif from the human pathogen Yersinia pseudotuberculosis and selected variants, and the position of Gln40 in the active site has allowed us to propose a catalytic mechanism for these enzymes.

• Publisher: National Academy of Sciences,CCSD,National Acad Sciences
• Subject: active sites / Amino Acid Sequence / Bacteria / Bacterial proteins / Bacterial Proteins - chemistry / Bacterial Proteins - genetics / Bacterial Proteins - metabolism / Biological Sciences / Catalytic Domain / Cell cycle / Crystal structure / Crystallization / deamidation / Enzymes / glutamine / Glutamine - genetics / HeLa Cells / Host-Parasite Interactions - physiology / Humans / Life Sciences / ligases / Molecular Sequence Data / Mutagenesis - physiology / Mutation / NEDD8 Protein / Oncogene Protein p21(ras) - metabolism / pathogens / Photorhabdus - enzymology / Photorhabdus - genetics / PNAS Plus / polyacrylamide gel electrophoresis / Polyubiquitin - metabolism / Protein Binding - physiology / Protein Structure, Tertiary / ubiquitin / Ubiquitins - chemistry / Ubiquitins - genetics / Ubiquitins - metabolism / Virulence Factors - chemistry / Virulence Factors - genetics / Virulence Factors - metabolism / Yersinia pseudotuberculosis / Yersinia pseudotuberculosis - enzymology / Yersinia pseudotuberculosis - genetics / Yersinia pseudotuberculosis Infections - metabolism / Yersinia pseudotuberculosis Infections - microbiology
• ISBN: 0003066411000