Institute of Structural Molecular Biology
Institute of Structural Molecular Biology
Von Loeffelholz O.,European Molecular Biology Laboratory |
Von Loeffelholz O.,University Grenoble Alpes |
Von Loeffelholz O.,Institute of Structural Molecular Biology |
Jiang Q.,European Molecular Biology Laboratory |
And 13 more authors.
Proceedings of the National Academy of Sciences of the United States of America | Year: 2015
signal recognition particle (SRP)-dependent pathway is essential for correct targeting of proteins to the membrane and subsequent insertion in the membrane or secretion. In Escherichia coli, the SRP and its receptor FtsY bind to ribosome-nascent chain complexes with signal sequences and undergo a series of distinct conformational changes, which ensures accurate timing and fidelity of protein targeting. Initial recruitment of the SRP receptor FtsY to the SRP- RNC complex results in GTP-independent binding of the SRP-FtsY GTPases at the SRP RNA tetraloop. In the presence of GTP, a closed state is adopted by the SRP- FtsY complex. The cryo-EM structure of the closed state reveals an ordered SRP RNA and SRP M domain with a signal sequence-bound. Van der Waals interactions between the finger loop and ribosomal protein L24 lead to a constricted signal sequence-binding pocket possibly preventing premature release of the signal sequence. Conserved M-domain residues contact ribosomal RNA helices 24 and 59. The SRP-FtsY GTPases are detached from the RNA tetraloop and flexible, thus liberating the ribosomal exit site for binding of the translocation machinery.
Petukhov M.,RAS Petersburg Nuclear Physics Institute |
Dagkessamanskaja A.,French National Center for Scientific Research |
Dagkessamanskaja A.,University Paul Sabatier |
Bommer M.,Institute of Structural Molecular Biology |
And 11 more authors.
Structure | Year: 2012
The TIP49a and TIP49b proteins belong to the family of AAA+ ATPases and play essential roles in vital processes such as transcription, DNA repair, snoRNP biogenesis, and chromatin remodeling. We report the crystal structure of a TIP49b hexamer and the comparative analysis of large-scale conformational flexibility of TIP49a, TIP49b, and TIP49a/TIP49b complexes using molecular modeling and molecular dynamics simulations in a water environment. Our results establish key principles of domain mobility that affect protein conformation and biochemical properties, including a mechanistic basis for the downregulation of ATPase activity upon protein hexamerization. These approaches, applied to the lik-TIP49b mutant reported to possess enhanced DNA-independent ATPase activity, help explain how a three-amino acid insertion remotely affects the structure and conformational dynamics of the ATP binding and hydrolysis pocket while uncoupling ATP hydrolysis from DNA binding. This might be similar to the effects of conformations adopted by TIP49 heterohexamers. © 2012 Elsevier Ltd.
Housden N.G.,University of Oxford |
Hopper J.T.S.,University of Oxford |
Lukoyanova N.,Institute of Structural Molecular Biology |
Rodriguez-Larrea D.,University of Oxford |
And 7 more authors.
Science | Year: 2013
Porins are β-barrel outer-membrane proteins through which small solutes and metabolites diffuse that are also exploited during cell death. We have studied how the bacteriocin colicin E9 (ColE9) assembles a cytotoxic translocon at the surface of Escherichia coli that incorporates the trimeric porin OmpF. Formation of the translocon involved ColE9's unstructured N-terminal domain threading in opposite directions through two OmpF subunits, capturing its target TolB on the other side of the membrane in a fixed orientation that triggers colicin import. Thus, an intrinsically disordered protein can tunnel through the narrow pores of an oligomeric porin to deliver an epitope signal to the cell to initiate cell death.
Sawa J.,Institute of Molecular Pathology |
Malet H.,Institute of Structural Molecular Biology |
Krojer T.,Institute of Molecular Pathology |
Canellas F.,Institute of Molecular Pathology |
And 2 more authors.
Journal of Biological Chemistry | Year: 2011
To react to distinct stress situations and to prevent the accumulation of misfolded proteins, all cells employ a number of proteases and chaperones, which together set up an efficient protein quality control system. The functionality of proteins in the cell envelope of Escherichia coli is monitored by the HtrA proteases DegS, DegP, and DegQ. In contrast with DegP and DegS, the structure and function of DegQ has not been addressed in detail. Here, we show that substrate binding triggers the conversion of the resting DegQ hexamer into catalytically active 12- and 24-mers. Interestingly, substrate-induced oligomer reassembly and protease activation depends on the first PDZ domain but not on the second. Therefore, the regulatory mechanism originally identified in DegP should be a common feature of HtrA proteases, most of which encompass only a single PDZ domain. Using a DegQ mutant lacking the second PDZ domain, we determined the high resolution crystal structure of a dodecameric HtrA complex. The nearly identical domain orientation of protease and PDZ domains within 12-and 24-meric HtrA complexes reveals a conserved PDZ1 → L3 → LD/L1/L2 signaling cascade, in which loop L3 senses the repositioned PDZ1 domain of higher order, substrate-engaged particles and activates protease function. Furthermore, our in vitro and in vivo data imply a pH-related function of DegQ in the bacterial cell envelope. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.
Bahi-Buisson N.,University of Paris Descartes |
Bahi-Buisson N.,French Institute of Health and Medical Research |
Souville I.,University of Paris Descartes |
Fourniol F.J.,Institute of Structural Molecular Biology |
And 20 more authors.
Brain | Year: 2013
X-linked isolated lissencephaly sequence and subcortical band heterotopia are allelic human disorders associated with mutations of doublecortin (DCX), giving both familial and sporadic forms. DCX encodes a microtubule-associated protein involved in neuronal migration during brain development. Structural data show that mutations can fall either in surface residues, likely to impair partner interactions, or in buried residues, likely to impair protein stability. Despite the progress in understanding the molecular basis of these disorders, the prognosis value of the location and impact of individual DCX mutations has largely remained unclear. To clarify this point, we investigated a cohort of 180 patients who were referred with the agyria-pachygyria subcortical band heterotopia spectrum. DCX mutations were identified in 136 individuals. Analysis of the parents' DNA revealed the de novo occurrence of DCX mutations in 76 cases [62 of 70 females screened (88.5%) and 14 of 60 males screened (23%)], whereas in the remaining cases, mutations were inherited from asymptomatic (n = 14) or symptomatic mothers (n = 11). This represents 100% of families screened. Female patients with DCX mutation demonstrated three degrees of clinical-radiological severity: a severe form with a thick band (n = 54), a milder form (n = 24) with either an anterior thin or an intermediate thickness band and asymptomatic carrier females (n = 14) with normal magnetic resonance imaging results. A higher proportion of nonsense and frameshift mutations were identified in patients with de novo mutations. An analysis of predicted effects of missense mutations showed that those destabilizing the structure of the protein were often associated with more severe phenotypes. We identified several severe-and mild-effect mutations affecting surface residues and observed that the substituted amino acid is also critical in determining severity. Recurrent mutations representing 34.5% of all DCX mutations often lead to similar phenotypes, for example, either severe in sporadic subcortical band heterotopia owing to Arg186 mutations or milder in familial cases owing to Arg196 mutations. Taken as a whole, these observations demonstrate that DCX-related disorders are clinically heterogeneous, with severe sporadic and milder familial subcortical band heterotopia, each associated with specific DCX mutations. There is a clear influence of the individual mutated residue and the substituted amino acid in determining phenotype severity. © (2012) The Author.
Fourniol F.J.,Institute of Structural Molecular Biology |
Moores C.A.,Institute of Structural Molecular Biology
Methods in Molecular Biology | Year: 2011
Kinesin motors couple ATP hydrolysis to movement along microtubules, which act both as tracks and as activators of kinesin ATPase activity. Cryo-electron microscopy and image processing enables generation of three-dimensional snapshots of kinesin motors on their tracks at different stages of their ATPase cycle, and can reveal their motor mechanisms at secondary structure resolution. Here, we describe in detail the methods and conditions employed in our lab to prepare high-quality frozen-hydrated samples, which yield structural insights into kinesin motor mechanisms. © 2011 Springer Science+Business Media, LLC.
PubMed | Institute of Structural Molecular Biology
Type: | Journal: Methods in molecular biology (Clifton, N.J.) | Year: 2011
Kinesin motors couple ATP hydrolysis to movement along microtubules, which act both as tracks and as activators of kinesin ATPase activity. Cryo-electron microscopy and image processing enables generation of three-dimensional snapshots of kinesin motors on their tracks at different stages of their ATPase cycle, and can reveal their motor mechanisms at secondary structure resolution. Here, we describe in detail the methods and conditions employed in our lab to prepare high-quality frozen-hydrated samples, which yield structural insights into kinesin motor mechanisms.