Conformational selection underlies recognition of a molybdoenzyme by its dedicated chaperone

• Published in: PloS one 2012-11, Vol.7 (11), p.e49523
• Main Authors: Lorenzi, Magali, Sylvi, Léa, Gerbaud, Guillaume, Mileo, Elisabetta, Halgand, Frédéric, Walburger, Anne, Vezin, Hervé, Belle, Valérie, Guigliarelli, Bruno, Magalon, Axel
• Format: Article
• Language: English
• Subjects: Analysis; Assembly; Binding; Biochemistry; Biochemistry, Molecular Biology; Biological activity; Biology; Cell Membrane - metabolism; Chaperones; Circular Dichroism; Complex formation; Crystallography; Electron Spin Resonance Spectroscopy - methods; Electrons; Elongation; Enzymes; Enzymes - chemistry; Escherichia coli - genetics; Escherichia coli - metabolism; Escherichia coli Proteins - metabolism; Experiments; Glycerol; Hydrophobicity; Ionic mobility; Iron; Kinases; Life Sciences; Mass spectrometry; Mass Spectrometry - methods; Mass spectroscopy; Metalloenzymes; Mobility; Models, Molecular; Molecular Chaperones - chemistry; Molecular Chaperones - metabolism; Molecular Conformation; Molybdenum; Molybdenum - chemistry; Mutagenesis, Site-Directed; N-Terminus; Nitrate reductase; Nitrate Reductase - metabolism; Peptides; Protein Conformation; Protein Structure, Tertiary; Proteins; Recognition; Scientific imaging; Spectrometry, Fluorescence - methods; Spectroscopy; Spin labeling; Spin Labels; Structural Biology; Temperature; Tryptophan - chemistry
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0049523

Molecular recognition is central to all biological processes. Understanding the key role played by dedicated chaperones in metalloprotein folding and assembly requires the knowledge of their conformational ensembles. In this study, the NarJ chaperone dedicated to the assembly of the membrane-bound respiratory nitrate reductase complex NarGHI, a molybdenum-iron containing metalloprotein, was taken as a model of dedicated chaperone. The combination of two techniques ie site-directed spin labeling followed by EPR spectroscopy and ion mobility mass spectrometry, was used to get information about the structure and conformational dynamics of the NarJ chaperone upon binding the N-terminus of the NarG metalloprotein partner. By the study of singly spin-labeled proteins, the E119 residue present in a conserved elongated hydrophobic groove of NarJ was shown to be part of the interaction site. Moreover, doubly spin-labeled proteins studied by pulsed double electron-electron resonance (DEER) spectroscopy revealed a large and composite distribution of inter-label distances that evolves into a single preexisting one upon complex formation. Additionally, ion mobility mass spectrometry experiments fully support these findings by revealing the existence of several conformers in equilibrium through the distinction of different drift time curves and the selection of one of them upon complex formation. Taken together our work provides a detailed view of the structural flexibility of a dedicated chaperone and suggests that the exquisite recognition and binding of the N-terminus of the metalloprotein is governed by a conformational selection mechanism.