Unraveling the interface of signal recognition particle and its receptor by using chemical cross-linking and tandem mass spectrometry.
Among the methods used to unravel protein interaction surfaces, chemical cross-linking followed by identification of the cross-linked peptides by mass spectrometry has proven especially useful in dynamic and complex systems. During the signal recognition particle (SRP)-dependent targeting of proteins to the bacterial plasma membrane, the specific interaction between Ffh (the protein component of SRP) and FtsY (the SRP receptor) is known to be essential for the efficiency and fidelity of this process. In this work, we studied the Escherichia coli and Thermus aquaticus Ffh.FtsY complexes by using chemical cross-linking and tandem mass spectrometry to identify nine intermolecular cross-linked peptides. This information was used in conjunction with a previously undescribed model-building approach that combines geometric restraint optimization with macromolecular docking. The resulting model of the Ffh.FtsY complex is in good agreement with the crystal structure solved shortly thereafter. Intriguingly, four of the cross-linked pairs involve the M domain of Ffh, which is absent from the crystal structure, providing previously undocumented experimental evidence that the M domain is positioned in close proximity to the Ffh.FtsY interface in the complex.
Chu
Feixia
F
Mass Spectrometry Facility, University of California, San Francisco 94143-0046, USA.
Shan
Shu-ou
SO
Moustakas
Demetri T
DT
Alber
Frank
F
Egea
Pascal F
PF
Stroud
Robert M
RM
Walter
Peter
P
Burlingame
Alma L
AL
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003432:Cross-Linking Reagents; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013058:Mass Spectrometry; D008958:Models, Molecular; D046912:Multiprotein Complexes; D017434:Protein Structure, Tertiary; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018000:Receptors, Peptide / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018271:Signal Recognition Particle / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013824:Thermus / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; C082931:Ffh protein, E coli; C087406:FtsY protein, Bacteria; D046912:Multiprotein Complexes; D018160:Receptors, Cytoplasmic and Nuclear; D018000:Receptors, Peptide; D011994:Recombinant Proteins; D018271:Signal Recognition Particle; C036318:signal peptide receptor