Identification of novel interactions in HIV-1 capsid protein assembly by high-resolution mass spectrometry.
The pleomorphic nature of the immature and mature HIV-1 virions has made it difficult to characterize intersubunit interactions using traditional approaches. While the structures of isolated domains are known, the challenge is to identify intersubunit interactions and thereby pack these domains into supramolecular structures. Using high-resolution mass spectrometry, we have measured the amide hydrogen exchange protection factors for the soluble capsid protein (CA) and CA assembled in vitro. Comparison of the protection factors as well as chemical crosslinking experiments has led to a map of the subunit/subunit interfaces in the assembled tubes. This analysis provides direct biochemical evidence for the homotypic N domain and C domain interactions proposed from cryo-electron microscopy image reconstruction of CA tubes. Most significantly, we have identified a previously unrecognized intersubunit N domain-C domain interaction. The detection of this interaction reconciles previously discrepant biophysical and genetic data.
Lanman
Jason
J
Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294-2170, USA.
Lam
TuKiet T
TT
Barnes
Stephen
S
Sakalian
Michael
M
Emmett
Mark R
MR
Marshall
Alan G
AG
Prevelige
Peter E
PE
D000577:Amides / Q000737:chemistry; D036022:Capsid Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D003432:Cross-Linking Reagents; D003903:Deuterium / Q000737:chemistry; D019281:Dimerization; D015497:HIV-1 / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D006859:Hydrogen / Q000737:chemistry; D046911:Macromolecular Substances; D013058:Mass Spectrometry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits
D000577:Amides; D036022:Capsid Proteins; D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D021122:Protein Subunits; D006859:Hydrogen; D003903:Deuterium