Structure of a low-population binding intermediate in protein-RNA recognition.
The interaction of the HIV-1 protein transactivator of transcription (Tat) and its cognate transactivation response element (TAR) RNA transactivates viral transcription and represents a paradigm for the widespread occurrence of conformational rearrangements in protein-RNA recognition. Although the structures of free and bound forms of TAR are well characterized, the conformations of the intermediates in the binding process are still unknown. By determining the free energy landscape of the complex using NMR residual dipolar couplings in replica-averaged metadynamics simulations, we observe two low-population intermediates. We then rationally design two mutants, one in the protein and another in the RNA, that weaken specific nonnative interactions that stabilize one of the intermediates. By using surface plasmon resonance, we show that these mutations lower the release rate of Tat, as predicted. These results identify the structure of an intermediate for RNA-protein binding and illustrate a general strategy to achieve this goal with high resolution.
Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, United Kingdom;
Aditi N
0000-0003-4361-6561
AN
Borkar
Department of Chemistry, University of Washington, Seattle, WA 98197-1700.
Michael F
MF
Bardaro
Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, United Kingdom;
Carlo
0000-0002-9923-8590
C
Camilloni
Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, United Kingdom;
Francesco A
FA
Aprile
Department of Chemistry, University of Washington, Seattle, WA 98197-1700.
Gabriele
G
Varani
Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, United Kingdom; mv245@cam.ac.uk.
Michele
M
Vendruscolo
D001665:Binding Sites; D056906:Cyclin T / Q000378:metabolism; D042863:Cyclin-Dependent Kinase 9 / Q000378:metabolism; D016325:HIV Long Terminal Repeat / Q000502:physiology*; D009682:Magnetic Resonance Spectroscopy; D056004:Molecular Dynamics Simulation; D012367:RNA, Viral / Q000378:metabolism*; D020349:Surface Plasmon Resonance; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000378:metabolism*
C503684:CCNT1 protein, human; D056906:Cyclin T; D012367:RNA, Viral; D054322:tat Gene Products, Human Immunodeficiency Virus; C467969:CDK9 protein, human; D042863:Cyclin-Dependent Kinase 9
NMR spectroscopy; RNA structure; exact RDC restraints; metadynamics; tensor-free method