HIV-1 Tat hijacks the human superelongation complex (SEC) to promote proviral transcription. Here we report the 5.9 A° structure of HIV-1 TAR in complex with HIV-1 Tat and human AFF4, CDK9, and CycT1. The TAR central loop contacts the CycT1 Tat-TAR recognition motif (TRM) and the second Tat Zn2+-binding loop. Hydrogen-deuterium exchange (HDX) shows that AFF4 helix 2 is stabilized in the TAR complex despite not touching the RNA, explaining how it enhances TAR binding to the SEC 50-fold. RNA SHAPE and SAXS data were used to help model the extended (Tat Arginine-Rich Motif) ARM, which enters the TAR major groove between the bulge and the central loop. The structure and functional assays collectively support an integrative structure and a bipartite binding model, wherein the TAR central loop engages the CycT1 TRM and compact core of Tat, while the TAR major groove interacts with the extended Tat ARM.
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We are grateful to Sarah Keane and Michael Summers for providing 5’UTR RNA and to Chris Jeans (Macrolab at UC Berkeley) for providing equipment for small-scale purification of proteins by size exclusion chromatography. We also thank James Holton for help with X-ray crystal data collection and processing at beamline 8.3.1 at the Advanced Light Source at LBNL. This work was supported by NIH grants P50GM082250 (JHH, JAD, and AS) and NIAID R01AI041757 and R01AI095057 (QZ). SAXS data were collected at the SIBYLS beamline (Lawrence Berkeley National Laboratory) which is funded by DOE/BER contract number DE- AC02-05CH11231 and NIGMS R01GM105404. Beamline 8.3.1 at the Advanced Light Source, LBNL, is supported by the UC Office of the President, Multicam- pus Research Programs and Initiatives grant MR-15-328599 and the Program for Breakthrough Bio- medical Research, which is partially funded by the Sandler Foundation. The Advanced Light Source is supported by the Director, Office of Science, Office of Basic Energy Sciences, of the US Department of Energy under Contract No DE-AC02-05CH11231. This research continues a program begun by the late Thomas Alber, and we dedicate this paper to him.
© Schulze-Gahmen et al.