title: An evolutionary conserved Hexim1 peptide binds to the Cdk9 catalytic site to inhibit P-TEFb.
abstract: The positive transcription elongation factor (P-TEFb) is required for the transcription of most genes by RNA polymerase II. Hexim proteins associated with 7SK RNA bind to P-TEFb and reversibly inhibit its activity. P-TEFb comprises the Cdk9 cyclin-dependent kinase and a cyclin T. Hexim proteins have been shown to bind the cyclin T subunit of P-TEFb. How this binding leads to inhibition of the kinase activity of Cdk9 has remained elusive, however. Using a photoreactive amino acid incorporated into proteins, we show that in live cells, cell extracts, and in vitro reconstituted complexes, Hexim1 cross-links and thus contacts Cdk9. Notably, replacement of a phenylalanine, F208, belonging to an evolutionary conserved Hexim1 peptide (202PYNTTQFLM210) known as the "PYNT" sequence, cross-links a peptide within the activation segment that controls access to the Cdk9 catalytic cleft. Reciprocally, Hexim1 is cross-linked by a photoreactive amino acid replacing Cdk9 W193, a tryptophan within this activation segment. These findings provide evidence of a direct interaction between Cdk9 and its inhibitor, Hexim1. Based on similarities with Cdk2 3D structure, the Cdk9 peptide cross-linked by Hexim1 corresponds to the substrate binding-site. Accordingly, the Hexim1 PYNT sequence is proposed to interfere with substrate binding to Cdk9 and thereby to inhibit its kinase activity.
authors: Ecole Normale Supérieure, Institut de Biologie de l'Ecole Normale Supérieure (IBENS), CNRS UMR 8197, INSERM U-1024, Université de Recherche Paris Sciences et Lettres, F-75230 Paris Cedex 05, France.
authors: Lydia
authors: L
authors: Kobbi
authors: Spectrométrie de Masse Biologique et Protéomique, Ecole Supérieure de Physique et Chimie Industrielle de la ville de Paris (ESPCI), CNRS Unité de Services et de Recherche (USR) 3149, Université de Recherche Paris Sciences et Lettres, F-75231 Paris Cedex 05, France.
authors: Emmanuelle
authors: E
authors: Demey-Thomas
authors: Ecole Normale Supérieure, Institut de Biologie de l'Ecole Normale Supérieure (IBENS), CNRS UMR 8197, INSERM U-1024, Université de Recherche Paris Sciences et Lettres, F-75230 Paris Cedex 05, France.
authors: Floriane
authors: F
authors: Braye
authors: Ecole Normale Supérieure, Institut de Biologie de l'Ecole Normale Supérieure (IBENS), CNRS UMR 8197, INSERM U-1024, Université de Recherche Paris Sciences et Lettres, F-75230 Paris Cedex 05, France.
authors: Florence
authors: F
authors: Proux
authors: Department of Integrated Structural Biology, Institut de Génétique et de Biologie Moléculaire et Cellulaire, INSERM U964, UMR 7104, CNRS/Strasbourg University, 67404 Illkirch, France.
authors: Olga
authors: O
authors: Kolesnikova
authors: Spectrométrie de Masse Biologique et Protéomique, Ecole Supérieure de Physique et Chimie Industrielle de la ville de Paris (ESPCI), CNRS Unité de Services et de Recherche (USR) 3149, Université de Recherche Paris Sciences et Lettres, F-75231 Paris Cedex 05, France.
authors: Joelle
authors: J
authors: Vinh
authors: Department of Integrated Structural Biology, Institut de Génétique et de Biologie Moléculaire et Cellulaire, INSERM U964, UMR 7104, CNRS/Strasbourg University, 67404 Illkirch, France.
authors: Arnaud
authors: A
authors: Poterszman
authors: Ecole Normale Supérieure, Institut de Biologie de l'Ecole Normale Supérieure (IBENS), CNRS UMR 8197, INSERM U-1024, Université de Recherche Paris Sciences et Lettres, F-75230 Paris Cedex 05, France; bensaude@biologie.ens.fr.
authors: Olivier
authors: O
authors: Bensaude
keywords: benzoyl phenylalanine; cyclin-dependent kinase inhibition; protein–protein cross-linking; regulatory noncoding RNA; transcription factor regulation