An evolutionary conserved Hexim1 peptide binds to the Cdk9 catalytic site to inhibit P-TEFb

• Published in: Proceedings of the National Academy of Sciences - PNAS 2016-11, Vol.113 (45), p.12721-12726
• Main Authors: Kobbi, Lydia, Demey-Thomas, Emmanuelle, Braye, Floriane, Proux, Florence, Kolesnikova, Olga, Vinh, Joelle, Poterszman, Arnaud, Bensaude, Olivier
• Format: Article
• Language: English
• Subjects: Amino acids; Binding sites; Biochemistry, Molecular Biology; Biological Sciences; Genes; Genomics; Life Sciences; Peptides; Proteins; RNA polymerase
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1612331113

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.