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

L Kobbi , E Demey-Thomas , F Braye , F Proux , O Kolesnikova , J Vinh (1, 2, 3) , A Poterszman , Oliver Bensaude

1 PSL - Université Paris Sciences et Lettres
2 ESPCI Paris - Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris
3 SMBP - Spectrométrie de Masse Biologique et Protéomique (USR3149 / FRE2032)

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 ((202)PYNTTQFLM(210)) 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.

Domaines: Génomique, Transcriptomique et Protéomique [q-bio.GN]

L Kobbi, E Demey-Thomas, F Braye, F Proux, O Kolesnikova, et al.. An evolutionary conserved Hexim1 peptide binds to the Cdk9 catalytic site to inhibit P-TEFb.. Proceedings of the National Academy of Sciences of the United States of America, 2016, ⟨10.1073/pnas.1612331113⟩. ⟨hal-01415487⟩