A Model of N‐Terminal Cyclin T1 Based on FRET Experiments

Sergio Pantano,Mauro Giacca,Alessandro Marcello,Aldo Ferrari,Fabio Beltram,Paolo Carloni,Vittorio Pellegrini,Arianna Sabò

doi:10.1080/10273660500149430

Abstract

Human Cyclin T1 is the cyclin partner of kinase CDK9 in the positive transcription elongation factor b (P‐TEFb). P‐TEFb is recruited by Tat, the transactivator of the human immunodeficiency virus type 1 (HIV‐1), to the viral promoter by direct interactions between Tat, Cyclin T1 and the cis‐acting transactivation‐responsive region (TAR) present at the 5′‐end of each viral mRNA. At present, no structural data for Cyclin T1 are available. Here, we build a structural model of an N‐terminus portion of Cyclin T1 (aa 27–263) based on the X‐ray structure of Cyclin H. The model is compared with site directed mutagenesis data from the literature and validated by fluorescence resonance energy transfer (FRET) using Tat as a probe in living cells. This model provides a first step towards the structural characterization of the CDK9–CycT1–Tat‐TAR complex, which is crucial for HIV‐1 replication and may constitute a promising target for pharmaceutical intervention.

Highlights

The cyclin family of proteins plays a fundamental role in the cell cycle functioning as regulatory subunits of the socalled cyclin dependent kinases (CDK), which act as catalytic subunits [1]
The model is compared with site directed mutagenesis data from the literature and validated by fluorescence resonance energy transfer (FRET) using Tat as a probe in living cells. This model provides a first step towards the structural characterization of the CDK9 –Cyclin T1 (CycT1) – Tat-transactivation-responsive region (TAR) complex, which is crucial for human immunodeficiency virus type 1 (HIV-1) replication and may constitute a promising target for pharmaceutical intervention
We considered a 236 residues long fragment of CycT1 spanning from Phe27 to Ala263 that covers the protein portion involved in HIV-1 Tat and CDK9 binding

Summary

Introduction

The cyclin family of proteins plays a fundamental role in the cell cycle functioning as regulatory subunits of the socalled cyclin dependent kinases (CDK), which act as catalytic subunits [1]. The common structural feature of the family is the cyclin box motif This is a characteristic two-repeats folding motif of , 100 amino acids long [3,4] that constitute a versatile scaffold for mediating protein– protein interactions. The helices within each repeat are spatially disposed with the very hydrophobic helix H3 surrounded by the other four This motif bears a high degree of structural conservation even at very low primary sequence identity. Cyclin boxes are usually inserted into a protein frame, with additional helical elements found at the N and C termini of the cyclin box. These segments are not conserved among the cyclin family and they likely provide binding specificity for protein– protein interactions

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Conclusion