Molecular dynamics simulation and experimental verification of the interaction between cyclin T1 and HIV-1 Tat proteins

Abstract

The viral encoded Tat protein is essential for the transcriptional activation of HIV proviral DNA. Interaction of Tat with a cellular transcription elongation factor P-TEFb containing CycT1 is critically required for its action. In this study, we performed MD simulation using the 3D data for wild-type and 4CycT1mutants3D data. We found that the dynamic structural change of CycT1 H2' helix is indispensable for its activity for the Tat action. Moreover, we detected flexible structural changes of the Tat-recognition cavity in the WT CycT1 comprising of ten AAs that are in contact with Tat. These structural fluctuations in WT were lost in the CycT1 mutants. We also found the critical importance of the hydrogen bond network involving H1, H1' and H2 helices of CycT1. Since similar AA substitutions of the Tat-CycT1 chimera retained the Tat-supporting activity, these interactions are considered primarily involved in interaction with Tat. These findings described in this paper should provide vital information for the development of effective anti-Tat compound.

Fig 1. Characteristic α-helices of CycT1 (3MI9) and Tat recognition residues (TRR).

(a) (Left panel) Crystal structure of CycT1-Tat complex obtained in the Tat/P-TEFb complex (PDBID:3MI9 [19]). Tat (blue) and Cyc T1 (red and green) are demonstrated in different colors. The locations of each α-helices are indicated: H1, 31–52aa; H2, 54–69aa; H3, 80–64aa; H4, 101–102aa; H5, 125–143aa; H1’, 153–163aa; H2’, 168–184aa; H3’, 193–207aa; H4’, 221–224aa; H5’, 231–245. (Right panel) Locations of Tat recognition residues in CycT1: Q40, D47, N53, V54, Q97, Q172, F176, N180, L184 and L245 (which side chains are shown in stick).

Fig 2. Molecular dynamics (MD) simulation.

(a) Time course of root mean square deviations (RMSDs) of wild-type CycT1 (left) and CycT1 mutants (Q46A and Q50A (middle) and Q46AQ50A and F176A (right)). (b) Root mean square fluctuations (RMSFs) of CycT1 molecules. (c) RMSF of TRR residues.

Fig 3. Two-dimensional PCA projections of trajectories obtained from MD simulations for various CycT1 proteins.

Initially, PCA calculations were carried out with all the CycT1 models including WT, and CycT1 mutants, Q46A, Q50A, Q46AQ50A and F176A, in order to obtain the coordinate for each component. Then, using the rmsd distance data for Cα atoms of TRR obtained from this coordinate, each trajectory data set was plotted in accordance with principal components 1 and 2 for abscissa and ordinate, respectively. 2-D (primary component 1 (PC1)-PC2) plots of trajectories obtained with WT (a) and various CycT1 molecules (b). The position of 3MI9 CycT1 is indicated as “*”. Probability distribution (right) of MD trajectories based on PCA was estimated using kernel density estimation method in R version 3.1.1.

Fig 4. Comparison of representative structures of various CycT1 models obtained by MD simulation.

(a) Comparison of CycT1 WT structures obtained by crystallographic analysis ((3MI9); green) and the representative structures predicted by MD simulation (Area1; cyan, Area 2; pink). Note the structural shifts of H1, H2, H1’ and H2’ helices upon superimposition. (b) The structural changes of Tat binding cavity circumscribed by TRR (red). Surface of TRR are shown in red and surrounded amino acids are shown in green (3MI9), cyan (Area 1) and pink (Area 2) in accordance with the color used in Fig. 4a. The position of Q50 is indicated by dotted circle. Lower panels indicate the positions of TRR with cylindrical helices for the backbone.

Fig 5. The transcriptional activity of CycT1 molecules in supporting the Tat-mediated HIV trans-activation: involvement of intra-molecular hydrogen bonds within CycT1, Q46:56 and N60:H183, forming the intra-molecular hydrogen bond network.

(a) Effects of Ala-substitution in CycT1 on the Tat-transactivation (CycT1 Q46A and Q56A mutants). (b) Effects of Ala-substitution in CycT1 on the Tat-transactivation (CycT1 N60A and H183A mutants). Transcriptional activities of Tat co-transfected with CycT1 (left panel) or those of CycT1-Tat chimera (right panel) are shown. Western blots shown below indicate that the equivalent amount of each protein was expressed in the transfected cells. These protein bands were originated from the same blot for each panel.

Fig 6. Effects of Ala-substitutions within Cyc T1 on the binding to Tat and CDK9 by immunoprecipitation.

HEK293 cells were transfected with various CycT1 mutants (FLAG-tagged), HA-tagged Tat and Myc-tagged CDK9. The cell extracts were immunoprecipitated with anti-FLAG(M2) beads or anti-HA antibody. The immunoprecipitated complex was then immunoblotted with indicated antibodies. Expression of FLAG-CycT1, HA-Tat and Myc-CDK9 proteins in the transfected cells was confirmed by immunoblotting with indicated antibodies (“Input” panels).