Molecular dynamics simulation of site-directed mutagenesis of HIV-1 Tat trans -activator

CUI Yan' , LING Lunjiang' , CHEN Runsheng' * , BAl ~ongchuan~ , YUAN Jiangang2 and QlANG Boqin2 * 1 . Institute of Biophysics, Chinese Academy of Sciences, Beiing 100101 , China ; 2 . Institute of Basic Medical Sciences , Chinese Academy of Medical Sciences , Beijing 100005, China * Corresponding authors

Abstract The Cys-rich domain, core region and basic domain are highly conserved and very important to the trans-activation activity of HIV-1 Tat trans-activator. The three-dimensional structures of 6 mutants of HIV-1 Tat protein were constructed with the methods of molecular dy- namics simulation. The variations of the structures of the mutants have been analyzed and the factors that led to abolishment of trans-activation activity have been discussed.

Keywords: human immunodeficiency virus type 1 ( HIV-1) , molecular dynamics simulation, site-directed mutagenesis, Tat protein.

HUMAN immunodeficiency virus type 1 (HIV-1) is the main pathogeny of AIDS. Besides encoding struc- tural proteins, the genome of HIV-1 encodes 6 regulatory proteins including trans-activator of transcrip- tion (Tat) protein, a very important regulator. Tat protein can combine with TAR RNA element ( trans- acting response element) which is transcripted by HIV LTR and enhance the transcription. HIV-1 Tat protein is composed of 86 amino acid residues. The 72 residues at the N-terminal have the complete trans-activation activity. Tat protein has three functional domains, they are cys-rich domain, core region and basic domain. We analysized the effects of the 6 kinds of amino acid residue mutations on the trans- activation activity with site-directed mutagenesis method and found that the trans-activation activity of the mutants are reduced greatly or abolished completely. These results indicate that the conservation of amino acid residues in these regions is crucial for the trans-activation function of Tat protein (table 1)"' .

Table 1 Trans-activation activities o f w i l d - t v ~ e and mutant Tat oroteins

Mutation Luciferase activity ( % ) Trans-activation activity ( % )

Wild-type Tat 100 100 Cys22-Gly 5 . 8 0 2 0 . 8 5 0 . 6 3 His33/Cys34+Ala/Ser 7 . 3 0 * 2 . 2 6 2 . 2 1 ThrrtO/Lys41 +Ala/Ala 4 . 8 7 k 0 . 4 7 x

Lys5O- STOP 5 . 9 7 * 1.21 0 . 8 1 Arg52+Leu 1 0 . 0 0 * 0 . 6 1 5 . 0 6 Thr40/Lys41/Lys5O-r 4 . 1 0 r 0 . 5 2 n

Ala/Ala/STOP

* , Trans-activation activities were abolished by amino acids substitutions.

We used molecular dynamics method to simulate these site-directed mutageneses. The structural variance of each mutant was analyzed. The reasons for loss of trans-activation activity were also explained according to the results of computer simulations.

1 Materials and methods

The structure of HIV-1 Tat protein (Thr40-tLys mutant) has been determined by NMR (PDB code: 1TIV) . There are 10 sets of coordinates in this PDB file. We optimized each structure with energy mini- mization method (the program we used was DISCOVER of MSI Inc . ) , and selected the structure with the lowest energy for the following simulation.

We changed the amino acid residues at the mutation site with molecular simulation program Insight11

(MSI Inc. ) . Then each of the 6 mutants was subjected to a 20 ps (1 ps = 10-j2 s ) molecular dynamic

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NOTES simulation. The force field was CVFF (consistent valence force field) . The time step of molecular dy-

manic integration is 1 fs ( 1 fs = 10- l5 s ) , and the temperature was kept at 300 K . After simulation, we analyzed the 6 trajectories of molecular dynamic simulation and selected the

structure with the lowest energy respectively. These 6 structures were used as representatives for analyzing the effect of the mutations on the trans-activation activity of Tat protein.

2 Results and discussion

The experimental data about the trans-activation activity of the 6 mutants of Tat protein are shown in table 1 . We give a summary of the results of MD simulation in table 2 , in which the position ( t ) on the molecular dynamic trajectory, energy and the root mean square errors of the mutations (using the NMR structure as reference) are listed.

Table 2 Summarv of the results of molecular dvnamics simulation

Mutation t/ps Energy/kJ. mol - ' RMS/nm

Cys224 Gly 16 .8 8 461.91 0 .325 8 His33/Cys34+Ala/Ser 1 4 . 4 8 578.53 0 .266 7 Thr40/Lys41+Ala/Ala 1 5 . 2 8 408.07 0 .398 0 Lys5O+STOP 1 9 . 6 4 777 .49 0 .433 2 Arg52- Leu 1 9 . 2 8 493.76 0 . 3 5 2 7 Thr40/Lys41/Lys50+ 1 9 . 6 4 445.18 0 .651 7 A l d A l d STOP

The effects of the 6 mutations on the structure and trans-activation activity of Tat protein were ana- lyzed as follows.

( i ) Cys22+Gly. This mutation is at the Cys-rich domain (Nos. 22-27 residues ) . The posi- tions of the residues from NOS. 10-19 changed obviously. This region is near the Cys-rich domain. It is

known that the combination of Cys and metal ions leads to the formation of the Tat dimer[21 . The stuckout of this region may prevent the formation of the Tat dimer and result in loss of trans-activation activity.

( ii ) His33/Cys34+Ala/Ser. This mutation is also at the Cys-rich domain. There is not obvious structural deformation, except that the residues from Nos. 54-58 move from their original positions slightly. Structural deformation is not the reason for losing activation. Cys34 is the critical residue for the formation of Tat dimer, while its substitute Ser cannot combine with metal ions. The substitution of Cys34 made the mutant unfeasible to form a Tat dimer. This is the reason for the loss of trans-activation activi-

ty. ( iii ) Thr40/Lys41-+Ala/Ala. This mutation is at the central domain and leads to a large defor-

mation of 3 fragments, which are residues from Nos. 14-17, residues from Nos. 40-55 and residues from Nos. 72-75 . The 3 fragments are on the surface of the HIV-1 Tat protein molecule (fig. 1 ) . From the results we infer that the deformation of the interaction site on the molecule surface is the main reason for losing trans-activation activity. Lys41 participates in the interaction between Tat protein and TFIID

directlyr3], at the same time, Lys41 is on the surface of the hydrophobic core and may take part in the

combination of Tat protein and TAR RNA. To substitute Lys41 with Ala will destroy this ~ombination'~]. ( iv ) LysSO+STOP. This mutant lost 36 residues at the C-terminal. Comparison of the simulated

structure of the mutant and the corresponding part of the NMR structure (residues from Nos. 1-50) is shown in fig. 2 . The overall structure is similar, though the RMS is a little bit larger (0.433 2 nm) . We infer that loss of the large portion of the domain (residues from Nos. 49-57) which is involved in nuclear

localizationr51 and TAR recognition[61 is responsible for losing trans-activation activity. ( V ) Arg52+Leu. The structural variance is not large (RMS is 0 .352 7 nm) . The mutation is at

the basic domain. The loss of trans-activation activity is because the mutation destroyed the function of nuclear localization and TAR recognition.

( ~i ) Thr40/Lys41/Lys50--+Ala/Ala/STOP. This mutation leads to a large structural deformation

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NOTES

Fig. 1 . Comparison behveen MD simulated structure (dark line) of ThM/Lys41~Ala /Ala mutant and the original NMR structure (gray line) .

Fig. 2 . Comparison between MD simulated structure of I,ys50+SM3P mutant (dark line) and the corresponding part of original NMR structure (gray line).

(fig. 3 ) . The RMS is 0.651 7 nm. The structural deformation and the loss of most residues of the basic domain is responsible for the loss of trans-activation activity.

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NOTES

Fig. 3 . Comparison between MD simulated structure of Thr40/Lys41/Lys50+A1a/Ala/STOP mutant (dark line) and the original NMR structure (gray line) .

References

1 Bai, L . , Yuan, J . , Chen, J . et a l . , Effect of site-directed mutagenesis on the trans-activation activity of human irnmunodefi- ciency virus type 1 Tat tram-activator, Chinese Biochem. J . ( in Chinese) , 1996, 12: 381 .

2 Frankel, A. D . , Bredt , D . S . , Pabo, C . D . , Tat protein from human immunodeficiency virus forms a metal-linked dimer, Science, 1988, 240 : 70.

3 Kashsnchi , F . , Direct interaction of human TFIID with the HIV-1 transactivator 'rat, Nature, 1994, 367 : 295. 4 Bayer, P. , Kraft, M . , Ejchart, A . et a l . , Structural studies of HIV-1 Tat protein, J . Mol. Bwl. , 1995, 247: 529. 5 Siomi, H . , Shida, H. , Maki, M . et a1 . , Effects of a highly basic region of human immunodeficiency virus Tat protein on nu-

cleolar localization, J . Virol . , 1990, 64 : 1803 . 6 Roy, S . , A bulge structure in HIV-1 TAR RNA is required for Tat binding and Tat mediated, Genes. Dev . , 1990, 4 : 1365.

( Received August 17, 1998)

Chinese Science Bulletin Vol .44 No. 8 April 1999