Subdomain motion in HIV-1 Reverse Transcriptase

1. Subdomain motion in HIV-1 Reverse Transcriptase Marcela Madrid Pittsburgh Supercomputing Center

2. Introduction • HIV-1 Reverse Transcriptase (RT) is the enzyme responsible for transcribing the single-stranded RNA of HIV-1 into double-stranded DNA (dsDNA). • RT is an important target for drugs used in the treatment of AIDS: the nucleoside RT inhibitors and the non-nucleoside RT inhibitors.

3. HIV-1 Reverse Transcriptase • RT is a dimeric protein consisting of a 66-kDA (p66) and a 51-kDa (p51) subunit. • Several crystal structures exist of RT in different ligation states. • Each subunit is composed of four subdomains: fingers, palm, thumb and connection. In addition, the p66 subunit includes the RNase H subdomain.

4. HIV-1 Reverse transcriptase

6. Molecular Dynamics • The crystal structures are snapshots of different conformational states. • Molecular dynamics provide an ideal technique to study at atomic detail the movements from one configuration to another.

7. Flexibility and function: why are we interested in studying flexibility? • Flexibility appears to play an important role by allowing the translocation of RT along the nucleic acid. • Non-nucleoside RT inhibitors might inhibit RT by a combination of factors, including allosteric distortion of the polymerase active site and/or impairment of RT mobility.

8. In vacuum molecular dynamics • Eight molecular dynamics simulations were run from the same minimized X-ray starting structure, using different random number generator seeds to generate different initial velocities for each run. • Explicit water was not included, the effect of the solvent was simulated using a dielectric function equal to the interatomic distance.

9. In vacuum-molecular dynamics • Starting structure: crystallographic structure of HIV-1 RT complexed with dsDNA from which the DNA was removed. • This starting configuration, with the p66thumb subdomain in the upright position, was chosen to see if, in the absence of ligands, the p66 thumb subdomain would close down over the p66 palm.

11. Results for in-vacuum simulations • Results: Starting from an open configuration, the p66 thumb subdomain closed down over the palm during six of the eight simulations performed, adopting a configuration very similar to the crystallographic structure of unliganded HIV-1 RT.

13. Biological significance • The fact that the thumb subdomain closes down in the majority of the simulations investigated indicates that, for unliganded RT, the closed down configuration is the favored one with this molecular dynamics model. This is consistent with two independently determined structures of unliganded RT.

14. MD simulations of RT unliganded and complexed to dsDNA in water • Simulations in a water bath, to more realistically simulate physiological conditions. Starting structures unliganded RT (Hsiou et.al, 1996 ) and RT/dsDNA (Jacobo-Molina et.al., 1993; Ding et.al., 1997). • Surrounded the structures by a 10 A water bath. • Using AMBER, constant T, P, PME.

15. Potential Energy as a function of time, RT/dsDNA system

16. RMS deviation from the RT/dsDNA crystallographic structure

17. Crystallographic B-factors and mean-square fluctuations Crystallographic B factors and mean-square fluctuations

18. Crystallographic B-factors and mean-square fluctuations

19. Comparison between B-factors and mean-square fluctuations, unliganded RT Comparison between B-factors and mean-square fluctuations, RT/dsDNA system

20. Comparison between B-factors and mean-square fluctuations, unliganded RT

21. Recently, the structure of RT/dsDNA and a deoxynucleoside Triphosphate (dNTP) was reported. (Huang, et.al, 1998). • In this structure, the tips of the p66 fingers subdomain are bent over the palm, with respect to the RT/dsDNA (Jacobo-Molina et.al, 1993; Ding et.al. 1997), which has the fingers in a more open position.

22. The non-nucleoside binding pocket does not exist in the structures that are not bound to a NNRTI. We observe an increased flexibility in some of the amino acid residues that would eventually form a binding pocket in the proximity of the NNRTI.

23. Correlated motions between the atoms calculated from the MD trajectory • The pattern of concerted motions between RT/dsDNA and the unliganded RT systems is different

24. Conclusions: • The flexibility of the p66 fingers, the tips of the p66 thumb and portions of the p51 thumb and RNase H subdomains, as calculated from the MD trajectories, is larger from the RT/dsDNA system than from the unliganded system, and larger than expected from the crystallographic B-factors.

25. Conclusions • The increased flexibility observed for the tips of the p66 fingers (we observe a sharp peak in the mean square fluctuations of residues 64-69)and p66 thumb subdomain is consistent with this ability of the “thumb open” or RT/dsDNA structure to alter its configuration upon binding of the dNTP or NNRTI. • We now know that the motions of the p66 thumb, p66 fingers and RNase H subdomain occur in a concerted fashion.

26. Conclusions: concerted motions The pattern of concerted motions is different between unliganded RT and RT/dsDNA. The p66 thumb subdomain is uncoupled in the unliganded system, the p66 connection subdomain is uncoupled in the RT/dsDNA system. In the RT/dsDNA, the p66 thumb is anticorrelated to the p66 fingers, the p66 palm, the p51 fingers and the RNase H subdomain.

27. Biological Significance • The concerted motions between the p66 fingers and p66 thumb subdomains might have relevance in the polymerization process of DNA, as HIV-1 RT slides DNA along the p66 subunit. These motions are concerted with those of the RNase H subdomain.

28. Collaborators • Alfredo Jacobo-Molina, Instituto Tecnologico de Monterrey. • Jianping Ding and Eddy Arnold, Center for Advanced Biotechnology and Medicine, and Rutgers University Chemistry Dep. • Jonathan Lukin, Carnegie Mellon University. • Jeffry Madura, Duquesne University.