The Interaction of RNA Helicase DDX3 with HIV-1 Rev-CRM1-RanGTP Complex during the HIV Replication Cycle

Molecular traffic between the nucleus and the cytoplasm is regulated by the nuclear pore complex (NPC), which acts as a highly selective channel perforating the nuclear envelope in eukaryotic cells. The human immunodeficiency virus (HIV) exploits the nucleocytoplasmic pathway to export its RNA transcripts across the NPC to the cytoplasm. Despite extensive study on the HIV life cycle and the many drugs developed to target this cycle, no current drugs have been successful in targeting the critical process of viral nuclear export, even though HIV’s reliance on a single host protein, CRM1, to export its unspliced and partially spliced RNA transcripts makes it a tempting target. Due to recent findings implicating a DEAD-box helicase, DDX3, in HIV replication and a member of the export complex, it has become an appealing target for anti-HIV drug inhibition. In the present research, we have applied a hybrid computational protocol to analyze protein-protein interactions in the HIV mRNA export cycle. This method is based on molecular docking followed by molecular dynamics simulation and accompanied by approximate free energy calculation (MM/GBSA), computational alanine scanning, clustering, and evolutionary analysis. We highlight here some of the most likely binding modes and interfacial residues between DDX3 and CRM1 both in the absence and presence of RanGTP. This work shows that although DDX3 can bind to free CRM1, addition of RanGTP leads to more concentrated distribution of binding modes and stronger binding between CRM1 and RanGTP.

[1]  C. Dominguez,et al.  HADDOCK: a protein-protein docking approach based on biochemical or biophysical information. , 2003, Journal of the American Chemical Society.

[2]  Douglas R. Houston,et al.  Consensus Docking: Improving the Reliability of Docking in a Virtual Screening Context , 2013, J. Chem. Inf. Model..

[3]  J. M. Izquierdo,et al.  RNA nuclear export is blocked by poliovirus 2A protease and is concomitant with nucleoporin cleavage , 2009, Journal of Cell Science.

[4]  M. Daugherty,et al.  Structural basis for cooperative RNA binding and export complex assembly by HIV Rev , 2010, Nature Structural &Molecular Biology.

[5]  D. Case,et al.  Exploring protein native states and large‐scale conformational changes with a modified generalized born model , 2004, Proteins.

[6]  M R Lee,et al.  Use of MM‐PB/SA in estimating the free energies of proteins: Application to native, intermediates, and unfolded villin headpiece , 2000, Proteins.

[7]  Michael J E Sternberg,et al.  Protein–protein docking using 3D‐Dock in rounds 3, 4, and 5 of CAPRI , 2005, Proteins.

[8]  N. Kudo,et al.  Leptomycin B inactivates CRM1/exportin 1 by covalent modification at a cysteine residue in the central conserved region. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[9]  Peter B. McGarvey,et al.  UniRef: comprehensive and non-redundant UniProt reference clusters , 2007, Bioinform..

[10]  C. del Rio,et al.  The Economic Burden of HIV in the United States in the Era of Highly Active Antiretroviral Therapy: Evidence of Continuing Racial and Ethnic Differences , 2006, Journal of acquired immune deficiency syndromes.

[11]  F. Ceccherini‐Silberstein,et al.  Genetic and Structural Analysis of HIV-1 Rev Responsive Element Related to V38A and T18A Enfuvirtide Resistance Mutations , 2011, Intervirology.

[12]  S. H. Mahboobi,et al.  The Interaction of CRM1 and the Nuclear Pore Protein Tpr , 2014, PloS one.

[13]  M. Mofrad,et al.  Nuclear pore complex: biochemistry and biophysics of nucleocytoplasmic transport in health and disease. , 2011, International review of cell and molecular biology.

[14]  Andrey Tovchigrechko,et al.  GRAMM-X public web server for protein–protein docking , 2006, Nucleic Acids Res..

[15]  P. Kollman,et al.  Binding of a diverse set of ligands to avidin and streptavidin: an accurate quantitative prediction of their relative affinities by a combination of molecular mechanics and continuum solvent models. , 2000, Journal of medicinal chemistry.

[16]  Mohammad R. K. Mofrad,et al.  An agent-based model for mRNA export through the nuclear pore complex , 2014, Molecular biology of the cell.

[17]  Tingjun Hou,et al.  Assessing the Performance of the MM/PBSA and MM/GBSA Methods. 1. The Accuracy of Binding Free Energy Calculations Based on Molecular Dynamics Simulations , 2011, J. Chem. Inf. Model..

[18]  P. Kollman,et al.  Computational Alanine Scanning To Probe Protein−Protein Interactions: A Novel Approach To Evaluate Binding Free Energies , 1999 .

[19]  S. Tugendreich,et al.  Major Binding Sites for the Nuclear Import Receptor Are the Internal Nucleoporin Nup153 and the Adjacent Nuclear Filament Protein Tpr , 1998, Journal of Cell Biology.

[20]  F. Bischoff,et al.  CRM1-mediated Recycling of Snurportin 1 to the Cytoplasm , 1999, The Journal of cell biology.

[21]  K. Jeang,et al.  Ring expanded nucleoside analogues inhibit RNA helicase and intracellular human immunodeficiency virus type 1 replication. , 2008, Journal of medicinal chemistry.

[22]  W. Fischer,et al.  Identification of a new vertebrate nucleoporin, Nup188, with the use of a novel organelle trap assay. , 2000, Molecular biology of the cell.

[23]  P. Kollman,et al.  Continuum Solvent Studies of the Stability of DNA, RNA, and Phosphoramidate−DNA Helices , 1998 .

[24]  Alexandre M J J Bonvin,et al.  Are scoring functions in protein-protein docking ready to predict interactomes? Clues from a novel binding affinity benchmark. , 2010, Journal of proteome research.

[25]  Marc F Lensink,et al.  Blind predictions of protein interfaces by docking calculations in CAPRI , 2010, Proteins.

[26]  Tal Pupko,et al.  Structural Genomics , 2005 .

[27]  Alexander D. MacKerell,et al.  An ab initio study on the torsional surface of alkanes and its effect on molecular simulations of alkanes and a DPPC bilayer. , 2005, The journal of physical chemistry. B.

[28]  U. Dietrich,et al.  A Motif Unique to the Human Dead-Box Protein DDX3 Is Important for Nucleic Acid Binding, ATP Hydrolysis, RNA/DNA Unwinding and HIV-1 Replication , 2011, PloS one.

[29]  K. Jeang,et al.  Requirement of DDX3 DEAD Box RNA Helicase for HIV-1 Rev-RRE Export Function , 2004, Cell.

[30]  M. Mofrad,et al.  Higher Nucleoporin-Importinβ Affinity at the Nuclear Basket Increases Nucleocytoplasmic Import , 2013, PloS one.

[31]  G. Degliesposti,et al.  Binding Estimation after Refinement, a New Automated Procedure for the Refinement and Rescoring of Docked Ligands in Virtual Screening , 2009, Chemical biology & drug design.

[32]  Dmitri I. Svergun,et al.  WeNMR: Structural Biology on the Grid , 2011, Journal of Grid Computing.

[33]  Sandor Vajda,et al.  ClusPro: a fully automated algorithm for protein-protein docking , 2004, Nucleic Acids Res..

[34]  Xiaojie Xu,et al.  Recent Advances in Free Energy Calculations with a Combination of Molecular Mechanics and Continuum Models , 2006 .

[35]  D. Stuart,et al.  Implications of the HIV-1 Rev dimer structure at 3.2 Å resolution for multimeric binding to the Rev response element , 2010, Proceedings of the National Academy of Sciences.

[36]  P. Kollman,et al.  Calculating structures and free energies of complex molecules: combining molecular mechanics and continuum models. , 2000, Accounts of chemical research.

[37]  J. Cowan,et al.  Targeted cleavage of HIV RRE RNA by Rev-coupled transition metal chelates. , 2011, Journal of the American Chemical Society.

[38]  Michael Sattler,et al.  NES consensus redefined by structures of PKI-type and Rev-type nuclear export signals bound to CRM1 , 2010, Nature Structural &Molecular Biology.

[39]  David E. Kim,et al.  Computational Alanine Scanning of Protein-Protein Interfaces , 2004, Science's STKE.

[40]  William R. Taylor,et al.  The rapid generation of mutation data matrices from protein sequences , 1992, Comput. Appl. Biosci..

[41]  R. Clark,et al.  Consensus scoring for ligand/protein interactions. , 2002, Journal of molecular graphics & modelling.

[42]  Alexandre M. J. J. Bonvin,et al.  CPORT: A Consensus Interface Predictor and Its Performance in Prediction-Driven Docking with HADDOCK , 2011, PloS one.

[43]  J. Bhattacharya,et al.  Evolutionary Constraints Acting on DDX3X Protein Potentially Interferes with Rev-Mediated Nuclear Export of HIV-1 RNA , 2010, PloS one.

[44]  C. David Andersson,et al.  Postprocessing of Docked Protein-Ligand Complexes Using Implicit Solvation Models , 2011, J. Chem. Inf. Model..

[45]  M. Mofrad,et al.  Biophysical coarse-grained modeling provides insights into transport through the nuclear pore complex. , 2011, Biophysical journal.

[46]  Aleksey A. Porollo,et al.  Prediction‐based fingerprints of protein–protein interactions , 2006, Proteins.

[47]  D. Guo,et al.  Knockdown of Cellular RNA Helicase DDX3 by Short Hairpin RNAs Suppresses HIV-1 Viral Replication Without Inducing Apoptosis , 2008, Molecular biotechnology.

[48]  Alexandre M J J Bonvin,et al.  A multidomain flexible docking approach to deal with large conformational changes in the modeling of biomolecular complexes. , 2011, Structure.

[49]  Ruth Nussinov,et al.  FireDock: Fast interaction refinement in molecular docking , 2007, Proteins.

[50]  N. Srinivasakumar.  Rev-Free HIV-1 Gene Delivery System for Targeting Rev-RRE-Crm1 Nucleocytoplasmic RNA Transport Pathway , 2011, PloS one.

[51]  Ruth Nussinov,et al.  HingeProt: Automated prediction of hinges in protein structures , 2008, Proteins.

[52]  P. Kollman,et al.  Combined molecular mechanical and continuum solvent approach (MM-PBSA/GBSA) to predict ligand binding , 2000 .

[53]  Ruth Nussinov,et al.  PatchDock and SymmDock: servers for rigid and symmetric docking , 2005, Nucleic Acids Res..

[54]  Yousef Jamali,et al.  Brownian Dynamics Simulation of Nucleocytoplasmic Transport: A Coarse-Grained Model for the Functional State of the Nuclear Pore Complex , 2011, PLoS Comput. Biol..

[55]  Tingjun Hou,et al.  Assessing the performance of the molecular mechanics/Poisson Boltzmann surface area and molecular mechanics/generalized Born surface area methods. II. The accuracy of ranking poses generated from docking , 2011, J. Comput. Chem..

[56]  Alexander D. MacKerell,et al.  An Improved Empirical Potential Energy Function for Molecular Simulations of Phospholipids , 2000 .

[57]  K Schulten,et al.  VMD: visual molecular dynamics. , 1996, Journal of molecular graphics.

[58]  F. Baldanti,et al.  Toward the Discovery of Novel Anti‐HIV Drugs. Second‐Generation Inhibitors of the Cellular ATPase DDX3 with Improved Anti‐HIV Activity: Synthesis, Structure–Activity Relationship Analysis, Cytotoxicity Studies, and Target Validation , 2011, ChemMedChem.

[59]  Laxmikant V. Kalé,et al.  Scalable molecular dynamics with NAMD , 2005, J. Comput. Chem..

[60]  M. Gilson,et al.  Proflavine acts as a Rev inhibitor by targeting the high-affinity Rev binding site of the Rev responsive element of HIV-1. , 2003, Biochemistry.

[61]  J. Yates,et al.  Host Cell Interactome of HIV-1 Rev Includes RNA Helicases Involved in Multiple Facets of Virus Production* , 2011, Molecular & Cellular Proteomics.

[62]  Y. Wang,et al.  Leptomycin B is an inhibitor of nuclear export: inhibition of nucleo-cytoplasmic translocation of the human immunodeficiency virus type 1 (HIV-1) Rev protein and Rev-dependent mRNA. , 1997, Chemistry & biology.