New pockets in dengue virus 2 surface identified b

Authors: Carlos A Fuzo Léo Degrève

Publish Date: 2012/11/30

Volume: 19, Issue: 3, Pages: 1369-1377

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Abstract

One of the factors limiting the search of new compounds based on the structure of target proteins involved in diseases is the limited amount of target structural information Great advances in the search for lead compounds could be achieved to find new cavities in protein structures that are generated using well established computational chemistry tools In the case of dengue the discovery of pockets in the crystallographic structure of the E protein has contributed to the search for lead compounds aimed at interfering in conformational transitions involved in the pHdependent fusion process This is a complex mechanism triggered by the acid pH of the endosomes that leads to the initial changes in the E protein assembly at the virus surface In the present work an arrangement of three ectodomain portions of the E protein present on the surface of the mature dengue virus was studied through long allatom molecular dynamics simulations with explicit solvent In order to identify new pockets and to evaluate the influence of the acid pH on these pockets the physiological neutral pH conditions and the acid pH of the endosomes that trigger the fusion process were modeled Several pockets presenting pHdependent characteristics were found in the contact regions between the chains Pockets at the proteinprotein interfaces induced by a monomer in another monomer were also found Some of the pockets are good candidates for the design of lead compounds that could interfere in the rearrangements in E proteins along the fusion process contributing to the development of specific inhibitors of the dengue diseaseDengue a viral disease is a major public health problem affecting more than 100 million people in tropical and subtropical areas annually 1 It is caused by four serotypes of the dengue virus DENV which pertains to the Flavivirus genus This is an enveloped virus of ∼500 Å diameter with an external shell consisting of 180 copies of the envelope E protein arranged as 90 homodimers over the viral surface in a herringbone pattern socalled protein rafts 2 3 4 Flaviviruses infect the host cell through a receptormediated endocytosis followed by the fusion between the endosome and virus membranes 5 6 7 The central event in the infection is the fusion that is a process mediated by E protein rearrangements due to the decrease of pH from neutral to acid in the endosomes 3 4 8 9 10 11 12 It was hypothesized that the E protein rearrangement is triggered by the histidine residues that change their ionization state from uncharged to positively charged in the lowpH environment of the endosome 13 14 15 16 17Many efforts to develop antivirals that act on the virus entry stage into the host cell are focused on the E protein 3 A hydrophobic pocket observed in the crystal structure of E protein of DENV 2 DENV2 which is occupied by a detergent molecule noctylβDglucoside βOG has been a primary potential binding site for small molecules that may inhibit the conformational changes in the E protein necessary to the fusion process 18 Several studies that focused on this pocket found candidates for drugs by searching compound libraries through virtual screening 9 10 11 Targeting another pocket in the crystal structure of DENV2 E protein located using a cavitydetection algorithm in virtual screening has also identified an inhibitor of DENV2 12 The knowledge or the detection of the pockets was the main factor that led to finding new drug candidates against dengue The use of the E protein as a target for antiviral therapy creates the possibility of acting on several routes through the identification of ligandbinding pockets such as those in the transition structures that occur in the E protein during the lifecycle of the virus in the fusionactive trimer and in E protein rafts 3 The E protein rafts densely populate the surface of the mature virus and are interesting starting points for the exploration of the cavities for the search of compounds able to interfere in the E protein rearrangements that lead to the fusion process 3 Inserted ligands in some of these cavities may act as allosteric modulators of the proteinprotein interactions 19 present at the ordered surface of the mature virus These questions reveal new possibilities because pockets can be found not only in protein crystals but also in computer modeled structures such as those found in intermediate states of the E protein computed using metadynamics 20 and in the structures obtained in molecular dynamics MD simulation studies The latter is extremely interesting since MD simulation methods are powerful and widely used techniques in understanding the dynamics and structures of proteins 21 22 since they are able to investigate the protein structures at the atomic levelMD simulation has been used mainly to study the influence of the histidine protonation on the E protein structure of DENV 13 14 20 23 24 In these studies the effects of the pH changes before and after the internalization of the virus in the endosome were modeled by studying the E protein in two extreme situations considering that the side chains of all the histidine residues were unprotonated or protonated and modeling something like the prefusion conditions and the acid conditions of the endosomes Together with the structural protein behavior information the MD simulations can also be helpful in the discovery of new pockets through the exploration of protein trajectory snapshots along the simulation time A large number of algorithms are available for predicting cavities on the surface of proteins 25 However the majority of the detection algorithms are only employed for evaluating the pockets in static protein structures and few computational tools have been developed for the identification of pockets and of their properties along the MD trajectory snapshots Eyrisch and Helms 26 deal with this problem of successfully detecting transient pockets on the surfaces of the proteins BCLXL IL2 and MDM2 with a proposed protocol for pocket detection with the application of the PASS algorithm 27 They also demonstrate the improved influence of the solvent polarity and of the backbone movements in the diversity and volume of the pockets by employing this same protocol 28 In another recent study Schmidtke et al 29 have identified by means of their pocket detection algorithm in MD snapshots named mdpocket open/closed conformations of the pockets along the trajectory These studies reveal the dynamic behavior of the protein pockets this behavior is important in the modern design of drugs since the consideration of multiple conformations of the pockets has been shown to improve the predictive power of docking over crystal structures 30 31 32I Biological assembly and II PDB structure of 1THD containing the Cα atoms 33 III Allatom dimer structure 18 The domains DI DII and DIII of E protein are shown within the circles in the A chain DI comprising the residue sequences Met1Asn52 Glu133Phe193 and Gly281Gly296 DII is the sequences Pro53Pro132 and Asn194Thr280 and DIII is the sequence Met297LYS394 IV Energy minimized T structure constructed from 1THD and 1OKE T In all panels the A B and C chains can be distinguished using different colorsThe monomer structure of the DENV E protein contains 394 amino acid residues spatially distributed in three domains namely DI DII and DIII Fig 1III The Cα structure of the DENV2 E protein deposited in the Protein Data Bank PDB 34 with identification code 1THD 33 was used as a template for the construction of the T initial structure for the simulation studies The 1THD structure of the trimer containing the A B and C chains was originally constructed by the superimposition of an allatom monomer structure PDB code 1TG8 33 onto the mature DENV2 CryoEM density map considering individually the agglomerates DI/DIII and DII as rigid bodies resulting in a difference of 029 nm in the Cα rootmeansquare deviation RMSD between the monomers of 1THD and 1TG8 However the monomers of the dimer DENV2 E protein structure under the PDB code 1OKE 18 present smaller RMSDs 025 nm and 026 nm for the A and B chains of 1THD The smaller RMSD and the advantage of the good intermonomer contacts in 1OKE are advantages taken into account in the T structure construction by means of the better Cα superposition of 1OKE on the dimer formed by the B and C chains of 1THD and by the best superposition of the A chain of 1OKE on the A chain of 1THD as shown in Fig 1Two target systems of interest were studied by MD simulation in the present work in order to mimic the physiological prefusion condition and the acid condition of the endosome These conditions were modeled as in previous simulation studies of E protein 13 14 20 23 24 considering the side chains of histidine residues respectively singly and doubly protonated In the first system namely T0 the histidine residue side chains were protonated only in position δ or ε with the aid of the MolProbity 35 In the second system the histidine residues were fully protonated in both the δ and ε positions T+ The objective of this strategy also used in simulations of the dimer of the E protein 13 14 23 was to observe the differences in the behavior of the T in different histidine residue protonation states due to their important role during the viral infection 8 The two systems were constructed by inserting T0 or T+ at the center of the parallel piped boxes of sides 210 120 and 120 nm The systems were completed using about 99000 water molecules and Na+ and Cl ions to neutralize the net charges of the proteins and to reach the ionic strength of 150 mM

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