Docking of HIV protease to silver nanoparticles

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Docking of HIV protease to silver nanoparticles C.G. Whiteley a,∗, C-Y Shing c, C-C Kuo c, Duu-Jong Lee b,c a

Graduate Institute of Applied Science & Technology, National Taiwan University of Science and Technology, Taipei, Taiwan Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei, Taiwan c Department of Chemical Engineering, National Taiwan University, Taipei, Taiwan b

a r t i c l e

i n f o

Article history: Received 26 June 2015 Revised 5 October 2015 Accepted 19 October 2015 Available online xxx Keyords: HIV protease Silver nanoparticles Docking Molecular dynamics simulation

a b s t r a c t This interaction of silver nanoparticles (AgNP) with human immune-deficiency virus aspartic protease (HIVPR) is examined by molecular dynamics simulation using the Colores (Situs) package and biophysical techniques using UV–vis spectroscopy, dynamic light scattering, transmission electron microscopy and circular dichroism. The ‘docking’ of AgNP with HIVPR creates a complex [AgNP–HIVPR] to initiate a hypochromic time-dependent red-shift for the surface plasmon resonance maximum. MD simulations reflect large perturbations to enzyme conformations by fluctuations of both rmsd and B-factors. Increase in changes to electrostatic potentials within the enzyme, especially, with chain B, suggest hydrophobic interactions for the binding of the AgNP. This is supported by changes to mainchain and sidechain dihedrals for many hydrophobic amino acid including Cys95 , Trp6 and Trp42 . Circular dichroism spectra reveal disappearance of α -helices and β sheets and increase in random coil first from chain B then chain A. During initial stages of the interactive simulation the enzyme is conformational flexible to accommodate the AgNP, that docks with the enzyme under a cooperative mechanism, until a more stable structure is formed at convergence. There is a decrease in size of the HIVPR–AgNP complex measured by changes to the gyration radius supporting evidence that the AgNP associates, initially, with chain B. © 2015 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

1. Introduction The unique, remarkable properties of nanoscale metal particles are being continuously explored and engineered within biomedical disciplines to resolve many medical challenges [1–10]. More specifically, nanomedicine and nanotechnology can be exploited in the preparation and properties of molecular diagnostics, metal nanoparticles, nanoproteomics, dendrimers, drug delivery, imaging and cardiac therapy [11–17]. Furthermore nanoparticles can influence changes to enzyme activities [18–20], protein conformations [21–24], thermodynamic instability [25] and hydrodynamic size [26]. Metal particles of less than 30 nm can pass through the blood-brain barrier to target specific organs [27–29] while those up to 50 nm can enter the cell nucleus and those around 100 nm are able to translocate into actual cells [30, 31]. Despite these great advances in nanobiotechnology it must be mentioned, that interactions on an in vitro scale are far from being a true reflection of what occurs in vivo [32]. This uncertain landscape in comparing the fate of nanoparticles from both an in vitro and in vivo window with reference to pharmacological,

∗

Corresponding author. Tel: +886 2 2737 6939; fax: +886 2 2730-3733. E-mail address: [email protected], [email protected] (C.G. Whiteley).

toxicological, immunological and mechanistic perspectives has led towards in-depth studies towards understanding the details, at a molecular level, of the effect of metal nanoparticle invasion on biological cellular infrastructure. Human immunodeficiency virus (HIV) is the pathogen that causes acquired immunodeficiency syndrome (AIDS) [33] and progressive damage to the immune system manifesting in serious opportunistic diseases. One of the biomedical target molecules identified in the fight against HIV/AIDS is HIV aspartic protease [HIVPR] which consists of two identical 99 amino acid sub-units and a gross molecular mass of 21.6 kDa [Fig. 1]. We recently reported not only the in vitro inhibition of HIVPR by silver nanoparticles (AgNP) but a fluorimetric quenching analysis [FRET] and mechanism for the interaction and binding [1]. From these fluorescent quenching studies the AgNP were bound 1.03 nm from a single tryptophan residue [Trp6 ] while Cys95 was juxtapositioned 1.05 nm from the reactive COO− of active site Asp25 and 1.07 nm from the Trp6 . It is well-known that sulphur atoms form strong associations with both gold and silver nanoparticles in various biotechnological applications [4, 5, 34] and therefore it is reasonable to suppose that the nanoparticle may link with the biomacromolecule via thiol groups on cysteine side chains. We proposed [1] that, in line with other reported evidence, the nanoparticles interact with the thiolate group of Cys95 and disrupt, not only its electronegativity but also a decrease in the nucleophilic potential of the

http://dx.doi.org/10.1016/j.jtice.2015.10.029 1876-1070/© 2015 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

Please cite this article as: C.G. Whiteley et al., Docking of HIV protease to silver nanoparticles, Journal of the Taiwan Institute of Chemical Engineers (2015), http://dx.doi.org/10.1016/j.jtice.2015.10.029

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Fig. 1. Orientation of AgNP and HIVPR juxtaposed towards the N- and C-terminal residues.

COO− of Asp25 towards water and/or substrate thereby inhibiting the mechanism of reaction. Bearing in mind the foregoing disclosure of the adverse effects of nanoparticles on the in vivo cellular machinery, that HIVPR was a crucial enzyme in the life cycle of HIV and that both Trp6 and Cys95 play crucial roles in the mechanism of HIVPR it was decided to investigate further the interaction of AgNP with this enzyme. In order to do this several biophysical and physicochemical techniques were followed: UV–vis spectrophotometry to investigate the spectral shift of the surface plasmon resonance peak from the AgNP on binding with the enzyme; zeta-potentials to estimate surface electrostatic potentials of amino acids, transmission electron microscopy (TEM) to study the formation of any AgNP–HIVPR complex; dynamic light scattering (DLS) to monitor the hydrodynamic size of the particle and circular dichroism to investigate the secondary structural integrity of the enzyme. Our findings were then corroborated with molecular dynamics simulations for the docking of AgNP, via rigid-body docking and flexible-fitting, through electron density maps from the Colores programme of the Situs commercial package [35]. Though this software suggests several plausible interactions and docking interfaces only one unique site is favored. Simulations are performed, initially to study the ‘docking’ mode of the AgNP to HIVPR, then to completely characterize the AgNP–HIVPR complex. It is anticipated that this paper will shed new light on the molecular driving forces that are involved in the binding of, not only HIVPR to AgNP, but any potential biomedical target with any functional metallic nanoparticle.

1.5 ml). After 1 min NaBH4 (20 mM, 0.5 ml) was added drop wise and the yellow solution stirred for a further 2 h. The particles were characterized by transmission electron microscopy (TEM), visible spectroscopy and their zeta potentials.

2. Materials and methods

2.3.1. Starting structures The X-ray crystallographic structure of HIVPR [PDB:1HXB] is used as the starting structure [36] with the pdb file being edited to remove all features of the hetero group. The AgNP were built with Inorganic Builder, using a unit-cell for Ag, followed by running a suitable script file, both from within the VMD programme [37], to create

2.1. Synthesis and characterization of silver nanoparticles The method was adopted from that reported [1]. Silver nitrate (0.25 mM, 45 ml) was treated, at 4 °C, with trisodium citrate (2.5%,

2.2. Interaction of HIVPR with silver nanoparticles 2.2.1. UV–vis spectrophotometry The binding of AgNP (5 μM) to HIVPR (0.5 ng μl−1 , 50 μl) in HEPES buffer (20 mM, pH 7.0) was investigated by measuring the surface plasmon resonance peak between 350–550 nm on a Cary 100 UV–vis (Agilent) spectrophotometer with 96 well plates, operated at 1 nm bandwidth using the Gen 5 software program. 2.2.2. TEM AgNPs (5 μM) were incubated (2 h) with HIVPR (0.5 ng μl−1 , 50 μl) in HEPES buffer (20 mM, pH 7.0), pipetted onto a Formvar copper grid, air-dried before being observed on a Jeol JEM 200X instrument. 2.2.3. DLS and zeta potentials Zeta potentials and hydrodynamic size of AgNP, HIVPR and HIVPR–AgNP complex were determined using a Zetasizer 2000 HAS (Malvern instruments). 2.3. Molecular dynamics simulations

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a ‘nanoparticle sphere’ of 236 silver atoms with a radius of approximately 1.06 nm. 2.3.2. General procedures and simulations All simulations for the interaction of AgNP with HIVPR were performed using the molecular dynamics package NAMD [38, 39] and version 22 of the CHARMM force field [40, 41]. The solvated protein was subjected to molecular dynamics simulations and energy equilibrations and minimization utilizing Langevin dynamics at constant temperature of 300 K for 10 ns [10,000,000 steps at 1 fs/step]. Molecules were visualized with the VMD program [37]. A cutoff coulombic force of 12 A˚ with switching function starting at 10 A˚ was used in all equilibrations and minimizations. The simulations, with the AgNP was initially orientated with HIVPR about 2–3 A˚ away from the enzyme surface [Fig. 1], were performed with periodic boundary conditions using Particle Mesh Ewald algorithms and a grid spacing ˚ The number of atoms represented in the simulations was 3122 of 1 A. from the enzyme (198 residues; 2 chains) and 236 Ag atoms in a single spherical nanoparticle; bond energies were computed at every time-step. Both rigid-body docking and flexible fitting molecular dynamics were performed using Colores – a Fourier-Transform accelerated 6D search [3-rotational; 3-translational] and correlation based docking programme [35, 42, 43] from the Situs suite of molecular dynamics packages run on a Windows platform with Cygwin. At a resolution of 5 A˚ using the Powell optimization algorithm [44] of 20 A˚ and 5° sampling steps the programme identifies several different specific sites within the HIVPR structure to simulate docking to the AgNP density map. Scaling factors of 0.1 and 1, and simulation times of 10 ns and 1 ns respectively, were used for the equilibration and minimization steps. Specific restraints to enforce secondary structural integrity, maintaining chirality and restricting cis-peptide bond formation were introduced during the running of the programme. 2.4. Analysis

 RMSD =

 [ri − ri ]2 Natom

Bfactor = 78.98[r2 ]

 Rg =

 mi [ri − rcom ]2  mi

θλ =  εi Sλi

(1) (2)

(3) (4)

where mi and ri are the mass and position of atom i and rcom is the center-of-mass position; This radius of gyration is the rmsd of all atoms from the enzymes center of mass; Natoms is the number of atoms used for comparison; (r) is the mean average atomic dis˚ between a simulated trajectory and the initial strucplacement (in A) ture for a particular amino acid, θ λ is the circular dichroism of the protein as a function of wavelength (λ), ε i is the fraction of each secondary structure (i) and Sλi is the ellipticity at each wavelength of each ith secondary element. The structural deviations of the enzymes from the initial x-ray crystal structure during each docking simulation were assessed by comparative analysis of the secondary structure, computational analysis of the root mean square deviations (RMSD) [45] (Eq. (1)), changes to the B-factors (Debye–Waller) (Eq. (2)), interactive bonding energies, dihedral angles ([phi; ϕ ; (OC)-NH-Cα -(CO); psi; ψ ; (NH)-Cα CO-(NH)] and side-chain dihedrals (χ 1 ) (NH)-Cα –Cβ -(Xγ ), radius of gyration (Rg ) (Eq. (3)), circular dichroism (CD) spectra according to typical analyses (Eq. (4)) with the on-line web server, DiChroWeb [46]

Fig. 2. Interactions of AgNPs with HIVPR: (a) UV–vis spectra. The spectrum of the complex displays a time-dependent red-shift of varying wavelengths and absorbance magnitude and (b) TEM image of AgNP–HIVPR [Scale bar = 50 nm]; (c) TEM image of AgNP.

and electrostatic potentials through the PBEQ Solver [47] and potential continuum electrostatics (PCE) web-server [48] 3. Results and discussion 3.1. Silver nanoparticles: synthesis, characterization and interaction with HIVPR The hydrodynamic size distribution and zeta potential of the AgNP–HIVPR complex, determined using a zetasizer and dynamic light scattering, are 11–12 nm and −19.4 mV, respectively. These values for HIVPR are 5.6 nm, 4.1 mV and for AgNP are 2.12 nm, −47.2 mV supporting our findings for an effective binding between the nanoparticle and the enzyme. It is extremely probable that HIVPR may replace the citrate molecules used during the preparation of AgNP since citrate has a very weak association with silver. The slight positive surface charge experienced by HIVPR and the change in this value on contact with AgNP suggests binding modes predominantly driven by van der Waals forces and electrostatic interactions. The formation of HIVPR–AgNP complex can be further confirmed by the UV–vis spectra [Fig. 2a]. The surface plasmon resonance of the AgNP’s appeared at 401 nm which shifted (red) about 9 nm on incubation with HIVPR; a small (8%) hypochromic absorbance peak is also observed. Over a period of 15 min and 24 h the red shift became 15 and 21 nm respectively with a further decrease in absorbance of about 35–65% [Fig. 2]. This change in dielectric constant must only result from the formation of a AgNP–HIVPR complex and, even though a careful scrutiny of the TEM image of the AgNP–HIVPR complex [Fig. 2b] illustrates a possible ‘halo’ of about 4–5 nm diameter surrounding the nanoparticle it is difficult to confirm whether this is the NP-enzyme corona. The size ratio (N) of HIVPR to AgNP is estimated (Eq. 5) while the number of silver atoms (S) on the surface of the spherical AgNP is estimated as 152 (Eq. 6) and the total number of silver atoms (T) within the nanoparticle is estimated as 236 (Eq. 7).

N = [4π (RAgNP + RHIVPR ) ]/π R2 HIVPR .

(5)

S = 4RAgNP2 /r2

(6)

T = RAgNP3 /r3

(7)

2

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˚ and B-factor deviations of HIVPR after equilibration and minimization, before and after interaction with AgNP (2.12 nm diameter). Dashed Fig. 3. Root mean square deviations (A) line represents HIVPR before incubation with AgNP. Relevant amino acids are noted. B-factor deviations represent cumulative values for all atoms for each amino acid residue.

where RAgNP and RHIVPR are the radii of the AgNP (1.06 nm) and HIVPR, respectively; r = VDW radius of the silver atom (0.172 nm). 3.2. Molecular dynamics simulations of AgNP–HIVPR interactions Prior to any docking studies conformational stability of the enzyme, after molecular dynamic equilibration and minimization, was confirmed from not only a comparison of the root mean square deviations (rmsd) of the backbone with the initial protein crystal structure but also from the B-factor as well (Fig. 3). The greatest deviations (with respect to rmsd) were no more than 1.4 A˚ reflecting no significant conformational change during any of the equilibrations and/or minimizations. The AgNP was initially positioned about 2–3 A˚ away from the enzyme surface [Fig. 1]. Docking of the AgNP with HIVPR was then performed using the Colores package from the Situs suite [35]. This correlation based docking molecular dynamic simulation programme undertakes both a rigid-docking and flexible-fit analysis (MDFF) for the binding of AgNP with HIVPR. Furthermore the programme creates several scenarios with different rotations and translations of the initial structure of HIVPR docked with AgNP. Analysis of each scenario of the ‘docking’ is scrutinized for amino acid–AgNP contacts by considering rmsd and B-factor variations between the ‘docked’ complex and initial X-ray structure [Fig. 3]. For chain B the amino acids in which ˚ Leu19 , 4.1 A; ˚ Ala28 , there are large deviations in rms are Trp6 , 9.4 A; 42 44 54 95 91 ˚ ˚ ˚ ˚ ˚ 4.0 A; Trp , 6.2 A; Ile –Ile , 7.9 A; Cys , 8.1 A and Thr , 4.9 A. For chain A the amino acids in which there are significant deviations ˚ Gly16 –Gln18 , 6.1 A; ˚ Trp42 , 6.6 A; ˚ Ile47 –Ile54 , in rms are Trp6 , 9.8 A; 78 82 91 95 ˚ ˚ ˚ 8.0 A; Gly –Val , 5.4 A; Thr –Cys , 6.3 A. It is obviously clear from these data that, apart from Trp6 , Trp42 and Cys95 mentioned earlier, the majority of amino acids affected in the docking of AgNP to HIVPR are hydrophobic. B-factors, otherwise called ‘temperature’ factors, reflect the motion of the atoms and/or amino acid residues during any ˚ (cumulative) of the enzyme perturbations. The relative distance (A) B-factors for the side-chains of all amino acid residues of HIVPR after docking of the AgNP is computed and represented [Fig. 3]. In support of the rmsd analysis the amino acid residues that need comment are:

˚ chain A – Trp6 , 1100 A; ˚ chain B – Trp6 , 3500 A˚ and Cys95 , 2150 A; 18 42 53 ˚ ˚ ˚ Gln , 750 A; Trp , 1900 A and Phe , 1410 A. Fluorescence resonance energy transfer [FRET] analysis of the binding of AgNP with HIVPR [1] revealed that the fluorescence of only one surface tryptophan within HIVPR was quenched and this was identified as either Trp6 or Trp42 . The proposed mechanism [1] suggested that it was Trp6 , since γ -S-Cys95 was only 1.07 A˚ away from Nε 1 -Trp6 while the distance between Nε 1 -Trp42 and γ -S-Cys95 was ˚ Our present findings, however, contradict this argument and, 3.68 A. due to large rmsd/B-factor perturbations support the involvement of Trp6 , Trp42 and Cys95 [Fig. 3]. A further qualifying point needs to be mentioned. Though phenylalanine has generally weak fluorescence quenching properties (relative to tryptophan) and may not be a major contributing factor to the fluorescence under FRET analysis [1] the presence of Phe53 within the amino acid envelope [Ile44 –Ile54 ] must also be considered as a possibility for interaction with the AgNP during the docking process. Though the AgNP from the present synthetic studies was complexed with several citrate molecules with a zeta potential of around −47 mV making the particle highly anionic this cannot be said for the AgNP used with the MD simulations. The atom surface of the nanoparticle is predominantly hydrophobic with zero charge and consequently are associated in hydrophobic-hydrophobic interactions. These are mainly through π –π interactions with aromatic moieties of tryptophan, phenylalanine and/or tyrosine as well as via the side chains of hydrophobic amino acids including the sulphur bearing residues of methionine and cysteine. This supports the present disposition for interaction of the AgNP with Trp6 , Trp42 , Phe53 and Cys95 [Fig. 4] The energies for the interaction of AgNP with HIVPR, such as bond interactive energies, van der Waals and electrostatic energies also need to be taken into consideration. These energies are measured as a difference between bound atoms of HIVPR to the silver and unbound atoms averaged over the whole molecule. One should be reminded that the simulation starts with a rigid-body docking in which the Colores programme develops a series of scenarios of AgNP docked with HIVPR that corresponds to a 6D Fourier-Transform search

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Fig. 4. Representations of HIVPR docked with AgNP: (a) HIVPR represented as an electron density map. (b) AgNP represented as an electron density map. Close proximity of Cys95 (with sulphur atom shaded yellow) and Trp6 from chain B are noted.

Fig. 5. Bond energy profiles before and after docking of AgNP with HIVPR.

(3-translational and 3-rotational) followed by a flexible-fitting analysis and orientation of the AgNP docking using the NAMD programme. The energy profiles for the binding of AgNP to HIVPR are shown [Fig. 5]. The start of the profile (ts = 0 ns) is the energy associated with the rigid-body docked complex [525 kcal/mol]. As the flexible-fit orientation of the AgNP within the docking site progresses a favorable minimum [96 kcal/mol] is eventually reached with convergence of

the system. As a control the HIVPR without docked AgNP was subjected to a similar simulation algorithm. The electrostatic potentials (EP) for all amino acids from chain A and chain B of HIVPR before and after docking with AgNP are computed and used to anticipate possible ‘patches’ to which the hydrophobic interaction with AgNP can be estimated. It follows that the lower the EP for any particular ‘patch’ of amino acid residues within HIVPR the greater the hydrophobicity and the greater the chance of AgNP binding. Residues that have electrostatic potential less than 1% that of the total EP for the protein are deemed to be hydrophobic and assigned a hydrophobic score. Four or more consecutive hydrophobic residues constitute a ‘hydrophobic patch’ which are then graded according to their respective total hydrophobic scores. After AgNP docking there is an overall increase in EP of −410 kcal mol−1 [chain A] and −131.4 kcal mol−1 [chain B] as more charged anionic side chains (glutamic and aspartic acids and arginine) became exposed [Fig. 6a/6b]. Since AgNP preferentially bind through hydrophobic interactions any ‘patches’ with highly positive or negative EP can be excluded as possible binding sites. These sites are indicated as major differences around Arg8 , Glu21 , Asp25 [the active site region], Asp29 , Glu34 , Arg41 , Arg57 , Asp60 , Glu65 and Arg87 . Furthermore in view of the difference in EP between chains A and B the AgNP would interact, preferentially, with chain B supporting the earlier diagnosis. The stability and flexibility of an enzyme during the docking with AgNP may be defined, respectively, as preserving a conformation and fluctuating oscillations about this conformation. These two

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-1 Electrostatic potential (kcal.mol )

600

Arg41 Chain A

500 400

Arg8

34 Asp25 Glu

Arg57

21

Glu

300

Arg87

Glu65

200 100 0 0

10

20

30

40 50 60 70 Residue number

80

90

100

Difference

150 50 -50

Electrostatic potential (kcal.mol -1)

-150 34

400 25

Asp

350 300

Arg8

Glu21

Glu

Chain B Asp60

41

Arg

Arg87

57

Arg

250 200 150 100 50 0 0

10

20

30 40 50 60 70 Residue number

80

90

100

Difference

150 50 -50

-150 Fig. 6. Electrostatic potentials of amino acid residues between HIVPR (chain A and chain B) in the absence [—] and presence of AgNP [––]. The differences between the potentials were also computed.

parameters can be considered from the changes to backbone dihedral angles [phi; ϕ ; (OC)-NH-Cα -(CO) and psi; ψ ; (NH)-Cα -CO-(NH)] and side-chain dihedrals (χ 1 ) (NH)-Cα –Cβ -(Xγ ). At the outset it was decided to report on the changes between the initial configuration (t = 0 ns), which is that immediately after rigid-body docking, and after the system has adopted a final configuration at convergence (t = 10 ns). Any major fluctuations to these dihedral angles must reflect configurational changes at that locus and would reflect both the enzymes stability and flexibility. It must be obvious that a change of 0° would be zero change in configuration while any other value would represent a respective percent change in configuration. Changes to phi; (ϕ ) and psi (ψ ) dihedrals were experienced for many hydrophobic residues (valine, leucine, isoleucine, alanine, glycine) as well as

Trp6 , Phe53 and Cys95 [Fig. 7, top and middle panels]. Though there was extensive change in conformation to chain B in comparison to that for chain A after docking with AgNP it is uncertain why there is a greater fluctuation in relative psi angles in comparison to the corresponding phi angles. Since this may be related to different thermodynamic potentials (internal energies) of rotation which, in turn, may be related to differences in steric clashes of specific sidechains our attention turned to the dihedrals (χ 1 ) (NH)-Cα –Cβ -(Xγ ) for each amino acid residue (chains A and B) [Fig. 7, lower panel]. Once again, not only do rotations about Cα –Cβ for many hydrophobic amino acid residues (Ile15 , Leu23 , Ile62 , Leu63 ) from chain B play a dominant role, but so do both Trp6 and Cys95 . It is worth mentioning that rotation about this bond occurs for two lysine residues – Lys45 , Lys55 – and, bearing in mind the propensity of sulphur for silver nanoparticles, for Met36 . All of these findings support our earlier results for, not only changes to rms, B-factors and electrostatic potentials for Trp6 , Trp42 and Cys95 and the many hydrophobic amino acids, but that AgNP preferentially interacts (docks) with chain B. An excellent analysis for studying conformational change to secondary structures including any fluctuations in folding of proteins and anticipated binding paradigms for AgNP is the one from circular dichroism (CD) spectra. Careful analysis of the CD spectra and trajectory profiles for HIVPR prior to docking with AgNP, immediately after rigid-docking with AgNP (t = 0 ns) and up to convergence (t = 10 ns) [Fig. 8] reveals the progressive disappearance of the two small α -helices (Arg87 –Ile93 ) in each subunit – first from chain B (t = 0.6 ns) and then from chain A (t = 1.7 ns). Several supporting claims can be inferred from this: first it supports the finding that chain B is the initial target for docking of AgNP; second the decrease in the α -helix suggests a large change in conformational structure around Arg87 – Ile93 endorsing the role of Cys95 ; third this time-scale (< 2.0 ns) implies high flexibility and low stability. It is also noted that during the simulation of docking with AgNP there was a gradual progressive decrease of β -sheet from 54% to 39% (relative decrease of 22.3%) concomitant with an increase of random coil. Despite the fact that HIVPR has over 39% random coil, even before AgNP docks, a further element of disorder is created within HIVPR–AgNP complex as witnessed by a low level of ellipticity above 210 nm in the CD spectrum. Furthermore bond rotation (as discussed above with changes to dihedral angles) can certainly contribute to the extent of random coil. Even though aromatic contributions to CD spectra are weak in comparison to those from backbone amides any protein with low α -helical content are known to yield detectable changes to CD signals. The high positive band at 193–195 nm and in the 225–235 nm region along with negative bands near 168, 175 and 215 nm points to aromatic amino acid π π ∗ transitions and/or the involvement of disulphide bridges.[Fig. 8]. Consequently these must be due to external perturbations of AgNP binding to Tyr, Phe, Trp side chains and/or disulphide bridges. Though a hydrophobic interaction with a cysteine sidechain may still be likely the involvement of the disulphide bond can be excluded since, after careful scrutiny of the structure for HIVPR–AgNP reveals that such bonds would be > 10 A˚ in length. With one tyrosine, two phenylalanine and two tryptophan residues per chain in HIVPR and with the contributions to the CD spectra from phenylalanine and tyrosine to be minimal must support the claim that changes to the CD spectra, on docking of the AgNP, arise through tryptophans – Trp6 , Trp42 . What remains unanswered, however, is which comes first: AgNP docking to cause changes to phi ϕ ; psi ψ and side-chain dihedral (χ 1 ) angles and conformation or the other way around with a conformational change that assists in AgNP docking. Attempts to answer this may arise from considering the radius of gyration Rg of HIVPR before and after integration of AgNP. The overall size of the enzyme decreases only slightly (∼4%) [Fig. 9] with respect to time during the docking of the AgNP which also supports enhanced configurational stability of the AgNP–HIVPR. Consequently, despite the small, limited conformational change to HIVPR before docking

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Fig. 7. Dihedral angle changes for chain A and chain B of HIVPR with docking by AgNP after 10 ns simulation (see text). Top (phi); middle (psi); bottom (chi-1). Relevant amino acids are represented.

Fig. 8. Changes to CD spectra of HIVPR on docking with AgNP. HIVPR = CD spectrum of enzyme prior to interaction with AgNP; 0 ns is CD spectrum immediately after rigidbody docking of AgNP to HIVPR; 10 ns is CD spectrum after convergence of the system.

with AgNP, the major changes to the conformation only occur after docking. Nevertheless, it is our opinion that, the interaction of the nanoparticle with HIVPR falls under a cooperative mechanism. Careful examination of change in radius of gyration [Fig. 9] reveals an ini-

Fig. 9. Change in radius of gyration of HIVPR during docking of AgNP. As a control HIVPR was subjected to the same simulation in the absence of AgNP.

tial increase in size over the first 2.0 ns of simulation that would correspond with enhanced flexibility of the system. The fluctuations in size between 2.0 ns and 6.0 ns must reflect changes in orientation of the docking site to allow an enhanced rate of access for AgNP and a more stable configuration. After 6.0 ns simulation a stable HIVPR– AgNP complex is formed and there is a steady gradual decrease in size

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up to convergence of the system at 10 ns. The overall dimensions of the HIVPR in the presence of AgNP decreases from 35.5 × 54 A˚ (t = 0 ns) to 34.92 × 51.8 A˚ (t = 5 ns) to 34.51 × 49.44 A˚ (t = 10 ns); interatomic distance between Pro1 [O] to Ile50 [O] is 29.53 A˚ (t = 0 ns) to 26.02 A˚ (t = 5 ns) to 23.87 A˚ (t = 10 ns) [chain B] during AgNP docking; interatomic distance between Pro1 [O] to Ile50 [O] chain A is 30.97 A˚ (t = 0 ns) to 30.3 A˚ (t = 5 ns) to 29.9 A˚ (t = 10 ns) during AgNP docking. A higher percent random coil that is experienced in the HIVPR–AgNP complex, however, must suggest more flexibility leading to a conclusion that HIVPR explores various conformations and/or configurations about its most stable structure. Indeed the enzymes flexibility/stability is paramount for the affinity and docking of AgNP. Though it may not be possible to examine the complete sequence of events during AgNP docking to HIVPR it may be safe to assume that initial time-scale analysis (t < 2.0 ns) would reflect flexibility while a time-scale at convergence (t = 10 ns) would reflect stability. 4. Conclusions This paper has attempted to study the interactive docking of AgNP with HIVPR. It has analyzed the findings by various physico-chemical and biophysical techniques such as UV–vis spectroscopy, dynamic light scattering (DLS), transmission electron microscopy and circular dichroism. Furthermore the results have been corroborated with an in silico molecular dynamic simulation of the docking. While the in vitro study of AgNP coated with citrate molecules, that involved a fluorimetric FRET analysis [1], implied a strongly anionic particle that interacted with HIVPR via van der Waals forces and electrostatic interactions this wasn’t the case with the in silico simulations. In the latter scenario the ‘naked’ AgNP had zero charge and interacted with HIVPR by π π ∗ hydrophobic-hydrophobic associations with tryptophan residues. Furthermore the FRET analysis [1] appeared limited in that only interactions with Trp6 and Cys95 could be identified. In contrast the present details for the in silico investigation have revealed that not only is Trp6 and Cys95 involved but Trp42 and many other hydrophobic amino acids as well. Perturbations to mainchain and sidechain dihedrals of the enzyme and the gradual changes in secondary structural elements (α -helix; β -sheet; random coil) reflect that chain B is the initial target for the docking of the AgNP. The findings also illustrate an initial flexibility of the HIVPR–AgNP complex within intial stages of its formation and a cooperative nature to the AgNP binding before a more stable structure is realized as the simulation approaches convergence. This paper has exploited approaches both from experimental and computational views that shed light on HIVPR and AgNP interactions which would not be observed by either the enzyme or the nanoparticle alone. Declaration of interest There is no ‘Conflict of interest’ Acknowledgments The authors wish to express gratitude towards the Ministry of Science and Technology (Taiwan) [Grant no: 103-2811-E-011-006] for financial support to undertake this work. Further thanks to Professor W Wriggers, Mechanical & Aerospace Engineering, Old Dominion University, Norfolk, Viginia, USA for a copy of the test Situs programme in order to undertake the molecular dynamic simulations. References [1] Shing C-Y, Whiteley CG, Lee D-J. HIV protease: Multiple fold inhibition by silver nanoparticles: spectrofluorimetric, thermodynamic and kinetic analysis. J Taiwan Inst Chem Eng 2014;45:1140–8.

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