Unfolding of Acinetobacter baumannii MurA proceeds through a metastable intermediate: A combined spectroscopic and computational investigation

Accepted Manuscript Unfolding of Acinetobacter baumannii MurA proceeds through a metastable intermediate: A combined spectroscopic and computational investigation

Amit Sonkar, Harish Shukla, Rohit Shukla, Jupitara Kalita, Timir Tripathi PII: DOI: Reference:

S0141-8130(18)33301-4 https://doi.org/10.1016/j.ijbiomac.2018.12.124 BIOMAC 11283

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

1 July 2018 10 December 2018 14 December 2018

Please cite this article as: Amit Sonkar, Harish Shukla, Rohit Shukla, Jupitara Kalita, Timir Tripathi , Unfolding of Acinetobacter baumannii MurA proceeds through a metastable intermediate: A combined spectroscopic and computational investigation. Biomac (2018), https://doi.org/10.1016/j.ijbiomac.2018.12.124

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ACCEPTED MANUSCRIPT Unfolding of Acinetobacter baumannii MurA proceeds through a metastable intermediate: A combined spectroscopic and computational investigation Amit Sonkar#, Harish Shukla#, Rohit Shukla#, Jupitara Kalita, and Timir Tripathi*

Molecular and Structural Biophysics Laboratory, Department of Biochemistry, North-Eastern

These authors contributed equally to the work

*

Corresponding author

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#

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Running title: Urea-induced unfolding of AbMurA

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Hill University, Shillong- 793022, India

Dr. Timir Tripathi, Assistant Professor, Department of Biochemistry, North-Eastern Hill

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University, Shillong- 793022, India. Email: [email protected], [email protected]; Tel:

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+91-364-2722141; Fax: +91-364-2550108.

ABSTRACT

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Peptidoglycan (PG) is the main constituent of the bacterial cell wall. The enzyme UDP-Nacetylglucosamine enolpyruvyl transferase (MurA) catalyzes the transfer of enolpyruvate from

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phosphoenolpyruvate to uridinediphospho-N-acetylglucosamine, which is the first committed step of PG biosynthesis. In this study, we have systematically examined the urea-induced

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unfolding of Acinetobacter baumannii MurA (AbMurA) using various optical spectroscopic techniques and molecular dynamic simulations. The urea-induced unfolding of AbMurA was a three-state process, where a metastable intermediate conformation state is populated between 3.0-4.0 M. Above 6.0 M urea, AbMurA gets completely unfolded. The transition from the native structure to the partially unfolded metastable state involves ~30% loss of native contacts but little change in the radius of gyration or core hydration properties. The intermediate-to-unfolded state transition was characterized by a large increase in the radius of gyration. Molecular dynamics trajectories simulated in different unfolding conditions suggest that urea destabilizes AbMurA structure weakening hydrophobic interactions and the hydrogen bond network. We observed a

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ACCEPTED MANUSCRIPT clear correlation between both in vitro and in silico studies. To our knowledge, this is also the first report on unfolding/stability analysis of any MurA enzyme.

Keywords Acinetobacter baumannii; UDP-N-acetylglucosamine enolpyruvyl transferase; unfolding;

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intermediate state; molecular dynamic simulation

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1 INTRODUCTION

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A well-defined 3D conformation of a protein is often necessary for its biological activity that is maintained by various covalent and non-covalent forces. Thus, the stability of the native state of

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any globular protein is critical. The covalent and non-covalent forces determine the folding pathways in a particular environment. Thus, the unfolding pathways are dependent on the

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solution conditions. Deciphering the dynamic unfolding events and the involved intermediates is important as it may give insight into the structural organization of the protein as well as the

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landscape that governs protein folding [1]. Urea and guanidine chloride (GdnHCl), which are important denaturing agents, are generally used for studying the unfolding pathway of proteins

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[2-5]. The folding of large proteins is generally populated with the formation of intermediates between the native and the unfolded states [6, 7]. Thus, the study of the intermediate states is

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valuable for determining the mechanism of folding of proteins [5, 6]. Acinetobacter baumannii is an opportunistic bacterial pathogen that is accountable for

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approximately 2–10% of all Gram-negative infections in hospitals [8]. It causes several severe skin and soft tissue infections, urinary tract infections, wound infections, and secondary

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meningitis [9-11]. The most effective drug against A. baumannii is colistin, but it has a number of side effects [12]; moreover, several incidents of colistin resistance has been reported worldwide [13, 14]. The emergence of multi-drug-resistant (MDR) strains of A. baumannii has created the need to develop novel drugs against these pathogens. The enzymes of peptidoglycan (PG) biosynthesis belong to the most important drug targets. PG is a key constituent of the prokaryotic cell wall that provides structural integrity to bacteria [15]. Its precursor is UDP-Nacetylmuramate (UNAM) that is synthesized by two enzymes: UDP-N-acetylglucosamine enolpyruvyl transferase (MurA, EC 2.5.1.7) and UDP-N-acetyl enolpyruvyl glucosamine reductase (MurB) [16]. Studies have reported several crystal structures of MurA [17-20]. A.

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ACCEPTED MANUSCRIPT baumannii MurA (AbMurA) exists as a monomer and consists of two domains connected by a flexible loop containing the catalytically important Cys residue (Supplementary Fig. S1). All reported MurA structures have a flexible surface loop (Pro111–Pro121, E. coli numbering) containing the catalytic Cys115 residue. This surface loop can adopt different conformations depending on the presence or absence of ligands [17-20]. The active site is present at the interface of these domains. Hence, MurA is considered as a potential drug target against this

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pathogen.

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In the present study, we have performed a combined spectroscopic and computational

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investigation to study the urea-induced unfolding of AbMurA. We observed that the unfolding of AbMurA is non-cooperative, where a metastable intermediate is populated during urea-induced

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unfolding. Till date there is no report available on unfolding and stability analysis of any MurA

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enzyme. We present the first report on unfolding studies of any MurA enzyme.

2 Material and methods

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2.1 Preparation of AbMurA

The overexpression and affinity purification of recombinant AbMurA was performed as reported

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earlier [21]. The protein was dialyzed against 20 mM Tris-Cl buffer, pH 8.0 containing 150 mM NaCl. Second step purification of AbMurA was performed using SuperdexTM 200 HR 10/300

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column on AKTA FPLC (GE Healthcare Biosciences). The purity and the integrity of the

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recombinant protein was determined using SDS-PAGE.

2.2 Denaturation/unfolding of AbMurA

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The equilibrium unfolding experiments with urea were performed in 20 mM Tris-Cl buffer, pH 8.0 containing 150 mM NaCl. Definite volumes of urea solution were added to 2 µM AbMurA to obtain an increasing urea concentration. The mixtures were incubated for 4 h at 25˚C before the measurements were made.

2.3 Fluorescence spectroscopy Fluorescence spectra were recorded with a PerkinElmer Life Sciences LS-55 fluorescence spectrophotometer in a 5 mm path length quartz cell at 25 °C. Excitation wavelength of 280 nm was used and the spectra were recorded between 300 and 400 nm.

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2.4 Circular dichroism measurements Circular dichroism (CD) measurements were made with a Jasco J800 spectropolarimeter calibrated with ammonium (+)-10-camphorsulfonate. The results are expressed as the CD ellipticity [θ]. The values acquired were normalized by subtracting the base line recorded for the

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buffer having the same concentration of denaturant under similar conditions.

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2.5 Analysis of spectral measurements

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All the transition curves created after plotting each spectral property vs [urea], were analyzed for estimating the stability parameters as reported earlier [22-24]. ΔG°H2O is the value of standard

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Gibbs free energy change (ΔGD) in the absence of urea; m is the slope (∂GD/∂[urea]); and Cm (=ΔG°H2O/m) is the midpoint of the unfolding curve where ΔGD=0. This evaluation is based

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upon a least-squares method.

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2.6 Fluorescence quenching

Fluorescence intensities were measured by a PerkinElmer LS-55fluorescence spectrometer at

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25 °C, using a 1 cm path length quartz cuvette. The excitation and emission wavelengths were 295 nm and 337 nm, respectively. Quenching of AbMurA with acrylamide (0-1 M) was

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performed by adding aliquots of the quencher stock solution (10 M acrylamide) to the protein (1 µM) in the appropriate reaction buffer. The experiments were repeated thrice, and the mean data

Volmer equation.

(9)

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Fₒ ⁄ F = 1+Ksv[Q]

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were considered for the study. The fluorescence quenching data were analyzed by the Stern-

where Fₒ and F are the fluorescence intensities in the absence and presence of the quencher (Q), respectively; Ksv is the dynamic quenching constants; and [Q] is the quencher concentration.

2.7 Molecular dynamics simulation Although several crystal structures of MurA have been reported, that of AbMurA has not been solved. The modeled structure of the AbMurA [21] was taken for molecular dynamics simulations (MDS). MDS were performed in an in-house supercomputer facility using

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ACCEPTED MANUSCRIPT GROMACS 4.6.5 [25, 26], with GROMOS 53a6 force field and SPC216 water model as previously mentioned [27-31]. A total of six systems (two for water and four with urea at 3.5 and 8.0 M) were created for MDS. A cubic simulation box was created and filled with water molecules and then allowed for padding around the protein; 16 Na+ ions were included to neutralize the system. The dimensions of the simulation boxes were 91.6 x 91.6 x 91.6 Å. Energy minimization was carried out using the steepest descent algorithm for removing the steric clashes

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of the systems. After that, all the systems were equilibrated in NVT and NPT for 1 ns for

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position restraint. The electrostatic interactions were calculated with particle mesh Ewald (PME)

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method [32, 33]. To compute Lennard-Jones and Coulomb interactions, 1.0 nm radius cut-off distance was used. The LINCS algorithm [34] was used to constrain the hydrogen bond lengths.

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To predict the short-range non-bonded interaction, 10 Å cut-off distance was used. 1.6 Å Fourier grid spacing was used for the PME method for long-range electrostatics. Three MD simulations

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were performed at 300 K, and the remaining three simulations were performed at 400 K temperature. MDS was performed with the integration time step 2 fs, period of 100 ns for each of

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the six systems. The coordinates and trajectories for velocity and energy were saved at the

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interval of 10 ps.

2.8 Analysis of MD data

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The analyses on the trajectories were performed by means of GROMACS utilities. To understand the conformational changes, root mean square deviation (RMSD) and radius of

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gyration (Rg) of all backbone Cα atoms were computed with respect to the native structure at 300 K and 400 K. The structural stability of the proteins was defined in terms of root mean

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square fluctuations (RMSF), hydrogen bond interaction, and solvent accessible surface area (SASA) to provide direct evidence of protein unfolding process. The RMSD and RMSF were calculated using g_rms and g_rmsf tools as mentioned earlier [25, 35] while the hydrogen bonds and SASA were calculated using g_hbond and g_sasa tool of GROMACS package[25, 35]. Secondary

structure

analysis

was

performed

using

the

do_dssp

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(https://swift.cmbi.umcn.nl/gv/dssp/). The g_energy tool was used for the calculation of potential energy of all the systems. To analyze the simulation results, time evolution snapshots were taken, and structures were drawn with Chimera 1.10.2 [36]. The trajectories were also analyzed using Chimera 1.10.2 [36], and the graphs were plotted by Origin 6.0 software.

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2.9 Principal component analysis The Principal component analysis (PCA) method was used for the calculation of eigenvectors and eigenvalues with their projection along the first two principal components (PCs) using g_covar and g_anaeig tools of the GROMACS package [37]. The concerted motions of proteins during MDS were extracted using PCA that are significant for biological functions. The

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positional covariance matrix C of atomic coordinates and its eigenvectors were used. The

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elements of the positional covariance matrix C were calculated by the following equation:

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𝐶𝑖𝑗 = 〈(𝑞𝑖 − ⟨𝑞𝑖 〉)(𝑞𝑗 − 〈𝑞𝑗 〉)⟩ (𝑖, 𝑗 = 1,2, … ,3𝑁)

(9)

where qi is the Cartesian coordinate of the ith Cα atom, and N is the number of Cα atoms.

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The average was calculated over the equilibrated trajectories after superimposition on a reference structure to remove overall translations and rotations by using a least-square fit procedure. The

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matrix was then diagonalized by an orthogonal coordinate transformation matrix Λ, to identify a set of eigenvectors and eigenvalues λi:

(10)

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Λ = 𝑇 𝑇 𝐶𝑖𝑗 𝑇

Here, the columns are the eigenvectors corresponding to the direction of motion relative

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to , and each eigenvector associated with an eigenvalue that represented the total meansquare fluctuation of the system along the corresponding eigenvector. The last 60 ns production

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runs were used to perform the analysis. The amplitude of eigenvectors and the displacement of atoms along each eigenvector show the concerted motions of protein along every direction and

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are represented by eigenvalues. The Cartesian trajectory coordinates were projected along the most important eigenvectors to identify the movements of structures in the essential subspace.

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Cα was selected for calculation.

3 Results and discussion 3.1 Purification of recombinant AbMurA The expression and purification of AbMurA were performed as mentioned earlier [21]. The purified protein displayed a single band of ~45 kDa on SDS-PAGE, and the SEC confirmed the monomeric status of AbMurA in solution as reported earlier [21].

3.2 Urea-induced unfolding of AbMurA studied by optical methods

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ACCEPTED MANUSCRIPT The intrinsic Trp fluorescence and far-UV CD were used to study the spectral changes associated with the unfolding of AbMurA. The spectral parameters of the Trp fluorescence emission, such as position, shape, and intensity, depend on the dynamic properties of the chromophore environment. Thus, the steadystate Trp fluorescence has been significantly used to obtain information about the structural and dynamic properties of proteins [38]. For AbMurA, the Trp fluorescence spectrum showed an

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maximum emission wavelength of ~337 nm [21]. In general, the buried Trp residues in a folded

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protein show maximum fluorescence emission between 330–335 nm [38]. The sequence of

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AbMurA encodes a single Trp residue at position 280 (C-terminal domain). In AbMurA, the Trp residue points towards the outer side of the protein (Figure 1A), which indicates that the Trp

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residue in AbMurA is partially exposed to the solution. We monitored the alteration in the microenvironment of Trp residue of AbMurA due to urea by studying the changes in the

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wavelength of emission maxima (Imax) of Trp fluorescence as a function of urea concentration. Changes in the Imax of Trp fluorescence of AbMurA with increasing urea concentration are

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shown in Figure 1B and supplementary figure 2. A sigmoidal loss of the fluorescence signal and shift in maximum emission wavelength from 337 nm to 355 nm was observed between 0 and 8

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M urea. At urea concentration above 6 M, the fluorescence emission maxima shifted to 355 nm, indicating that the enzyme is completely unfolded under these conditions. These observations

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indicate that urea induces cooperative unfolding of the protein. Protein containing α-helices and β-sheets show characteristic far-UV CD spectrum, with

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α-helical proteins having two minima at 222 and 208 nm and β-sheets proteins having a single minimum at 216 nm [39]. The far-UV CD spectrum of AbMurA reveals the presence of mixed

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α/β type secondary structure [21]. Thus, far-UV CD studies on the urea-induced unfolding of AbMurA were performed to study the effect of urea on the secondary structure of the enzyme. The effect of increasing urea concentration on the ellipticity at 222 nm is summarized in Figure 1B and supplementary figure 2. A gradual decrease in ellipticity at 222 nm was observed up to a urea concentration of ~3 M, followed by a static phase between 3 M to 4 M. However, between 4–6 M urea, there was a gradual reduction in CD ellipticity with complete loss of ellipticity above 6 M urea. The change in the CD ellipticity at 222 nm of AbMurA samples as a function of increasing concentration of urea showed a three-state unfolding with stabilization of an intermediate state between 3-4 M urea. At a urea concentration above 6 M, an almost complete

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ACCEPTED MANUSCRIPT unfolding of the protein was observed (Figure 1B). Thus, changes in the CD ellipticity suggest a non-cooperative unfolding of AbMurA with the stabilization of an intermediate conformation of AbMurA (Figure 1B). The positions of the equilibrium unfolding curves measured by intrinsic fluorescence and far-UV CD were found to be different. The unfolding curve measured by intrinsic fluorescence showed a biphasic transition while the curve measured by far-UV CD indicated a triphasic

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transition. This is because AbMurA exists as a monomer and encodes a single Trp residue in the

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C-terminal domain. This suggests that the unfolding of C-terminal domain starts first, followed

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by the N-terminal domain. Also, the intermediate state has partly open conformation of the Cterminal domain while the structure of the N-terminal domain is still intact.

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We further estimated the conformational stability of AbMurA in terms of Gibbs free energy (GD) by using the linear least square fit method [40]. The stability parameters-GD, and

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Cm were estimated by analyzing [θ]222 and transition curve (Figure 1C and 1D and Table 1). GD

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of (N↔X) and (X↔D) was used to calculate the GD of AbMurA (3.4.0.17 Kcal.mol-1).

3.3 Fluorescence quenching by acrylamide

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Quenchers are sensitive extrinsic probes that can monitor conformational changes in proteins [40]. Acrylamide-induced quenching provides additional information about the solvent

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accessibility of Trp residues. Stern-Volmer plots for native AbMurA at varying concentrations of acrylamide are presented in supplementary Fig. S3. The relative solvent exposure of different

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fluorophores was assessed using these data [41]. The quenching constants (KSV) calculated for AbMurA at 0 M urea (hereafter, AbMurAH2O), 3.5 M (hereafter, AbMurA3.5), and 8.0 M

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(hereafter, AbMurA8.0) urea were found to be 0.676, 0.833, and 1.00 M−1, respectively. The KSV value for the intermediate state (AbMurA3.5) was found to be higher than that of the native state (AbMurAH2O). This quenching is accompanied by a red shift in the Imax of emission. The KSV value of the denatured state of AbmurA (AbMurA8.0) was significantly higher than both the native and intermediate states. KSV can reflect the accessibility of the Trp residues in the protein when its values are correlated with denaturant concentrations. The results indicate that the Trp accessibility was less in native AbMurA and increased during the denaturation process due to structural changes in the protein. The Trp accessibility of AbMurA8.0 was effectively quenched by acrylamide, indicating the higher accessibility of Trp to quencher in AbMurA8.0, i.e., its

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ACCEPTED MANUSCRIPT exposure to the solvent [41]. The Stern–Volmer plot indicates that the Trp residue in AbMurA8.0 is more exposed to the solvent as compared to AbMurAH2O and AbMurA3.5. Thus, monitoring the exposure of Trp residue and KSV values provided additional information for the unfolding pathway of AbMurA.

3.4 Molecular dynamics simulation

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A detailed description of protein dynamics at the atomic-level is required for complete

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understanding of protein unfolding processes. This can be achieved using the all-atom MDS tool.

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MDS can provide precise details regarding the motion of individual atom as a function of time and thus, aid in the calculation of the dynamic properties of molecules. The advantage of MDS is

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that it provides information about the unfolding pathways, the final folded structure, the time dependence of these events, and the inter-residue interactions that underlie these processes. MDS

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has been employed in several studies to understand the effect of urea on protein structure and dynamics [42-45].

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To investigate the conformational changes during the unfolding of the AbMurA, six systems were created and employed for 100 ns MDS study. The AbMurAH2O, AbMurA3.5 and

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AbMurA8.0 at 300 K and 400 K temperature were used for investigating the unfolding pathway

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of AbMurA.

3.4.1 Root mean square deviation

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The stability of each system during the simulation was monitored by plotting the Root mean square deviation (RMSD) of AbMurA at that concentration of urea with respect to the initial

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structure. RMSD was used to measure the scalar distance between atoms in the structure. MDS was performed for all the three systems (AbMurAH2O, AbMurA3.5 and AbMurA8.0) at 300 K, and the RMSD values were accordingly determined (Figure 2A).. Following MDS, AbMurAH2O attained the equilibration state quickly and remained stable till 100 ns. A sharp increase in RMSD value was observed for an AbMurA3.5 system that attained equilibrium after 40 ns of simulation. The RMSD of AbMurA8.0 showed an abrupt pattern till 65 ns, and then it got stable. The average RMSD values for AbMurAH2O, AbMurA3.5, and AbMurA8.0 were 0.41, 0.71, and 1.30 nm, respectively, at 300 K. These values suggest that increasing concentration of urea

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ACCEPTED MANUSCRIPT leads to instability of the structure, and the deviation from the native structure increased during simulation. Similarly, MDSs were performed for all the three systems at 400 K. It was observed that the RMSD values of AbMurAH2O slowly increased till 20 ns, but then it attained stability until the end of the simulation. The RMSD values of AbMurA3.5 showed an abrupt pattern till 40 ns, and then the values got stable. Lastly, we observed that AbMurA8.0 showed the highest RMSD

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values as compared to all other systems. An abrupt pattern of RMSD was observed till 40 ns, but

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then it got stabilized till 100 ns. The average RMSD values for AbMurAH2O, AbMurA3.5 and

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AbMurA8.0 were 0.93, 1.36, and 2.18 nm, respectively, at 400 K temperature. The average results suggested that increasing the urea concentration and temperature

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induces unfolding of the structure. Thus, for the prediction of the unfolding pathway, we calculated different structural properties such as RMSF, Rg, SASA, and number of hydrogen

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bonds for all the systems. Principal component analysis (PCA) was also carried out for understanding the correlation dynamics of the systems. In addition, the secondary structure and

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potential energy analysis of the systems were also calculated. All these calculations were

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performed on the last 60 ns equilibrated trajectories.

3.4.2 Root mean square fluctuations

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Root mean square fluctuation (RMSF) was used for predicting the fluctuation of each residue during simulation. The rigid parts of protein like helices and sheets showed less fluctuation while

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loop and turns showed higher fluctuations. Upon addition of urea or increasing the temperature, the atoms moved from their original position, and it was recorded by RMSF. The average RMSF

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of residues in water and urea solutions were examined for all Cα atoms and plotted in Figure 2B for 300 K and 400 K.

The RMSF values of AbMurAH2O, AbMurA3.5, and AbMurA8.0 were calculated at 300 K. As can be seen in the plot, AbMurAH2O showed lower RMSF value as compared to the proteins with urea (AbMurA3.5 and AbMurA8.0). The RMSF value for AbMurA8.0 was much higher, indicating several structural changes occurred due to the addition of urea. AbMurA3.5 showed RMSF values higher than that of AbMurAH2O and lower than that of AbMurA8.0. The average RMSF for AbMurAH2O, AbMurA3.5 and AbMurA8.0 were 0.17, 0.31, and 0.94 nm, respectively, at 300 K. For residues, 115-125 AbMurAH2O showed higher RMSF values

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ACCEPTED MANUSCRIPT between 0.23 nm to 0.78 nm. The RMSF plot for AbMurA3.5 showed greater fluctuations as compared to the AbMurAH2O. Lastly, the RMSF value for AbMurA8.0 showed the highest and abrupt pattern compared to AbMurAH2O and AbMurA3.5. It showed an RMSF value >0.5 nm for all the residues. For residues 33-77, the RMSF value was greater than 2 nm while the RMSF values for residues 330-370 and 392-403 was >1.5 nm. All other regions of AbMurA8.0 showed RMSF value between 0.5 to 1 nm. Thus, it was observed that AbMurA 8.0 showed very high

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RMSF values, indicating the loss of structure in AbMurA8.0. Also, the overall RMSF of

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AbMurA3.5 was observed to be between AbMurAH2O and AbMurA8.0, which showed that

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AbMurA3.5 exists as the intermediate state.

The RMSF values of AbMurAH2O, AbMurA3.5 and AbMurA8.0 were calculated at 400 K;

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the obtained values were higher as compared to the values at 300 K. The average RMSF for all the residues for AbMurAH2O, AbMurA3.5, and AbMurA8.0 were 0.38, 0.60, and 1.14 nm,

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respectively, at 400 K. The AbMurAH2O showed an average fluctuation peak between 0.2 to 0.5 nm. Some residues like 35-49, 66-70, 323-352, and 411-418 showed RMSF values of >0.5 nm.

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In case of AbMurA3.5, most of the residues showed RMSF values between 0.5 to 1.0 nm. Some residues like 35-77, 113-134, 184-227, 252-269, 312-389, and the C-terminal residues showed

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RMSF values of >1 nm. In AbMurA8.0, we observed very high fluctuations that showed abrupt RMSF peaks. Here, most residues showed RMSF values between 0.5 to 1.5 nm. Some residues

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like 34-47, 113-132, 157-170, and 364-376 showed RMSF values between 1.5 to 2 nm. These values of RMSF suggest that after addition of urea and increasing the temperature to 400 K, the

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protein gets unfolded more easily than at 300 K. We plotted the snapshot of the proteins at different time steps for all the systems during

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both the temperatures. The figure shows how the proteins lose their structures at different time steps (Figures 3 and 4).

3.4.3 Radius of gyration Radius of gyration (Rg) is an important parameter to study the compactness of a protein. The compactly folded and expanded protein showed low and high Rg values, respectively. Backbone Rg of all the systems was calculated for the last 60 ns of trajectories at 300 and 400 K and plotted in Figure 5A. At 300 K, AbMurAH2O was found to be more stable as compared to AbMurA3.5 and AbMurA8.0 systems. AbMurA3.5 showed a metastable state. The average value of

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ACCEPTED MANUSCRIPT Rg of AbMurAH2O, AbMurA3.5, and AbMurA8.0 were 2.27, 2.35, and 3.02 nm, respectively. The high Rg value of AbMurA8.0 indicates the formation of an unfolded structure. The Rg values of AbMurAH2O, AbMurA3.5, and AbMurA8.0 were also analyzed at 400 K. The AbMurA8.0 showed much higher Rg value as compared to other two systems (AbMurAH2O, AbMurA3.5) as shown in Figure 5A. One interesting observation was that the AbMurA3.5 showed slightly lesser Rg value after 60 ns than AbMurAH2O. The average value for AbMurAH2O,

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AbMurA3.5, and AbMurA8.0 was 2.30, 2.25, and 3.19 nm, respectively. This may be due to that

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the intermediate state of AbMurA3.5 is partly compact at 400 K. The overall results at 300 K and

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400 K suggest that the protein gets unfolded on addition of 8 M urea. The Rg results also revealed that AbMurA3.5 is a metastable state. The Rg analysis of the unfolding at different urea

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concentration is in accordance with our acrylamide quenching experiments as both the data suggest the opening of structure with increasing urea concentration, and this opening of structure

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facilitates the binding of acrylamide.

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3.4.4 Hydrogen bonds

The hydrogen bonds are important for providing the stability of the protein structure. They are

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mainly involved in the stabilization of helices and sheets. We calculated the number of hydrogen bonds for AbMurAH2O, AbMurA3.5, and AbMurA8.0 at 300 K and 400 K (Figure 5B) for

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predicting the urea-induced changes in hydrogen bonding patterns of the proteins. At 300 K, the number of hydrogen bonds decreases in the order of AbMurAH2O>AbMurA3.5>AbMurA8.0. This

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suggests that due to the addition of urea, the hydrogen bonds break, and the structures also get unfolded. The result at 300 K clearly indicated that AbMurA8.0 exists in expanded conformation,

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suggesting that the protein is in the unfolded state at 8 M urea. After that, the number of hydrogen bonds was calculated for all the three systems at 400 K. It was observed that after 80 ns, the number of hydrogen bonds is similar for all the systems. The overall result suggests that AbMurA8.0 showed the lowest number of hydrogen bonds as compared to other systems. The average number of hydrogen bonds for AbMurAH2O, AbMurA3.5, and AbMurA8.0 was 270, 264, and 258, respectively. The results at both temperatures suggest that after adding the 8 M urea, the protein lose the hydrogen bonds network and attain the unfolded state.

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ACCEPTED MANUSCRIPT 3.4.5 Residue solvent accessible surface area We examined the time-dependent change in the solvent accessible surface area (SASA) to quantify the urea-induced unfolding process of AbMurA. The hydrophobic SASA was calculated for the last 60 ns of trajectories and shown in Figure 5C. From Figure 5C, it can be clearly seen that the addition of urea induces unfolding as AbMurA8.0 showed much higher SASA value as compared to the other two systems. The average SASA value for AbMurAH2O, AbMurA3.5, and

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AbMurA8.0 was 247, 258, and 268 nm, respectively, at 300 K. The values indicated that the

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structure loses its dynamic stability. The average SASA value for AbMurAH2O, AbMurA3.5, and

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AbMurA8.0 was 255, 265, and 266 nm, respectively, at 400 K. The SASA values for AbMurA8.0 showed very abrupt patterns, suggesting that in 8 M urea, the structure is lost and the residues of

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the hydrophobic core are completely exposed to the solvent.

In the modeled structure of AbMurA, one Trp is present in the 280 position, which is

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partially exposed towards the solvent. We plotted the time evolution graph of the surface area of residues accessible to solvent as shown in Figure 5D and supplementary Fig. S4. From this

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SASA plot, it can be seen that Trp280 is gradually exposed to solvent in the presence of urea. The value of SASA for Trp280 gradually increased in AbMurA3.5 and AbMurA8.0. The value of

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SASA for AbMurAH2O, AbMurA3.5, and AbMurA8.0 was 0.552, 0.566, and 0.657 nm, respectively, at 300 K. This increasing value for Trp280 indicates that the structure is lost and the

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Trp280 is exposed towards the solvent at high urea concentration at 300 K. The average SASA value for Trp280 was also calculated at 400 K. The average value for AbMurAH2O, AbMurA3.5,

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and AbMurA8.0 was found to be 0.557, 0.614 and 0.682 nm, respectively. All these results indicate that the addition of urea leads to the loss hydrophobic interactions and the hydrogen

process.

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bond network. Furthermore, the loss of hydrophobic contact provides clear evidence of unfolding

3.4.6 Potential energy The potential energies for AbMurAH2O, AbMurA3.5, and AbMurA8.0 were calculated for predicting the stability of proteins. AbMurAH2O, AbMurA3.5, and AbMurA8.0 showed -1.03×106, -8.58×105, and -6.48×105, KJ/mol energy, respectively, at 300 K. The energy values suggest that upon addition of urea, the structure gets unfolded. The potential energy for AbMurA H2O, AbMurA3.5, and AbMurA8.0 was also calculated at 400 K that showed values of -8.98×105, -

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ACCEPTED MANUSCRIPT 7.22×105, and -5.19×105 KJ/mol energy, respectively. From the results, it can be clearly concluded that in 8 M urea, the protein is completely unfolded while in 3.5 M urea, the protein is partially unfolded, revealing the formation of an intermediate state.

3.5 Principal component analysis To check the presence of correlated motions in AbMurAH2O, as compared to AbMurA3.5 and

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AbMurA8.0, and to validate our previous results, the large-scale collective motions of

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AbMurAH2O, AbMurA3.5, and AbMurA8.0 were determined using PCA. It is well known that the

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first few eigenvectors play an important role in motions in the protein. Figure 6A shows a plot of the eigenvalues obtained from the diagonalization of the covariance matrix of the atomic

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fluctuations for the first 50 eigenvectors. The first few eigenvalues are relative to concerted motions and quickly decreased in amplitude to reach a number of constrained, more localized

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fluctuations. We selected the first 50 eigenvectors for analysis. The first five principal components (PCs) account for 84.29%, 72.93%, and 80.20% motions in the last 60 ns trajectory

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for AbMurAH2O, AbMurA3.5, and AbMurA8.0, respectively. In Figure 6A, AbMurA8.0 showed large-amplitude motions, indicating that the addition of urea induces unfolding in the structure;

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thus, the structure involves more correlated motions and getting expanded. The result also suggested that each system has different concerted motions.

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The correlated motions were also calculated for 400 K for all the systems. Here, Figure 6B clearly indicated that the AbMurA8.0 M showed the highest correlated motions as compared to

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other systems. The first five PCs account for 76.84%, 72.33%, and 75.30% motions in the last 60 ns trajectory for AbMurAH2O, AbMurA3.5, and AbMurA8.0, respectively. The result suggests that

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due to the addition of urea, the magnitude of PCs is increased. We then calculated the 2D projection plot for PC1 and PC2 since the phase space behavior is best characterized by the dynamics of the system. The level of fluctuation in a 2D plot was obtained by the spectrum of corresponding eigenvalues at 300 and 400 K temperatures. At 300 K, we can clearly see that, due to the addition of urea, the protein do not show stable cluster in the phase space and AbMurA8.0 occupied much larger phase space while AbMurAH2O showed stable cluster. AbMurA3.5 also showed stable cluster, which revealed that at 3.5 M of urea, the structure is not completely lost and the protein exists in metastable state. Then, we analyzed the results at 400 K. Here, we also predicted similar results as 300 K, except that

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ACCEPTED MANUSCRIPT AbMurA3.5 showed more expanded form and correlated motions as compared to AbMurAH2O. This result indicates that AbMurA3.5 makes large-amplitude motions, and urea induces structural plasticity in the protein, resulting in the partly expanded form of the structure. All these results clearly suggest that the first PC is mainly involved in overall motions of the protein. So, we predicted the specific motions residues-wise for different regions of protein, where it induces the correlated motions. The average value of fluctuations for AbMurAH2O,

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AbMurA3.5, and AbMurA8.0 was 0.11, 0.17 and 0.59 nm, respectively. Figure 6C indicated that

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the urea induces large-amplitude motions, leading to unfolding of the structure. The AbMurA8.0

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showed >0.5 nm value for regions 33-99 and 328 -428 residues. The average values for AbMurAH2O, AbMurA3.5, and AbMurA8.0 at 400 K were 0.21, 0.32, and 0.64 nm, respectively.

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The average values and figure indicate that after the addition of 8 M urea, the protein lost its

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structure completely. This result is consistent with the RMSF analysis.

3.5.1 Secondary structure analysis

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Time evolutionary of secondary structure was predicted for the last 40 to 100 ns trajectories. During unfolding, the coils, bends and turn increased while alpha-helices and beta-sheets

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disappeared. The secondary structure is shown in Figure 7 and 8 for all the systems at 300 and 400 K.

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Firstly, we analyzed the secondary structure result at 300 K. AbMurAH2O showed a stable pattern of structure with several alpha-helices and beta-sheets. The AbMurA3.5 showed some

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increment in bend, coils, and turns in the structure. More number of turns, coils, and turns were increased in AbMurA8.0. Residues 1-130 completely lost the secondary structures and the rigid

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structures like were converted into bends and coils. The overall structure also showed more number of coils and bends (Figure 7). Then we analyzed secondary structural changes at 400 K temperature. Here, we also saw a drastic change; when urea was added, the structure was totally distorted. The AbMurAH2O showed stable structure with several helices and sheets; additionally, due to an increase in temperature, some bends also appeared. AbMurA3.5 showed much structural change at 400 K temperature. Though it showed stable structure with some helices and sheets, it showed more number of coils, bends and turns that were increased indicating that the structure is more unfolded than at 300 K. Lastly, we predicted the secondary structure of AbMurA8.0 at 400 K. It

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ACCEPTED MANUSCRIPT showed total distortion and loss in secondary structure. Only few beta-sheets were observed in the structure, and no helix was seen in the secondary structure. AbMurA8.0 totally lost the secondary structures, and urea completely unfolded its structure (Figure 8). These results are in agreement with RMSD, RMSF, Rg, number of hydrogen bonds, SASA, and PCA results. Thus, from the overall MDS results, we can suggest that at 8 M urea, the AbMurA is completely unfolded while at 3.5 M urea, the protein exists in an intermediate

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state.

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4 Conclusion

In spite of the tremendous improvements in computations, protein folding is a slow and complex

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process to be accurately described via MDS. In the present work, MDS and spectroscopic investigations were performed to gain insights into the unfolding process of AbMurA. The data

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revealed the stabilization of the intermediate conformation and transition of AbMurA on the way

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to unfolding. The results offer insights into the molecular mechanism of AbMurA unfolding.

Acknowledgements

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Authors thank the Sulekor Supercomputing facility of NEHU installed at computer Center. AS (PDF/2016/001711) and HS (PDF/2017/000458) thanks SERB, New Delhi, for providing

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Competing interest

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National Postdoctoral Fellowship, while RS thank UGC, New Delhi for providing fellowship.

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The authors declare that there are no competing interests.

Authors' contributions AS, HS, RS, and JK carried out the experiments. AS, HS, RS, and TT analyzed the data. HS, RS and TT conceived the study, participated in its design and coordination and drafted the manuscript. All authors read and approved the final manuscript.

Abbreviations Acinetobacter

baumannii

MurA,

AbMurA;

Phosphoenol

pyruvate,

PEP;

UDP-N-

acetylglucosamine, UNAG; UDP-N-acetylmuramate, UNAM; UDP-N-acetylglucosamine

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ACCEPTED MANUSCRIPT enolpyruvate, UNAGEP; Size exclusion chromatography, SEC; Root mean square deviation, RMSD; Root mean square fluctuation, RMSF; Principal component analysis, PCA; Solvent accessible surface area, SASA.

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ACCEPTED MANUSCRIPT Table 1. Thermodynamic parameters obtained from urea-induced denaturation of AbMurA.

Transition

ΔGI or ΔGII (kcal mol-1)

m (kcal mol-1 M-1)

Cm [M]

[θ]222

N↔X

1.44  0.072

~0.56

~1.8

X↔D

1.68  0.084

~1.16

~5.1

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Probe

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ACCEPTED MANUSCRIPT Figure Legends Figure 1. (A) Molecular model of AbMurA showing the position of Trp280. (B) Fraction unfolding of AbMurA with increasing urea concentration. (C) The linear free energy extrapolation curve with respect to [Urea] up to 3.5 M. (D) The linear free energy extrapolation curve with respect to [Urea] between 3.5 M to 5.5 M. The ΔGI and ΔGII were the intercepts on

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the Y-axis, obtained using the linear extrapolation method.

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Figure 2. RMSD and RMSF plots of AbMurA in water, 3.5 M and 8.0 M urea. (A) The

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RMSD plot of AbMurA in water, 3.5 and 8.0 M urea as a function of time at 300 K. (B) RMSF values of AbMurA during MDS in water, 3.5 and 8.0 M urea at 300 K. The plots were color-

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coded: 300 K in water, black; 400 K in water, blue; 300 K in 3.5 M urea, red; 300 K in 8 M urea,

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green; 400 K in 3.5 M urea, cyan; 400 K in 8 M urea, magenta.

Figure 3. Snapshots at different time frames. Structural characterization of AbMurA by MDS

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for 100 ns with different concentrations of urea at 20 ns time interval at 300 K.

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Figure 4. Snapshots at different time frames. Structural characterization of AbMurA by MDS

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for 100 ns with different concentrations of urea at 20 ns time interval at 400 K.

Figure 5. Radius of gyration, number of hydrogen bonds and solvent accessible surface

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area of AbMurA in water, 3.5 M, and 8.0 M urea. (A) Time evolution of radius of gyration (Rg) values during the last 60 ns of MDS. (B) Number of hydrogen bonds with respect to time

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for last the 60ns of MD simulation. (C) The SASA plot for AbMurA in water and different concentrations of urea with respect to time. (D) The SASA for AbMurA with respect to each residue. The plots were color-coded: 300 K in water, black; 400 K in water, blue; 300 K in 3.5 M urea, red; 300 K in 8 M urea, green; 400 K in 3.5 M urea, cyan; 400 K in 8 M urea, magenta.

Figure 6. Principal component analysis of AbMurA in water, 3.5 M and 8.0 M urea. (A) The eigenvalue vs. first 50 eigenvector was calculated from the last 60 ns of the trajectories. (B) The 2D projection plot in various conditions. (C) The EigenRMSF plot for all the residues.

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ACCEPTED MANUSCRIPT Figure 7. Secondary structure evolutions of AbMurA in water, 3.5 and 8.0 M urea at 300 K. Time evolution of secondary structural elements during the unfolding processes at 300 K with water and various urea concentrations calculated from last 60 ns trajectory.

Figure 8. Secondary structure evolutions of AbMurA in water, 3.5 and 8.0 M urea at 400 K. Time evolution of secondary structural elements during the unfolding processes at 400 K with

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water and various urea concentrations calculated from last 60 ns trajectory.

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ACCEPTED MANUSCRIPT Highlights 

We examined the urea-induced unfolding of AbMurA using spectroscopic techniques and molecular dynamic simulations.



The unfolding of AbMurA was a three-state process, where a metastable intermediate conformation state is populated. The transition from the native-to-metastable state involves ~30% loss of native contacts

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

The intermediate-to-unfolded state transition was characterized by a large increase in the radius of gyration.



MD trajectories suggest that urea destabilizes AbMurA structure weakening hydrophobic

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interactions and the hydrogen bond network.

To our knowledge, this is the first report on unfolding and stability analysis of any MurA

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M

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enzyme.

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

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

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but little change in the radius of gyration.

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Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8