Structural and Functional Significance of the N-and C-Terminal

Feb 25, 2016 - Department of Biochemistry, University of Delhi South Campus, New Delhi 110021, India. •S Supporting Information. ABSTRACT: Plant ...
0 downloads 0 Views 3MB Size
Article pubs.acs.org/biochemistry

Structural and Functional Significance of the N- and C‑Terminal Appendages in Arabidopsis Truncated Hemoglobin Nitika Mukhi,† Sonali Dhindwal,‡ Sheetal Uppal,§ Abhijeet Kapoor,§ Richa Arya,§ Pravindra Kumar,‡ Jagreet Kaur,*,† and Suman Kundu*,§ †

Department of Genetics, University of Delhi South Campus, New Delhi 110021, India Department of Biotechnology, Indian Institute of Technology, Roorkee, Uttarakhand 247667, India § Department of Biochemistry, University of Delhi South Campus, New Delhi 110021, India ‡

S Supporting Information *

ABSTRACT: Plant hemoglobins constitute three distinct groups: symbiotic, nonsymbiotic, and truncated hemoglobins. Structural investigation of symbiotic and nonsymbiotic (class I) hemoglobins revealed the presence of a vertebrate-like 3/ 3 globin fold in these proteins. In contrast, plant truncated hemoglobins are similar to bacterial truncated hemoglobins with a putative 2/2 α-helical globin fold. While multiple structures have been reported for plant hemoglobins of the first two categories, for plant truncated globins only one structure has been reported of late. Here, we report yet another crystal structure of the truncated hemoglobin from Arabidopsis thaliana (AHb3) with two water molecules in the heme pocket, of which one is distinctly coordinated to the heme iron, unlike the only available crystal structure of AHb3 with a hydroxyl ligand. AHb3 was monomeric in its crystallographic asymmetric unit; however, dimer was evident in the crystallographic symmetry, and the globin indeed existed as a stable dimer in solution. The tertiary structure of the protein exhibited a bacterial-like 2/2 α-helical globin fold with an additional N-terminal α-helical extension and disordered C-termini. To address the role of these extended termini in AHb3, which is yet unknown, N- and C-terminal deletion mutants were created and characterized and molecular dynamics simulations performed. The C-terminal deletion had an insignificant effect on most properties but perturbed the dimeric equilibrium of AHb3 and significantly influenced azide binding kinetics in the ferric state. These results along with the disordered nature of the C-terminus indicated its putative role in intramolecular or intermolecular interactions probably regulating protein−ligand and protein−protein interactions. While the Nterminal deletion did not change the overall globin fold, stability, or ligand binding kinetics, it seemed to have influenced coordination at the heme iron, the hydration status of the active site, and the quaternary structure of AHb3. Evidence indicated that the N-terminus is the predominant factor regulating the quaternary interaction appropriate to physiological requirements, dynamics of the side chains in the heme pocket, and tunnel organization in the protein matrix.

G

bacterial trHbs. In contrast to bacterial trHbs, which typically have shorter sequences, ptrHbs have longer polypeptide chains, the size sometimes being larger than those of even class I and II nsHbs but still retaining the putative truncated 2/2 α-helical globin fold.9−11 Arabidopsis thaliana encodes a single copy for each of the three classes of nonsymbiotic globin genes: AHb1 (class I), AHb2 (class II), and AHb3 (truncated hemoglobin).11,12 AHb1 and AHb2 share 60% sequence homology between them, and both display a classical 3/3 α-helical globin fold.8−12 With differences in expression pattern and tissue localization, these globins have been proposed to play diverse physiological roles.13−15 AHb1 displays a relatively high oxygen binding affinity16 and is primarily believed to be engaged in NO binding

lobins are pervasive in all kingdoms of life, ranging from unicellular bacteria to higher eukaryotes.1−3 Apart from the well-characterized symbiotic hemoglobins, plant genomes are endowed with multiple nonsymbiotic hemoglobins (nsHbs), the significance of which can be anticipated by their ubiquitous occurrence in almost all land plants.4,5 The nsHbs are further categorized into three separate classes: class I (nsHb1), class II (nsHb2), and class III or truncated hemoglobins (trHbs).6 Members of each class bear unique kinetic and structural fingerprints, suggestive of their distinct physiological role.7 Structurally, land plant hemoglobins (Hbs) are divided into two distinct lineages: those that hold a classical 3/3 α-helical globin fold (like symbiotic and class I and class II nsHbs) and the rest with 2/2 α-helical fold globins, like the class III Hbs.6−8 TrHbs, marked by the presence of a typical 2/ 2 globin fold, are found to be ubiquitously present in plants, bacteria, and unicellular eukaryotes, with a typical size of 110− 140 amino acid residues.9,10 Plant truncated hemoglobins (ptrHbs) (class III) share 40−45% sequence similarity with © XXXX American Chemical Society

Received: September 14, 2015 Revised: February 25, 2016

A

DOI: 10.1021/acs.biochem.5b01013 Biochemistry XXXX, XXX, XXX−XXX

Article

Biochemistry because of its NO dioxygenase activity.16−18 AHb2, in comparison, has an oxygen affinity lower than that of AHb1 and has been proposed to facilitate the supply of oxygen to developing tissues.19 In Arabidopsis, class II nsHb have been proposed to participate in fatty acid metabolism and shoot organ development.20,21 AHb3 is expressed in both Arabidopsis roots and shoots and was found to be downregulated by hypoxia.11,22 In contrast to class I and class II nsHbs, much less is known about plant trHbs. Structures of class I and class II nsHbs were obtained for several plant Hbs.8,23−25 However, only recently was a solitary ptrHb structure reported.26 Characterizing the three-dimensional structure is fundamental to gaining insights into the function of a protein, with structures of Hbs having played a significant role in understanding their functions and evolutionary relationship. Every three-dimensional structure of novel Hbs deciphered in the past two decades has ushered in new insights about this ubiquitous protein family. Due to a lack of multiple crystal structures for ptrHbs, their physiological role in land plants is still under contest; their structural characteristics and functional potential are not fully recognized or explored. Attempts were thus made to crystallize trHb from the model plant A. thaliana, and we could successfully determine the three-dimensional structure of AHb3 [Protein Data Bank (PDB) entry 4C44]. Simultaneously and independently, however, Reeder et al. also determined the structure of AHb3 at 1.7 Å (PDB entry 4C0N).26 PDB records are testimony to the fact that our coordinates were submitted almost at the same time (PDB entry 4C0N, August 6, 2013; PDB entry 4C44, August 30, 2013), but for technical reasons, we were late in reporting the crystal structure. To overcome this deficiency, we extended our investigation beyond the three-dimensional structure for additional insight into the structure−function relationship of the novel globin, especially in solution. As in the previous crystal structure,26 the tertiary architecture of the protein exhibited a bacterial-like 2/2 α-helical globin fold in its core with an added N-terminal α-helical extension and disordered C-termini. However, subtle differences were also seen in the two structures, like the presence of different ligands in the heme pocket. For insight into the functional and structural significance of the N- and C-terminal appendages in ptrHbs, N- and C-terminal deletion mutants, AHb3-Δ25N and AHb3-Δ25C, respectively, as well as the double mutant AHb3Δ25NΔ25C were investigated. It was observed that the removal of 25 residues from the N- or C-terminus did not cause any significant perturbation of the overall core structure or globin fold of the protein. Molecular dynamics (MD) simulations of the crystal structure of AHb3 or models of AHb3-Δ25C and AHb3-Δ25N displayed stable trajectories throughout the simulation, further precluding their role in the maintenance of the structural architecture or integrity of the protein. AHb3-Δ25C was also similar to wild-type AHb3 in most of its biochemical properties, suggesting the functional irrelevance of the C-terminus in general characteristics of the globin. However, the C-terminally truncated mutant displayed a significant difference in its azide binding kinetics in the ferric state. The C-terminus, thus, might be vital for recognition of interacting or binding ligands and proteins, which is reminiscent of intrinsically disordered domains or proteins.27 However, it was evident that the N-terminus played a significant role in the dimerization of the globin, the coordination of heme iron, the hydration state of the heme pocket, and the tunnel topology in the protein matrix as judged

from its spectroscopic, structural, and other biochemical properties.



MATERIALS AND METHODS Protein Expression and Purification. cDNA (Unigene clone U83861) for Arabidopsis truncated hemoglobin (AHb3) was amplified via polymerase chain reaction (PCR) and cloned in bacterial expression vector pET21c (Novagen, Darmstadt, Germany) between BamHI and XhoI restriction sites. For expression of wild-type AHb3, transformed BL21(DE3) bacterial cells were initially grown in Terrific broth at 37 °C to an optical density of 0.6 (at 600 nm) followed by induction at 25 °C (without IPTG) for 16 h at 200 rpm. Cells were then harvested and lysed by both chemical and mechanical treatment as described previously for other globins.8,28 Purification of sixHis-tagged recombinant protein was performed by NiSepharose (GE Healthcare, Little Chalfont, United Kingdom) affinity chromatography. Protein was further purified by employing DEAE Sephadex anion exchange (GE Healthcare) and S-200 Sephacryl (GE Healthcare) size exclusion chromatography. An ASoret/A280 absorbance ratio of ≥3.5 indicated the purity of the protein, which was also confirmed by sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS−PAGE). The purified recombinant protein was then subjected to reduction and oxidation of heme iron by sodium dithionite and potassium ferricyanide, respectively, following desalting on a G-25 Sephadex (GE Healthcare) column to obtain a homogeneous population of ferric globin. Pure protein was stored at −80 °C for further use. Crystallization and Data Collection. Before crystallization trials, the quality and oxidation state (ferric) of AHb3 were assessed by examining the absorbance spectrum, and if required, it was converted completely into the ferric state using potassium ferricyanide at pH 7.0. Crystallization screens (Hampton) were used to identify suitable crystallization conditions. Crystals of ferric AHb3 were grown using the sitting drop vapor diffusion method at 20 °C by mixing equal volumes of a 2 mM protein solution and a precipitant solution containing 1.2 M sodium potassium tartarate tetrahydrate in 0.1 M Tris (pH 8.5). Well-defined crystals were observed in approximately a few weeks. Before data collection, the protein crystals were cryoprotected by being directly transferred to the mother liquor drop containing 10% (v/v) ethylene glycol as a cryoprotectant. The diffraction data were collected at 100 K using Cu Kα X-ray radiation generated by a rotating-anode generator (Bruker-Nonius Microstar H, Billerica, MA) equipped with a MAR345 imaging plate detector on an inhouse facility at the Indian Institute of Technology Roorkee (India). Structure Determination and Refinement. Diffraction data, obtained as described above, were reduced and scaled using the HKL2000 program suite.29 Initial phases for the structure were obtained by the molecular replacement method with the crystal structure of Geobacillus stearothermophilus truncated hemoglobin (PDB entry 2BKM) as a search model (35% sequence homology) using MOLREP from the CCP4.6.3 software suite.30,31 Crystallographic refinement was performed using REFMAC 5.32,33 COOT was used for electron density map and model building.34 The PyMol visualization tool was utilized for structural analysis and production of figures.35 The atomic coordinates and structure have been submitted to the PDB36 as entry 4C44. B

DOI: 10.1021/acs.biochem.5b01013 Biochemistry XXXX, XXX, XXX−XXX

Article

Biochemistry

flow spectrometer using an SFM400 module equipped with four syringes in association with an MPS70 syringe controller and an MOS50 spectrophotometer from BioLogic Science Instruments (Bio-Logic SAS, Claix, France). The kinetic traces were monitored using absorbance measurements at 414 nm for O2 dissociation and 431 nm for CO association. Laser flash photolysis was conducted to determine the O2 association rate constant for the proteins. Oxygenated protein samples were prepared as described above, and the oxy samples were collected directly into gastight syringes from the desalting column. Subsequently, they were transferred to a 3 mL quartz cuvette with a path length of 1 cm sealed with a rubber septum. The O2-bound protein samples were then subjected to flash photolysis using an LKS.60 flash system (Applied Photophysics Ltd., Leatherhead, Surrey, United Kingdom) containing a Nd:YAG pulsed laser at 1064 nm, frequency doubled to 532 nm with an energy of 10 or 20 Hz. White light from a Xe lamp was used to probe the samples. The kinetic traces corresponding to changes in the Soret peak wavelength for either formation of oxy-bound globin or depletion of deoxyglobin were recorded and rates extracted as described elsewhere.41,42 Azide Binding. Azide binding in the ferric proteins was performed with a stopped-flow spectrometer using an SFM400 module equipped with four syringes in association with an MPS70 syringe controller and an MOS50 spectrophotometer from BioLogic Science Instruments (Bio-Logic SAS). The kinetic traces were monitored using absorbance measurements at 409 nm for ligand association. The azide binding in the respective ferric proteins was spectroscopically confirmed by recording the shift in the Soret peak and Q-bands upon ligand binding. Biochemical Characterization. Autoxidation Measurements. Autoxidation rates were measured by monitoring the absorbance changes at 581 nm for the proteins investigated, as described previously.28 Briefly, 0.3 mM oxyprotein was diluted into ∼100 mM potassium phosphate (pH 8.0) supplemented with 1 mM EDTA and 3 mmol/mol of heme catalase and superoxide dismutase. Changes in the entire visible spectra were recorded using a Cary Varian 100 Bio UV−vis spectrophotometer (Varian Inc.) in scanning kinetics mode. Igor Pro (Wavemetrics Inc.) was used for data plotting and analysis. Stability Investigations and pH Titration Profiles. The stability of the proteins against changes in pH was probed using buffers in the pH range of 2.0−11.0.28 Prior to spectroscopic measurements, appropriate protein at 3 μM was diluted in a buffer at the desired pH and incubated for 3−4 h at 25 °C. To generate pH titration profiles, ferric proteins (wild-type AHb3 and its mutants) were subjected to the pH range mentioned above while recording their optical spectrum between 260 and 700 nm. The apparent pKa was calculated by plotting the difference in absorbance at 424 and 406 nm against the change in pH as reported by Reeder et al.26 The globins tend to aggregate and precipitate at pH 5.5 and hence the titration profiles included the pH range of 5.5−11. Thermal stability was probed at pH 7.0 in the range of 25−80 °C with a 5 min incubation at each temperature followed by absorbance measurement. Heme Dissociation Assay. To assess the effect of N- and Cterminal deletions on the heme retention capability of AHb3, if any, heme was extracted from the globins using Teale’s method.43 Briefly, the pH of the sample was adjusted to 2.0

Cloning, Expression, and Purification of Truncated Recombinant AHb3 Proteins. AHb3-Δ25N, AHb3-Δ25C, and AHb3-Δ25NΔ25C deletion mutants (from which 25 amino acids were deleted from either the N-terminus, the Cterminus, or both) were generated using a PCR-based approach. The primer pairs used for the amplification of the deleted variants were 5′-CACGGATCCAGGAGTCCAATCTGTTCG-3′ and 5′-TGCCTCGAGTTCTGCTGGTTTATTG3′, 5′-CACGGATCCAGATGCAATCGCTGCAAG-3′ and 5′TGCCTCGAGCAGCTCGTTTCCAGCCAC-3′, and 5′-CACGGATCCAGGAGTCCAATCTGTTCG-3′ and 5′-TGCCTCGAGCAGCTCGTTTCCAGCCAC-3′. The PCR-amplified genes were then independently cloned into the pET21c bacterial expression vector between BamHI and XhoI sites and the clones verified by DNA sequencing. Subsequently, individual variants were expressed and purified in the same way as for wild-type AHb3 protein. The purified proteins were then reduced, oxidized, and stored at −80 °C for further use as described above. Quaternary Structure Determination of AHb3 and Its Truncated Derivatives. HPLC Method. For protein quaternary structure determination, size exclusion chromatography was performed using the HPLC system from Waters (model 2489, Milford, MA) fitted with a UV−vis detector. Prior to the analysis, the column (Biosuite 125, 4 μm UHR SEC, 4.6 mm × 300 nm) was pre-equilibrated using 0.1 M phosphate buffer (pH 8.0). Protein concentrations ranging from 200 μM to 1 mM were investigated at a flow rate of 0.5 mL/min. The retention time was recorded while the absorbance at 280 nm was monitored to obtain the chromatogram. Proteins of known molecular masses were used as markers for reference (ribonuclease A, ∼14 kDa; carbonic anhydrase, ∼29 kDa; ovalbumin, ∼45 kDa; conalbumin, ∼76 kDa). Cross-Linking Assay. To investigate the oligomerization potential of AHb3 and AHb3-Δ25C, the glutaraldehydedependent cross-linking assay was used as described previously.8 In brief, 100 μg of appropriate protein was incubated with varying concentrations of a freshly prepared solution of glutaraldehyde ranging from 0.01 to 1% for 5 min at 37 °C. The reaction was terminated by adding 10 μL of 1 M Tris-HCl (pH 8.0). Cross-linked protein was analyzed using SDS−PAGE. Spectroscopic Characterization of AHb3-Δ25N, AHb3-Δ25C, and AHb3-Δ25NΔ25C. All the absorbance measurements were taken using a Cary Varian 100 Bio UV−vis spectrophotometer (Varian Inc.) in the range from 260 to 700 nm with protein samples at a concentration of ∼0.5 mg/mL, as determined by Bradford’s assay. For spectral measurement of ferrous globin and ligand-bound proteins, deoxy samples were generated by reducing ferric protein with sodium dithionite. Following reduction, CO-bound protein was prepared by directly purging CO gas in the deoxy sample. Oxygen-bound samples were prepared by desalting the reduced hemoglobin over a G-25 column. CD spectra were recorded on a JASCO J815 spectropolarimeter (JASCO Corp., Tokyo, Japan) using a cylindrical quartz cell with a path length of 1 mm for the far-UV region between 190 and 260 nm. Ligand Binding Kinetic Studies. Stopped-flow and laser flash photolysis-based spectroscopic methods were employed to determine the effect of deletions on the gaseous ligand binding kinetics of AHb3 as described elsewhere for pentacoordinated Hbs.37−40 O2 dissociation rates and CO association rates for AHb3 and its mutants were measured at 25 °C in a stoppedC

DOI: 10.1021/acs.biochem.5b01013 Biochemistry XXXX, XXX, XXX−XXX

Article

Biochemistry using ice-chilled 0.1 M HCl. An equal volume of cold ethyl methyl ketone was added to the sample immediately, followed by brief shaking. The samples were then incubated for 5 min on ice to separate the two phases. Usually, heme partitions in the top organic phase and the resulting apoglobin in the bottom aqueous phase. The rate constants for heme dissociation were measured as described by Hargrove et al.44 The transfer of heme from 3 μM holoprotein of interest to 30 μM H64Y/V68F apomyoglobin was measured at pH 7.0 by monitoring changes in absorbance at 410 and 600 nm. Curve fitting to a singleexponential equation was accomplished using Igor Pro (Wavemetrics Inc.), and rate constants for heme dissociation were calculated accordingly.28 Modeling of AHb3 To Include or Exclude Terminal Amino Acid Side Chains. The crystal structure of wild-type AHb3 was used as the template to model the derivatives. The model to include the C-terminus, because its electron density was missing in the crystal structure, was built using ITASSER.45 The N-terminally truncated model was obtained simply by deleting the coordinates for the corresponding side chains from the PDB file of the crystal structure of AHb3. Molecular Dynamics (MD) Simulation. MD simulation studies were conducted for the X-ray crystal structure of AHb3 (with missing C-terminal density) and AHb3 with a deleted Nterminus (pre-A helix). All the simulations were executed using NAMD46 and the CHARMM2247 force field with CMAP correction. MD simulations were performed using periodic boundary conditions and TIP3P water, and the system was neutralized by adding counterions. A 2 fs time step was used with a 12 Å cutoff for VDW interactions and full particle-mesh Ewald electrostatics. All simulations were initiated by first minimizing the structure followed by constant volume heating (to 310 K) for 10 ps. This was followed by constanttemperature and constant-pressure (1 atm) dynamics for 50 ns, including a 1 ns equilibration run. Trajectory analysis was done using Visual Molecular Dynamics (VMD).48 The average rootmean-square deviation (rmsd) on all protein atoms was calculated compared to the X-ray structure for the wild-type and mutated protein structures over their entire trajectory simulations using the VMD rmsd trajectory application. All protein structures were visualized, displayed, and analyzed using PyMol.35 Analysis of Protein Matrix Tunnels. Tunnel analyses of the crystal structure of AHb3 and bacterial truncated hemoglobins were conducted using online molecular channel analysis tool MOLE 2.0.49 MD pocket calculations were performed on MD simulated trajectories of AHb3 and AHb3ΔN25.50 The tunnels and pockets were analyzed and visualized using PyMol.35 Some paradigm 3/3 globins were also analyzed similarly and compared with the truncated hemoglobins.

Table 1. Crystallographic Data for Arabidopsis Hemoglobin 3 Crystallographic Data space group wavelength (Å) resolution cell dimensions a = b = c (Å) α = β = γ (deg) no. of unique reflections completeness (%) (last shell) Rsym (%) (last shell) I/σ (last shell) multiplicity (last shell) Refinement no. of reflections (working/test) no. of residues no. of water molecules resolution range (Å) Rcryst (%) Rfree (%) average B factor (Å2) water atoms all atoms rmsd for bond lengths (Å) rmsd for bond angles (Å) Ramachandran plot favored (%) allowed (%) outliers (no. of residues)

P4332 1.54 50.0−2.65 122.08 90 9881 100.0 (100.0) 0.09 (0.64) 14.56 (2.0) 5.68 (5.7) 9020/504 150 37 43.20−2.65 20.27 25.21 40.12 40.20 40.0 0.009 1.44 98.0 99.8 3

reported recently by Reeder et al.26 with a disordered Cterminus, an extended N-terminus, and a 2/2 α-helical sandwich fold similar to those of other bacterial truncated hemoglobins (Figure 1A). As reported earlier, the electron density map at the active site displayed additional density in the distal pocket, best fitted with two water or hydroxide molecules stabilized by H-bonding and electrostatic interactions via GlnE11 in assistance with TrpG8 and TyrCD1. The heme pocket, described in detail by Reeder et al.,26 is endowed with an array of polar and hydrophobic residues, with mainly TyrB10, PheCD1, GlnE11, and TrpG8 as putative ligand stabilizing partners, similar to bacterial truncated hemoglobins.51−55 The primary structure of plant truncated hemoglobins displayed long terminal extensions on either side of the protein core, as reflected in the crystal structures, as well, which aligned with the sequence of bacterial truncated Hbs.11 In contrast to the C-terminal region, the sequence of the Nterminal region is highly conserved across all plant truncated hemoglobins (Supplementary Figure 1). In AHb3, the 25 residues at the N-terminal extension formed a pre-A helix composed of two short helical regions, which lay almost perpendicular to each other. The N-terminal helical extension (pre-A helix) was anchored to the protein core via electrostatic contacts between residues in the pre-A helix and the H-helix, as described before26 (Figure 1B). What was not emphasized in the earlier report of the AHb3 crystal structure26 was the presence of an almost continuous tunnel through the protein matrix connecting the heme distal pocket to the external surface, thereby providing direct access of ligand from solvent to the central heme cavity, and vice versa, as depicted by the flow of water molecules from the



RESULTS Crystal Structure of Arabidopsis Truncated Hemoglobin Displayed a 2/2 Globin Fold, an Extended Polypeptide Terminus, and a Heme Pocket Architecture As Reported Previously, with Distinctive Observations of an Open Tunnel, a C-Terminal Orientation, and a “ϕ” Helix. The three-dimensional structure of AHb3 was determined to 2.65 Å resolution by the molecular replacement method. The crystallographic data collection and refinement statistics are summarized in Table 1. The protein was found to be a monomer in the crystallographic asymmetric unit. The overall structure of AHb3 was found to be similar to that D

DOI: 10.1021/acs.biochem.5b01013 Biochemistry XXXX, XXX, XXX−XXX

Article

Biochemistry

Figure 1. Structural features of Arabidopsis hemoglobin 3 (AHb3). (A) The X-ray crystallographic structure of AHb3 displays a 2/2 globin fold with pentacoordinated heme and an extended N-terminus. (B) Electrostatic interactions anchoring the N-terminal helical extension of AHb3 to the globin core. (C) The surface representation of AHb3 displays an open cavity occupied by an array of water molecules from the external surface to the heme pocket. (D) Overlay of the crystal structure of AHb3 (blue) with the I-TASSER-modeled structure (green) display extended and exposed C-termini primarily constituted of loop regions. (E) Superposition of the I-TASSER-modeled structure of AHb3 (blue) with Hell’s gate HbIV (red) showing similar C-terminal tails. (F) Cartoon representation of the characteristic “ϕ” helix, present between helices E and F hosting Tyr85 and Lys89 for interaction with heme propionates and certain other distal/proximal pocket residues.

II truncated hemoglobins, AHb3 retains Phe at both positions E14 and E15 (Table 2), similar to Mycobacterium trHbN, suggestive of a similar regulatory role. While the density of 25

solvent to the heme (Figure 1C). Such an open tunnel may influence the binding of the ligand to the truncated hemoglobins. Interestingly, unlike its homologues from group E

DOI: 10.1021/acs.biochem.5b01013 Biochemistry XXXX, XXX, XXX−XXX

Article

Biochemistry

stabilize bound gaseous ligands like O2 or CO, as well, influencing kinetics. Reeder et al.26 considered hydroxyl ligand in their crystal structure based on the optical spectrum of AHb3 and the nature of the acid−alkali transition of the ferric globin monitored by probing the difference spectra at 424 and 406 nm. For comparative purposes, similar investigations were performed here, and the absorbance spectra of AHb3 in the buffer that crystallized the protein in the report presented here and in the buffer that crystallized the globin in ref 26, were seen to be distinctly different in both the Soret wavelength and the Q-bands (Figure 2D). The blue shift in the Soret peak (413 nm) in the crystallization condition of Reeder et al. compared to ours (409 nm) clearly indicated the possibility of hydroxyl as the ligand in the former. The presence of a shoulder at ∼620 nm (closer to 630 nm) in our globin spectrum also indicates a higher probability of aquo-met Hb. The pKa between the Fe3+H2O and Fe3+-OH transition of wild-type ferric AHb3 under our experimental conditions was found to be ∼7.8 (Figure 2E), unlike the value of 7.17 determined by Reeder et al.,26 supporting the inclusion of hydroxyl ligand in the earlier structure and water in the present structure. Structural Conservation among Plant and Bacterial Truncated Hemoglobins. Despite significant variation in the amino acid sequence, comparative structural analysis of AHb3 with the bacterial truncated hemoglobins exhibited conservation across the family. Figure 3 displays the structural overlay of AHb3 with representative crystal structures of bacterial truncated hemoglobins from each class of the family. The average rmsd value with respect to AHb3 varies from 3.3 Å for group I to 0.97 Å for group II to 3.8 Å for group III. Clearly, the tertiary structure of AHb3 was more similar with that of members of group II truncated hemoglobins with a rmsd ranging from 0.7 to 1.2. The only major difference was the presence of the extra terminal extensions in AHb3, of which the C-terminus significantly protruded from the core domain (Figure 3). A similar kind of N- and C-terminal extensions has previously been reported for some bacterial truncated hemoglobins (MtrHbN and HGbIV) and protoglobins, suggesting their evolutionary significance.56−58 AHb3 shares seven conserved residues with the bacterial truncated Hbs in the heme pocket at topological positions: B9, B10, CD1, E7, E11, E14, and F8 (Figure 3). Except HGbIV, residues lining the distal pocket of AHb3 were conserved across members of the trHbO group (Table 2), with minor differences in their spatial orientation (Figure 3), which may lead to variation in ligand binding regulation mechanisms. Comparative analyses of tunnel systems (well-documented for truncated hemoglobins in the literature by Bolognesi et al.59,60) in the crystal structure of AHb3 and the bacterial truncated hemoglobins as well as some established 3/3 globins showed significant differences in their topology (Supplementary Figure 2) across the groups, suggestive of diverse ligand migration pathways for entry and/or exit, which probably evolved to play the physiological role of a particular globin in the context in which it functions. The classical 3-on-3 plant globins like soybean leghemoglobin (Figure 2A) display a wellconserved ligand migration route via the E7 His gate and associate on the distal side of the heme pocket, required for their role in oxygen transport. However, alternate tunnel systems exist in dimeric plant nonsymbiotic hemoglobins. For example, we had shown in an earlier study8 that the 3-on-3 globin AHb1 has a novel tunnel connecting the distal heme

Table 2. Residues Lining the Active Site of Plant and Bacterial Truncated Hemoglobins PDB entrya

B10

CD1

E7

E11

E14

E15

G8

4C44 1IDR 1DLY 1NGK 2BKM 4NK1 1UX8 21G3

Tyr Tyr Tyr Phe Tyr His Tyr Tyr

Phe Phe Phe Phe Phe Phe Phe Phe

Ala Leu Gln Ala Thr Ser Gln His

Gln Gln Gln Leu Gln Arg Gln Ile

Phe Phe Ala Phe Phe Asp Phe Phe

Phe Phe Phe Leu Leu Phe Leu Trp

Trp Val Val Trp Trp Trp Ala Trp

a

4C44, A. thaliana AHb3; 1IDR, Mycobacterium tuberculosis trHbN; 1DLY, Chlamydomonas eugamentos trHb; 1NGK, M. tuberculosis trHbO; 2BKM, Geobacillus stearothermophilus trHb; 4NK1, Hell’s gate globin IV; 1UX8, Bacillus subtilis trHb; 2IG3, Campylobacter jejuni trHb.

residues at the C-termini was found missing in the diffraction data, the I-TASSER (ab initio)-modeled structure of C-termini predicted predominantly loop regions, whose flexibility might have precluded proper X-ray diffraction (Figure 1D). A comparison of the crystal structure of AHb3 with the Cterminal inclusive model demonstrated the N- and C-terminal appendages to be external to the core and available for intramolecular or intermolecular interactions (Figure 1A,B,D). The conformation of the predicted C-terminus was found to be similar to that of the I-helix of Hell’s gate hemoglobin (PDB entry 4NK1) (Figure 1E), suggesting a similar yet unknown role.56 Another distinctive feature observed in the present crystal structure (Figure 1F) was the presence of an extra “ϕ” helix between residues 84 and 89 (between helices E and F), with the helix positioning Tyr85 and Lys89 for interaction with heme propionate (Figure 2D). This feature is characteristic of group II truncated hemoglobins. The AHb3 Crystal Structure Reveals Coordinated Heme Pocket Water Molecules. The crystal structure of AHb3 reported here was compared to the one reported recently,26 and both structures displayed common signatures with a similar fold, and characteristics as mentioned above, with similar active site architecture (Figure 2A). The major difference lay in the presence of different putative ligands in the active sites of the two (Figure 2A, red circles), which were believed to be water molecules in the former but hydroxyl ion in the latter (Figure 2B). Considering the resolution of the crystal structure and the electron densities in the distal heme pocket, both hydroxide and water molecules could probably fit, and ambiguity cannot be ruled out. However, our structure best fitted with two water molecules. Two hydroxyls in the distal pocket did not seem likely in the absence of any literature evidence of the same. On the contrary, the presence of ordered water molecules in the heme pocket has been reported previously for other heme proteins, including myoglobin and heme oxygenase. One of the water molecules was 2.06 Å from the iron atom and displayed unambiguous electron density, indicating coordination at the heme iron (Figure 2C). Water molecules found in the heme pockets of hemoglobins are usually more than 6 Å from iron; AHb3 thus presents a novelty in its use of water molecules in the heme pocket. The hydroxyl group in the previously reported crystal structure seems also to be coordinated to the heme iron at a similar distance, acting as a ligand. The water/hydroxide molecules seemed to favor a network of H-bonding interactions (Figure 2B,C), which might F

DOI: 10.1021/acs.biochem.5b01013 Biochemistry XXXX, XXX, XXX−XXX

Article

Biochemistry

Figure 2. Water molecules are coordinated in the distal pocket of the AHb3 crystal structure. (A) Overlay of the two crystal structures that displays no significant differences in the backbone conformation or overall fold: blue for PDB entry 4C44 and green for PDB entry 4C0N. (B) Magnified view of the heme pockets in the two structures, with PDB entry 4C44 displaying two water molecules forming a H-bonding network. In PDB entry 4C0N, a hydroxyl ligand was observed. (C) Fo − Fc electron density map in the distal site of AHb3 (4C44) best fitted with a water molecule tightly coordinated to the heme iron in the active site. (D). Comparative spectral imprints of ferric AHb3 wild-type protein under the crystallization conditions of 4C44 (black) and 4CON (red). (E) pH titration of ferric AHb3 displaying the acid−alkaline transition of met AHb3. The observed pKa between aqua and hydroxyl derivatives was found to be ∼7.8.

on the protein surface.58,59 The long tunnel (∼20 Å) connects the region between the AB and GH corners to the distal site, whereas the short tunnel branch (∼8 Å) holds the regions between the G- and H-helices to the heme. The tunnel is composed of mainly hydrophobic residues with a tunnel volume of 330−360 Å3.58,59 Similar to other bacterial truncated hemoglobins, AHb3 hosts a two-branch protein matrix tunnel

pocket of both the monomers. Such tunnels in nsHbs may function as an intermediate docking site for the incoming ligand that may also support NO dioxygenase activity. The tunnel system in bacterial truncated hemoglobins is however complex and is composed of two orthogonal branches centered at the distal face of the heme (Bolognesi et al. and Supplementary Figure 2). The tunnel opens up at two distinct accessible sites G

DOI: 10.1021/acs.biochem.5b01013 Biochemistry XXXX, XXX, XXX−XXX

Article

Biochemistry

Figure 3. Structural comparison of AHb3 with bacterial truncated hemoglobins. Overlay of the crystal structure of AHb3 (blue) with group I (Mycobacterium trhbN; yellow), group II (Mycobacterium trhbO; green), and group III (C. jejuni trHbP; orange) truncated hemoglobins that display conserved globin fold architecture. The extended N-termini of Mtb trHbN and AHb3 were observed distinctly. The magnified view of the heme pockets of the globin displays conserved side chains that differ only in their spatial orientation.

system. However, the branches seem to exist on only one side of the heme center. The residues lining the tunnel are mainly hydrophobic with a tunnel volume of ∼400−700 Å3. Such differences suggest that AHb3 may not have a role in O2 transport or NO dioxygenase function via these tunnels. Deletion of the N- and C-Terminal Extensions in Arabidopsis Truncated Hemoglobin. To understand the functional and structural relevance of the N- and C-terminal extensions of AHb3, we generated truncated versions of AHb3 lacking 25 residues from the N-terminus (AHb3-Δ25N), the Cterminus (AHb3-Δ25C), or both (AHb3-Δ25NΔ25C). All the deleted variants expressed as soluble red protein, suggesting that the mutants were capable of holding the heme. However, the expression yields of AHb3-Δ25N and AHb3-Δ25NΔ25C were very low and were frequently observed to undergo facile aggregation and precipitation during downstream processing. Quaternary State of AHb3 and Its Mutants in Solution. HPLC Analysis Indicated AHb3 Is a Dimeric Globin. It was necessary to inspect the quaternary state of the protein in solution, because such states affect the function of globins. HPLC analysis revealed AHb3 to exhibit an elution time equivalent to that of a protein with a molecular mass of ∼40 kDa, indicating that the globin is a dimer (Figure 4). The

deletion of 25 residues from the C-terminus resulted in a HPLC profile that indicated the mutant protein to have a molecular mass of ∼29 kDa, indicating an equilibrium mixture of monomeric and dimeric populations (Figure 4A). However, AHb3-Δ25N and AHb3-Δ25NΔ25C derivatives displayed protein peaks corresponding to ∼90 kDa, suggesting the presence of the proteins in the higher oligomeric state (Figure 4A). The absence of the N-terminus makes the globin sticky and aggregation-prone, as also suggested by the broad HPLC elution peaks and the associated shoulders (Figure 4A). A Chemical Cross-Linking Assay Validated the Dimeric Nature of AHb3. To further confirm the effect of the polypeptide terminal deletions on the quaternary structure of AHb3, we performed a chemical cross-linking assay. A glutaraldehyde cross-linking assay is one of the methods widely used for obtaining preliminary information about the quaternary association of proteins.62 Purified proteins were incubated with varying concentrations of glutaraldehyde with subsequent separation by SDS−PAGE (12%). It was observed that AHb3 predominantly existed in the dimeric state (Figure 4B), while AHb3-Δ25C existed as an equilibrium mixture of monomer and dimer in solution (Figure 4B), with a higher population of the monomeric state. AHb3-Δ25N and AHb3H

DOI: 10.1021/acs.biochem.5b01013 Biochemistry XXXX, XXX, XXX−XXX

Article

Biochemistry

Figure 4. Analysis of the oligomeric state of AHb3 and its truncated derivatives. (A) Chromatographic elution profiles of AHb3 (green), AHb3Δ25C (blue), AHb3-Δ25N (magenta), and AHb3-Δ25NΔ25C (cyan) from the HPLC gel filtration column. The graph displays the absorbance at 280 nm as a function of retention time in minutes. In the absence of the N-terminus, the globin appears to have attained an oligomeric state. Proteins of known molecular masses were used as markers for reference (I, ribonuclease A, ∼14 kDa; II, carbonic anhydrase, ∼29 kDa; III, ovalbumin, ∼45 kDa; IV, conalbumin, ∼76 kDa). (B and C) The oligomerization potential of AHb3 and AHb3-Δ25C was also assessed using glutaraldehyde as a cross-linking agent followed by electrophoresis on a 12% SDS−polyacrylamide gel. A concentration-dependent cross-linking assay was performed for (B) wild-type AHb3 and (C) AHb3-Δ25C using varied concentrations of glutaryldehyde ranging from 0.1 to 1%. While AHb3 is predominantly in its dimeric state, AHb3-Δ25C represented a mixed population of monomer and dimer with a higher population of the monomeric state. (D) Surface representation of dimeric AHb3 in crystallographic symmetry. (E) Close-up of the crystallographic contacts between the two monomers in the dimeric interface that includes H-bonding and electrostatic interactions between residues in the H-helix, F-helix, and G-helix.

Δ25NΔ25C, however, precipitated heavily in the presence of the cross-linker and hence did not migrate successfully on SDS−PAGE. Nonetheless, this observation indicated the innate ability of these mutants to form higher-order oligomers. Due to the high aggregation potential observed for the doubly truncated mutant, AHb3-Δ25NΔ25C, it was not investigated

further in detail. The N-terminus thus played a distinct role in maintaining AHb3 in a dimeric state. Crystallographic Evidence of Dimerization. The crystal structure of AHb3 is a monomer in the crystallographic asymmetric unit, but a stable dimeric assembly was observed in the crystallographic symmetry (Figure 4C). This was also I

DOI: 10.1021/acs.biochem.5b01013 Biochemistry XXXX, XXX, XXX−XXX

Article

Biochemistry Table 3. Spectral (Soret peak) and Ligand Binding Kinetic Parameters for AHb3 and Its Mutants O2 and CO (gaseous ligand) Binding 3+

ferric (Fe ) AHb3 wt AHb3-Δ25N AHb3-Δ25C myoglobin soybean Lba AHb1 AHb2 rice Hb1

408 411 413 408 408 411 412 409

2+

ferrous (Fe )

nm nm nm nm nm nm nm nm

430 426 424 434 434 424 424 430

nm nm nm nm nm nm nm nm

2+

Fe -CO 421 421 418 417 417 417 417 421

Soret peak (nm)

nm nm nm nm nm nm nm nm

k′CO (μM−1 s−1)

Fe3+-O2

a

408 411 413 408 411 412 409

a

411 nm 0.013 (0.014 ) 412 nm 0.016 411 nm 0.014 416 nm 0.51 417 nm 16b 416 nm 0.55a 416 nm 22a 412 nm 72c Azide Binding

Q band (nm)

0.28 (0.2 ) 0.28 0. 27 17 130b 74a 86a 68c

Soret peak (nm)

ferric (Fe3+) AHb3 wt AHb3-Δ25N AHb3-Δ25C myoglobin AHb1 AHb2 rice Hb1

k′O2 (μM−1 s−1)

a

419 417 414 421 418 418 411

a

0.35 (0.3 ) 0.40 0.31 15 5.6b 0.12a 0.14a 0.04c

KO2 (μM−1) 0.8 0.7 0.87 1.1 23b 616 614 1800

Q band (nm) k′azide (mM−1 s−1)

Fe3+-azide

542/580 540/578 540/570 503 and minor peaks 542/578 540/578 532/580

kO2 (s−1)

540/570 541/568 538/562 540/568 540/571 540/571 542/557

8 15 0.06 3 0.03 0.04 0.11

Reported kinetic value from ref 11. bReported kinetic value from ref 75. cReported kinetic value from ref 76.

Figure 5. Spectroscopic properties of AHb3 and its truncated mutants. Overlay of Fe3+ (red), Fe2+ (blue), and Fe2+-CO (green) absorbance spectra of (A) wild-type AHb3, (B) AHb3-Δ25C, and (C) AHb3-Δ25N displaying a single broad Q-band at 554 nm in the ferrous state for wild-type AHb3 and AHb3-Δ25C while split Q-bands (535 and 561 nm) for AHb3-Δ25N. The spectral characteristics indicate potential hexacoordination of heme iron by external/internal ligand in the N-terminal deletion mutant in the ferrous form. pH titration curves displaying the acid−alkaline transition in (D) AHb3-Δ25C and (E) AHb3-Δ25N, with an apparent pK of ∼8.2.

between the N-terminus and H-helix (Figure 4C) as reported in detail by Reeder et al.26 and hence not described here. The N- and C-Terminal Extensions Do Not Influence the General Biochemical Properties of AHb3, but the NTerminal Extension Induces Hexacoordination at the Heme Iron. CD Spectroscopy. In an attempt to address the

predicted by PISA analysis (http://www.ebi.ac.uk/pdbe/prot_ int/pistart.html)61 encompassing the N-terminal α-helical extension in the dimeric interface. The total buried surface area in dimeric AHb3 was predicted to be 4841.8 Å2, which is on the order of that seen in other dimeric globins (data not shown). The dimerization interface involved interaction J

DOI: 10.1021/acs.biochem.5b01013 Biochemistry XXXX, XXX, XXX−XXX

Article

Biochemistry

Figure 6. Structural features of MD-simulated AHb3 and its mutant (AHb3-ΔN25). (A) Superposition of the backbone for snapshots collected across the trajectories, with the wild-type crystal structure (dark blue) as the starting point. Salt bridges stabilizing the pre-A-helix to the protein core in (B) the crystal structure of AHb3 and (C) the MD-simulated structure remain unaffected. (D) Stereoview of the superposed crystal structure (dark blue) and snapshots of MD-simulated trajectories of pre-A-helix-deleted AHb3. The similar backbone conformations indicated that the deletion of the N-terminus had no significant influence on the overall globin fold or tertiary structure.

the heme−polypeptide interaction was assessed by measuring autoxidation and heme dissociation rates for AHb3 and its mutants. All the mutants displayed autoxidation rates (AHb3Δ25C, 0.91 h−1; AHb3-Δ25N, 0.96 h−1) comparable to that of the wild-type protein (0.92 h−1) and much higher than that of wild-type Mb (0.141 h−1),28 indicating that AHb3 might not play a role in oxygen transport or storage (Supplementary Figure 3A). Both AHb3-Δ25N and AHb3-Δ25C readily lose heme in the upper organic layer, which is similar to the case for

influence of the extended N- and C-termini, if any, on the overall structural integrity and folding of the protein, CD spectra for the wild type and its mutants were recorded in the far-UV region (data not shown). Similar CD spectra for the wild-type and mutants proteins, characteristic of all α-helical folds, preclude the role of both N- and C-terminal extension in the overall structural integrity or folding of the protein. Influence of the Mutants on Autoxidation and Heme Dissociation Rates. The influence of the terminal deletions on K

DOI: 10.1021/acs.biochem.5b01013 Biochemistry XXXX, XXX, XXX−XXX

Article

Biochemistry

Figure 7. Dynamics of the active site residues in MD-simulated structures of AHb3 and AHb3-ΔN25. (A) Structural dynamics of the residues in the active site of the wild-type simulated structure (pink) (B) Pre-A-helix-deleted MD simulated trajectories (green), in comparison to the AHb3 crystal structure (blue). It is evident that in the absence of the N-terminus, AHb3 displays significant changes in the orientations of the key side chains, some of which are indicated by arrows.

wild-type protein (Figure 2E), pH titration of ferric AHb3ΔN25 and AHb3-ΔC25 (Figure 5 D,E) displayed a pKa of ∼8.2 between the Fe3+-H2O and Fe3+-OH transition, indicating a higher proportion of distal pocket hydration in these globins. Polypeptide Terminal Deletions Do Not Influence the Gaseous Ligand Binding Kinetics of AHb3 but Affect Azide Binding Kinetics. UV−vis absorption spectra of O2and CO-bound forms of AHb3-Δ25N and AHb3-Δ25C were similar in nature to that of wild-type AHb3, suggesting that these termini did not affect equilibrium ligand binding. To investigate the effect on the rate of ligand binding, kinetic measurements of O2 and CO binding were also taken using stopped-flow and laser flash photolysis. The rates determined for O2 and CO binding for AHb3, AHb3-Δ25N, and AHb3Δ25C are listed in Table 3 along with the rates for several other globins. All the kinetic parameters determined for the mutants were similar to those of the wild-type globin, indicating that the hexacoordination observed for AHb3-Δ25N was too weak to influence kinetics and was probably brought about by water molecules in the pocket. Further kinetic investigation in this regard is necessary to delineate such an influence, but it is obvious that the corresponding N- and C-terminal deletions had no influence on the O2/CO binding rate constants of AHb3. In fact, it is interesting to note that the kinetic parameters of AHb3 and its mutants are unique compared to all others (Table 3) in that both the association and dissociation rates constants are similar and quite low. The two rate constants are also similar for Mb but much higher in numbers. Moreover, the oxygen binding affinity of AHb3 was also similar to that of Mb unlike those of the other plant globins, though the low oxygen dissociation rate constant would preclude a role in oxygen transport unlike Mb. On the other hand, the azide ligand binding kinetics presented an interesting finding (Table 3). This ligand, the prototype for ferric globins, binds with AHb3 at rates similar to that of Mb but unlike that of AHb1, AHb2, or rice Hb1, which are hexacoordinate globins. The N-terminal mutant binds twice

the wild-type protein when a heme dissociation assay was performed (data not shown). The mutants displayed dissociation rates similar to that of the wild-type protein (Supplementary Figure 3B), indicating that the truncations did not significantly alter the heme−polypeptide interaction. Stability Studies. The influence of the terminal truncations on the conformational stability of the globin fold was evaluated by exposing the wild-type protein and its derivatives to different denaturing conditions such as pH and temperature. The change in the Soret peak in response to different pHs ranging from 2 to 11 was monitored, and no significant difference was observed in their profiles or midpoints of transitions (Supplementray Figure 4). Similarly, CD measurements at 222 nm suggested that pH influenced the changes in secondary structure to the same extent in all three proteins (Supplementray Figure 4). Thermal stability studies of the wild type and its deleted variants displayed a similar thermal denaturation profile as well when probed by CD (Supplementray Figure 4), indicating that these deletions did not affect the overall stability of the protein. UV−Vis Spectroscopy. UV−visible absorbance spectra for AHb3 and its derivatives were measured from 260 to 700 nm. Absorbance spectra of AHb3, AHb3-Δ25C, and AHb3-Δ25N showed Soret peak at 408, 409, and 412 nm, respectively (Table 3 and Figure 5). The Q-bands for AHb3 and AHb3Δ25C were similar, displaying a single asymmetric absorbance peak at 556 nm in ferrous form and a weak and broad absorbance band near 635 nm and between 500 and 550 nm for the ferric protein, typical of pentacoordinate Hbs (Figure 5A,B). On the other hand, AHb3-Δ25N showed distinct splitting in the visible region Q-bands in ferrous form (535 and 561 nm), suggesting the presence of some internal or external ligand occupying the active site of ferrous protein making the heme iron hexacoordinated (Figure 5C). It is thus evident that the N-terminus of AHb3 helps the globin to attain its pentacoordinate heme iron state that is just sufficiently hydrated by water molecules (Figure 2A), its absence giving rise to hexacoordinate heme iron. Interestingly, unlike that of L

DOI: 10.1021/acs.biochem.5b01013 Biochemistry XXXX, XXX, XXX−XXX

Article

Biochemistry

Figure 8. Structural illustration of the tunnel systems in MD-simulated trajectories. Representation of dynamic tunnels (green mesh) for (A) wildtype AHb3 and (B) AHb3-Δ25N. The absence of the pre-A-helix in the mutant changes the topology of tunnels.

AHb3-ΔN25, across the trajectories (Figure 7). Similar opening and closing events for PheE15 was previously reported for M. tuberculosis hemoglobin N, where it has been implicated to regulate access of the ligand to the heme pocket and in turn its function.63 To further gain insights into the dynamics of ligand migration cavities imposed by deleting the pre-A-helix in AHb3, extensive cavity analysis was conducted for both the simulated structures in comparison to the crystal structure. Our analysis showed that the deletion of the pre-A-helix introduced significant alterations into the ligand diffusion pathway by providing alternate pathways to the heme active site (Figure 8). Such dynamics in ligand migration cavities upon deletion of the pre-A-helix suggests its functional significance in regulating ligand entry and/or exit.

as fast. However, the C-terminal mutant hardly binds to azide, as also observed for AHb1 and AHb2. This indicates that the Cterminal extension of AHb3 indeed plays a very significant role in ligand−protein (and maybe protein−protein) interaction, especially in the ferric state. MD Simulation Studies for Investigating the Roles of the N-Terminus of AHb3. To explore the role of the pre-Ahelix in protein dynamics, extensive MD simulations of the wild type (with 25 residues of the C-terminus deleted) and AHb3 with a deleted pre-A-helix were conducted. The X-ray crystal structure of AHb3 was used as a starting point for the simulations. The stability of the trajectories was calculated in comparison to the crystal structure of AHb3. The rmsd of the last snapshot of the trajectories and the X-ray crystallographic structure varied from 1 to 1.3 Å for the wild type and from 1.9 to 2.6 Å for AHb3-Δ25N. For wild-type simulations, analysis of the MD trajectories showed that the relative movement of the protein backbone does not change significantly with a stable conformation of the pre-A-helix across the trajectories (Figure 6A). Salt bridges between residues in the pre-A-helix and the H-helix remained unaffected throughout the simulations, which may contribute to its conformational rigidity (Figure 6B,C). In comparison to the wild-type simulations, minor changes in the protein backbone was observed when the pre-A-helix was deleted from the protein core (AHb3-Δ25N) (Figure 6D). Significant changes in the dynamics of protein structure were observed around E- and F-helices and CD and EF loop regions (Figure 6D). Fluctuations in these regions could be important for the regulation of access of the ligand to the heme pocket by modulating ligand migration cavities and the heme pocket environment. The changes might even be of further significance in the quaternary interactions of the mutant protein because AHb3-Δ25N was observed experimentally to oligomerize and aggregate. To identify the local changes in the heme pocket architecture through the course of simulations, series of snapshots collected at different intervals for wild-type and AHb3-Δ25N simulations were compared to that of the crystal structure of AHb3. Interestingly, deletion of the pre-A-helix introduced a noticeable change in the dynamics of residues shielding the heme pocket. Major fluctuations were observed for GlnE11, TyrB10, and PheE15 (Figure 7). In comparison to the wild type, several transitions of open and closed events were observed for Phe36, Phe74 (E14), Phe75 (E15), and Phe143 in



DISCUSSION Ever since the discovery of nonsymbiotic hemoglobins in plants, a large fraction of research has been directed to gain deeper insights into their physiological role. A plethora of information has been generated for class I and class II nonsymbiotic hemoglobins; these have been implicated in performing diverse physiological functions such as oxygen sensing, NO detoxification, etc.16,64 Detailed crystallographic investigations of class I nonsymbiotic hemoglobins supported NO dioxygenase activity.8 In contrast to class I and class II nonsymbiotic hemoglobins, research on plant truncated hemoglobins is still in its infancy. Detailed structural and functional investigation for this class of protein is therefore the need of the hour to understand their biological role. Our crystallographic investigation of truncated hemoglobin from the model plant A. thaliana attempted to provide new insights into its biological role from a structural perspective. The tertiary structure of the protein displayed a bacterial-like 2/2 α-helical truncated globin fold with pentacoordinated heme. In unison, Reeder et al.26 in 2014 had also determined and reported the crystal structure of Arabidopsis truncated hemoglobin at 1.7 Å. Detailed structural comparison of the two structures displayed identical globin folds and active site architectures. In the case of the previous structure, however, the heme iron was coordinated to a hydroxyl ligand, whose role in biological function or ligand binding is not yet understood. Spectroscopically, however, we observed AHb3 to be pentacoordinated in solution, and only M

DOI: 10.1021/acs.biochem.5b01013 Biochemistry XXXX, XXX, XXX−XXX

Article

Biochemistry

intrinsically disordered proteins27,71 and predicted for other globins with such extensions. The survival of the C-terminus in AHb3 over the course of evolution suggests that it must have an important physiological role to play, though it seems to have a minimal influence on the general structural properties of the globins. We show here that the C-terminus influences ligand binding in the ferric state and hence indeed plays a role in ligand−protein interaction. The floppy C-terminus may help in regulating the entry and/or exit to the open water cavity. The fact that the C-terminus influences the monomer−dimer equilibrium of AHb3 also indicates its role in protein−protein interaction. The fact that it protrudes out of the core of the globin fold [modeled structure (Figures 1E and 3)] also lends support to its putative role as an interacting domain. These observations present an immense scope of investigation in AHb3. Flanking IDRs of plant proteins are often correlated with the presence of multiple PTM sites in these regions.27,72 In agreement with these findings, in silico analysis of Arabidopsis truncated hemoglobins indeed displayed the occurrence of acetylation and other post-translational modification sites in both disordered N- and C-termini (data not shown). In fact, Johnson et al.73 recently showed the presence of acetylation sites at the N-terminal peptides of Chlamydomonas truncated hemoglobin 1, further supporting the hypothesis, though the physiological significance of these modifications is still not well understood. We have performed MD simulations with supportive experimental evidence to address the role of these extended termini in Arabidopsis truncated hemoglobin. A detailed biochemical and biophysical fingerprint of AHb3-Δ25N and AHb3-Δ25C obliterated their role in maintaining the globin fold and structural integrity of the protein. Both the deleted mutants displayed similar gaseous ligand binding fingerprints, further precluding their role in gaseous ligand binding. Interestingly, AHb3-Δ25N displayed distinct splitting in the visible region of Q-bands in the ferrous form, suggesting the presence of some ligand occupying the active site of ferrous protein forming a weak covalent bond. The kinetic investigation, however, indicated that the active site was occupied by hydroxyl ligand or weakly coordinated water molecules and not any heme pocket side chain. In spite of the presence of an open heme pocket, the association rate constant for ligand binding was found to be low, which could be due to the tightly bound water molecule in the active site of the protein acting as a barrier to ligand binding. A large number of potential amino acid side chains in the distal pocket (Results) may help in such stabilization. At the same time, once a ligand binds, because of the strong stabilization of the ligand by distal pocket residues, the dissociation rate constant of the bound ligand was also found to be low. These factors provide AHb3 with a unique kinetic characteristic. Even though the association of the ligand in the ferric state was found to be low in the C-terminally deleted mutant, the N- and C-terminally deleted mutants did not influence ligand binding of the gaseous ligands. The gaseous ligand binding may play a more significant role under abnormal conditions like stress. The C-terminus thus may play a role in preventing overhydration of the active site, probably by blocking tunnels in the protein matrix that facilitate facile solvent entry of certain ligands like azide that bind in the ferric state. The most significant influence of the N-terminus was its effect on the quaternary state of AHb3. In its absence, the

N-terminal deletion resulted in a spectrum typical of hexacoordinated globin. The active site of AHb3 in the current structure was occupied by two water molecules stabilized by Hbonding and electrostatic interactions with GlnE11 in assistance with TrpG8 and TyrCD1. The spectroscopic property of the globin was thus more in tune with what we observed in the structure presented here. The presence of such loosely bound solvent molecules in the heme pocket, together with the possibility of the presence of hydroxide as shown by Reeder et al.,26 may rationalize the transient observations of different coordination states in the globin reported previously.11 Moreover, these observations indicate that the appearance of absorbance spectra typical of hexacoordinate globins may not always relate to an internal amino acid side chain resulting in hexacoordination; it could simply arise due to the coordination of extrinsic solvent molecules, as seen here for the N-terminal mutant of AHb3. Water molecules or hydroxyl ligand in the heme pocket of AHb3, as described above, and their rearrangement and electrostatic interaction with heme pocket side chains might contribute to the biological function of AHb3. Such water molecules have been implicated in the regulation of ligand binding in other heme proteins, including myoglobin, hemeoxygenase, and a few truncated hemoglobins.52 Comparative analysis of the crystal structure of AHb3 with bacterial truncated hemoglobins in the investigation presented here indeed displayed high structural conservation across the family with well-conserved distal active site residues, suggesting a similar evolutionary trail and a similar function. The bacterial truncated hemoglobins were implicated as playing a role in nitric oxide detoxification, oxygen or nitric oxide sensing, and long-term ligand storage,10,65,66 and the same might be true for plant truncated hemoglobins, as well. In contrast to bacterial truncated hemoglobins, plant truncated hemoglobins displayed extra N- and C-terminal appendages flanking the truncated globin core. The structural and functional significance of these appendages in plant hemoglobins in not known, though they have been implicated in ligand sensing, nitric oxide scavenging, and interaction with a partner molecule in vivo in protoglobins and other globins.56,67−69 The N-terminal α-helical extension is highly conserved across plant truncated hemoglobins (Supplementary Figure 1), suggestive of its physiological significance. We have shown that this extension mainly influences the quaternary structure of the globin and its distal pocket hydration. This, thus, presents a unique feature common to all novel plant hemoglobins. In the investigation presented here, the Cterminus was found to influence the ligand (azide) binding properties of ferric AHb3 to a larger extent than the Nterminus; however, MD simulations suggested the probable role of the N-terminus in regulating NO detoxification analogous to M. tuberculosis truncated hemoglobin N. Computational analysis of the protein sequence of AHb3 and other plant truncated hemoglobins predicted both N- and Ctermini to be structurally disordered, which could explain the missing density of C-termini in the crystal structure. Such proteins with intrinsically disordered regions (IDRs) represent a broad class of polypeptides, mostly inhabiting eukaryotic genomes.27,70,71 These proteins are widespread in the plant kingdoms and are predicted to play important role in several regulatory and environmental signals.27 The disordered Cterminus of AHb3 may be essential for the recognition and binding of interacting protein partners as is known for several N

DOI: 10.1021/acs.biochem.5b01013 Biochemistry XXXX, XXX, XXX−XXX

Biochemistry



globin cannot maintain itself in the dimeric state and tends to be polymeric in nature. The N-terminal extension thus might play a crucial role in dimeric interaction of the globin. Dimerization of globins has returned as a recurring theme for many of the novel globins and must be important for their physiological function and regulation.23,74 Comparative analysis of MD-simulated trajectories of wildtype AHb3 and AHb3 with deleted N-termini displayed major fluctuations around E- and F-helices. A distinct change in the conformation of residues lining the heme pocket was observed when the pre-A-helix was deleted from the protein core. Several transitions of open and closed events were observed for Phe36, Phe74 (E14), Phe75 (E15), and Phe143. Similar shifting of the open and closed conformations of these residues was previously reported for M. tuberculosis hemoglobin N, where these have been implicated in the regulation of access of the ligand to the heme pocket and in turn its function.63,69 The importance of the PheE15 gate in modulating the NOD function of MtrHbN is well-documented, in which it plays a key role in mediating the access of NO to the oxygenated protein. Analysis of ligand migration dynamics across the simulated models displayed significant divergence in the migration routes that may influence its biological role. In agreement to the previous leads from Mycobacterium trHbN, the pre-A-helix could be essential for maintaining NOD activity in AHb3, though detailed experimental validation is required to further validate this role.



ACKNOWLEDGMENTS

The Macromolecular Crystallography Unit (MCU), Institute Instrumentation Centre, IIT Roorkee (PK), is acknowledged for X-ray diffraction experiments. The Central Instrumentation Facility at University of Delhi South Campus is appreciated for the help with circular dichroism. Dr. Monica Sundd’s laboratory at the National Institute of Immunology (New Delhi, India) is acknowledged for help with HPLC.



ABBREVIATIONS Hb, hemoglobin; wt, wild type; nsHbs, nonsymbiotic hemoglobins; trHbs, truncated hemoglobins; ptrHbs, plant truncated hemoglobins; 3/3Hb, 3-on-3 globin; 2/2Hb, 2-on-2 globin; AHb1, Arabidopsis hemoglobin 1; AHb2, Arabidopsis hemoglobin 2; AHb3, Arabidopsis hemoglobin 3; MD, molecular dynamics; CD, circular dichroism; HPLC, highperformance liquid chromatography; IDRs, intrinsically disordered regions; NO dioxygenase, nitric oxide dioxygenase.



REFERENCES

(1) Hardison, R. C. (1996) A brief history of hemoglobins: plant, animal, protist, and bacteria. Proc. Natl. Acad. Sci. U. S. A. 93, 5675− 5679. (2) Hardison, R. (1998) Hemoglobins from bacteria to man: evolution of different patterns of gene expression. J. Exp Biol. 201, 1099−1117. (3) Kundu, S., Trent, J. T., 3rd, and Hargrove, M. S. (2003) Plants, humans and hemoglobins. Trends Plant Sci. 8, 387−393. (4) Arredondo-Peter, R., Hargrove, M. S., Moran, J. F., Sarath, G., and Klucas, R. V. (1998) Plant hemoglobins. Plant Physiol 118, 1121− 1125. (5) Sowa, A. W., Guy, P. A., Sowa, S., and Hill, R. D. (1999) Nonsymbiotic haemoglobins in plants. Acta Biochim Pol 46, 431−445. (6) Garrocho-Villegas, V., Gopalasubramaniam, S. K., and Arredondo-Peter, R. (2007) Plant hemoglobins: what we know six decades after their discovery. Gene 398, 78−85. (7) Garrocho-Villegas, V., Bramaniam, S. K., and Arredondo-Peter, R. (2007) Plant Hemoglobins:What we know six decades after their discovery. Gene 398, 78−85. (8) Mukhi, N., Dhindwal, S., Uppal, S., Kumar, P., Kaur, J., and Kundu, S. (2013) X-ray crystallographic structural characteristics of Arabidopsis hemoglobin I and their functional implications. Biochim. Biophys. Acta, Proteins Proteomics 1834, 1944−1956. (9) Wittenberg, J. B., Bolognesi, M., Wittenberg, B. A., and Guertin, M. (2002) Truncated hemoglobins: a new family of hemoglobins widely distributed in bacteria, unicellular eukaryotes, and plants. J. Biol. Chem. 277, 871−874. (10) Kumar, A., Nag, M., and Basak, S. (2013) Truncated or 2/2 hemoglobins: A new class of globins with novel structure and function. Journal of proteins and proteomics 4, 45−64. (11) Watts, R. A., Hunt, P. W., Hvitved, A. N., Hargrove, M. S., Peacock, W. J., and Dennis, E. S. (2001) A hemoglobin from plants homologous to truncated hemoglobins of microorganisms. Proc. Natl. Acad. Sci. U. S. A. 98, 10119−10124. (12) Trevaskis, B., Watts, R. A., Andersson, C. R., Llewellyn, D. J., Hargrove, M. S., Olson, J. S., Dennis, E. S., and Peacock, W. J. (1997) Two hemoglobin genes in Arabidopsis thaliana: the evolutionary origins of leghemoglobins. Proc. Natl. Acad. Sci. U. S. A. 94, 12230− 12234. (13) Hoy, J. A., Robinson, H., Trent, J. T., 3rd, Kakar, S., Smagghe, B. J., and Hargrove, M. S. (2007) Plant hemoglobins: a molecular fossil record for the evolution of oxygen transport. J. Mol. Biol. 371, 168− 179. (14) Hoy, J. A., and Hargrove, M. S. (2008) The structure and function of plant hemoglobins. Plant Physiol. Biochem. 46, 371−379.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.5b01013. Multiple-sequence alignment of the N-terminal extension of multiple plant truncated hemoglobins from different species (Supplementary Figure 1), a cartoon representation of the ligand migration protein matrix tunnels in a few 3-on-3 and 2-on-2 globins (Supplementary Figure 2), the effect of N- and C-terminal deletions on the physicochemical properties of AHb3 determined by recording the rate of change in autoxidation and heme dissociation of AHb3 and its deleted variants (Supplementary Figure 3), and pH- and temperature-dependent stability profile of wild-type AHb3 with respect to the deleted mutants (Supplementary Figure 4) (PDF)



Article

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Telephone: +91-1124117460. Fax: +91-11-24115270. *E-mail: [email protected]. Funding

Financial aid from the University of Delhi and the Department of Science and Technology, Government of India, under the PURSE program [Dean(R)/2009/868] (J.K. and S.K.) is acknowledged. The University of Delhi is duly acknowledged for R&D funding (DRCH/R&D/2013-14/4155) (J.K. and S.K.). Fellowship support from UGC, Government of India, to N.M. and R.A. and CSIR, Government of India, to S.U. are also appreciated. Notes

The authors declare no competing financial interest. O

DOI: 10.1021/acs.biochem.5b01013 Biochemistry XXXX, XXX, XXX−XXX

Article

Biochemistry (15) Smagghe, B. J., Hoy, J. A., Percifield, R., Kundu, S., Hargrove, M. S., Sarath, G., Hilbert, J. L., Watts, R. A., Dennis, E. S., Peacock, W. J., Dewilde, S., Moens, L., Blouin, G. C., Olson, J. S., and Appleby, C. A. (2009) Review: correlations between oxygen affinity and sequence classifications of plant hemoglobins. Biopolymers 91, 1083−1096. (16) Perazzolli, M., Dominici, P., Romero-Puertas, M. C., Zago, E., Zeier, J., Sonoda, M., Lamb, C., and Delledonne, M. (2004) Arabidopsis nonsymbiotic hemoglobin AHb1 modulates nitric oxide bioactivity. Plant Cell 16, 2785−2794. (17) Hebelstrup, K. H., van Zanten, M., Mandon, J., Voesenek, L. A., Harren, F. J., Cristescu, S. M., Möller, I. M., and Mur, L. A. (2012) Haemoglobin modulates NO emission and hyponasty under hypoxiarelated stress in Arabidopsis thaliana. J. Exp. Bot. 63, 5581−5591. (18) Hebelstrup, K. H., and Jensen, E. O. (2008) Expression of NO scavenging hemoglobin is involved in the timing of bolting in Arabidopsis thaliana. Planta 227, 917−927. (19) Spyrakis, F., Bruno, S., Bidon-Chanal, A., Luque, F. J., Abbruzzetti, S., Viappiani, C., Dominici, P., and Mozzarelli, A. (2011) Oxygen binding to Arabidopsis thaliana AHb2 nonsymbiotic hemoglobin: evidence for a role in oxygen transport. IUBMB Life 63, 355−362. (20) Wang, Y., Elhiti, M., Hebelstrup, K. H., Hill, R. D., and Stasolla, C. (2011) Manipulation of hemoglobin expression affects Arabidopsis shoot organogenesis. Plant Physiol. Biochem. 49, 1108−1116. (21) Vigeolas, H., Huhn, D., and Geigenberger, P. (2011) Nonsymbiotic hemoglobin-2 leads to an elevated energy state and to a combined increase in polyunsaturated fatty acids and total oil content when overexpressed in developing seeds of transgenic Arabidopsis plants. Plant Physiol. 155, 1435−1444. (22) Hebelstrup, K. H., Hunt, P., Dennis, E., Jensen, S. B., and Jensen, E. O. (2006) Hemoglobin is essential for normal growth of Arabidopsis organs. Physiol. Plant. 127, 157−166. (23) Kakar, S., Sturms, R., Tiffany, A., Nix, J. C., DiSpirito, A. A., and Hargrove, M. S. (2011) Crystal structures of Parasponia and Trema hemoglobins: differential heme coordination is linked to quaternary structure. Biochemistry 50, 4273−4280. (24) Goodman, M. D., and Hargrove, M. S. (2001) Quaternary structure of rice nonsymbiotic hemoglobin. J. Biol. Chem. 276, 6834− 6839. (25) Hargrove, M. S., Brucker, E. A., Stec, B., Sarath, G., ArredondoPeter, R., Klucas, R. V., Olson, J. S., and Phillips, G. N. (2000) Crystal Structure of a nonsymbiotic plant hemoglobin. Structure 8, 1005− 1014. (26) Reeder, B. J., and Hough, M. A. (2014) The structure of a class 3 nonsymbiotic plant haemoglobin from Arabidopsis thaliana reveals a novel N-terminal helical extension. Acta Crystallogr., Sect. D: Biol. Crystallogr. 70, 1411−1418. (27) Kurotani, A., Tokmakov, A. A., Kuroda, Y., Fukami, Y., Shinozaki, K., and Sakurai, T. (2014) Correlations between predicted protein disorder and post-translational modifications in plants. Bioinformatics 30, 1095. (28) Uppal, S., Salhotra, S., Mukhi, N., Zaidi, F. K., Seal, M., Dey, S. G., Bhat, R., and Kundu, S. (2015) Significantly enhanced heme retention ability of myoglobin engineered to mimic the third covalent linkage by nonaxial histidine to heme (vinyl) in synechocystis hemoglobin. J. Biol. Chem. 290, 1979−1993. (29) Otwinowski, Z., and Minor, W. (1997) Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307−326. (30) Potterton, E., McNicholas, S., Krissinel, E., Cowtan, K., and Noble, M. (2002) The CCP4 molecular-graphics project. Acta Crystallogr., Sect. D: Biol. Crystallogr. 58, 1955−1957. (31) Collaborative Computational Project, No. 4 (1994) The CCP4 suite: programs for protein crystallography. Acta Crystallogr., Sect. D: Biol. Crystallogr. 50, 760−763. (32) Murshudov, G. N., Skubak, P., Lebedev, A. A., Pannu, N. S., Steiner, R. A., Nicholls, R. A., Winn, M. D., Long, F., and Vagin, A. A. (2011) REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr., Sect. D: Biol. Crystallogr. 67, 355−367.

(33) Winn, M. D., Murshudov, G. N., and Papiz, M. Z. (2003) Macromolecular TLS refinement in REFMAC at moderate resolutions. Methods Enzymol. 374, 300−321. (34) Emsley, P., and Cowtan, K. (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr., Sect. D: Biol. Crystallogr. 60, 2126−2132. (35) DeLano, W. L. (2002) The PyMOL molecular graphics system, DeLano Scientific, San Carlos, CA. (36) Bernstein, F. C., Koetzle, T. F., Williams, G. J., Meyer, E. F., Jr., Brice, M. D., Rodgers, J. R., Kennard, O., Shimanouchi, T., and Tasumi, M. (1977) The Protein Data Bank: a computer-based archival file for macromolecular structures. J. Mol. Biol. 112, 535−542. (37) Olson, J. S. (1981) Stopped-flow, rapid mixing measurements of ligand binding to hemoglobin and red cells. Methods Enzymol. 76, 631−651. (38) Hargrove, M. S., and Olson, J. S. (1996) The stability of holomyoglobin is determined by heme affinity. Biochemistry 35, 11310−11318. (39) Hargrove, M. S. (2000) A flash photolysis method to characterize hexacoordinate hemoglobin kinetics. Biophys. J. 79, 2733−2738. (40) Draghi, F., Miele, A. E., Travaglini-Allocatelli, C., Vallone, B., Brunori, M., Gibson, Q. H., and Olson, J. S. (2002) Controlling ligand binding in myoglobin by mutagenesis. J. Biol. Chem. 277, 7509−7519. (41) Kundu, S., Snyder, B., Das, K., Chowdhury, P., Park, J., Petrich, J. W., and Hargrove, M. S. (2002) The leghemoglobin proximal heme pocket directs oxygen dissociation and stabilizes bound heme. Proteins: Struct., Funct., Genet. 46, 268−277. (42) Kundu, S., and Hargrove, M. S. (2003) Distal heme pocket regulation of ligand binding and stability in soybean leghemoglobin. Proteins: Struct., Funct., Genet. 50, 239−248. (43) Teale, F. W. (1959) Cleavage of the haem-protein link by acid methylethylketone. Biochim. Biophys. Acta 35, 543. (44) Hargrove, M. S., Singleton, E. W., Quillin, M. L., Ortiz, L. A., Phillips, G. N., Jr., Olson, J. S., and Mathews, A. J. (1994) His64(E7)– > Tyr apomyoglobin as a reagent for measuring rates of hemin dissociation. J. Biol. Chem. 269, 4207−4214. (45) Yang, J., Yan, R., Roy, A., Xu, D., Poisson, J., and Zhang, Y. (2014) The I-TASSER Suite: protein structure and function prediction. Nat. Methods 12, 7−8. (46) Phillips, J. C., Braun, R., Wang, W., Gumbart, J., Tajkhorshid, E., Villa, E., Chipot, C., Skeel, R. D., Kale, L., and Schulten, K. (2005) Scalable molecular dynamics with NAMD. J. Comput. Chem. 26, 1781− 1802. (47) Brooks, B. R., Brooks, C. L., 3rd, Mackerell, A. D., Jr., Nilsson, L., Petrella, R. J., Roux, B., Won, Y., Archontis, G., Bartels, C., Boresch, S., Caflisch, A., Caves, L., Cui, Q., Dinner, A. R., Feig, M., Fischer, S., Gao, J., Hodoscek, M., Im, W., Kuczera, K., Lazaridis, T., Ma, J., Ovchinnikov, V., Paci, E., Pastor, R. W., Post, C. B., Pu, J. Z., Schaefer, M., Tidor, B., Venable, R. M., Woodcock, H. L., Wu, X., Yang, W., York, D. M., and Karplus, M. (2009) CHARMM: the biomolecular simulation program. J. Comput. Chem. 30, 1545−1614. (48) Humphrey, W., Dalke, A., and Schulten, K. (1996) VMD: visual molecular dynamics. J. Mol. Graphics 14, 27−38. (49) Sehnal, D., Svobodova Varekova, R., Berka, K., Pravda, L., Navratilova, V., Banas, P., Ionescu, C. M., Otyepka, M., and Koca, J. (2013) MOLE 2.0: advanced approach for analysis of biomacromolecular channels. J. Cheminf. 5, 39. (50) Schmidtke, P., Bidon-Chanal, A., Luque, F. J., and Barril, X. (2011) MDpocket: open-source cavity detection and characterization on molecular dynamics trajectories. Bioinformatics 27, 3276−3285. (51) Droghetti, E., Nicoletti, F. P., Bonamore, A., Boechi, L., Arroyo Manez, P., Estrin, D. A., Boffi, A., Smulevich, G., and Feis, A. (2010) Heme pocket structural properties of a bacterial truncated hemoglobin from Thermobifida fusca. Biochemistry 49, 10394−10402. (52) Ouellet, Y. H., Daigle, R., Lague, P., Dantsker, D., Milani, M., Bolognesi, M., Friedman, J. M., and Guertin, M. (2008) Ligand binding to truncated hemoglobin N from Mycobacterium tuberculosis P

DOI: 10.1021/acs.biochem.5b01013 Biochemistry XXXX, XXX, XXX−XXX

Article

Biochemistry is strongly modulated by the interplay between the distal heme pocket residues and internal water. J. Biol. Chem. 283, 27270−27278. (53) Ouellet, H., Milani, M., LaBarre, M., Bolognesi, M., Couture, M., and Guertin, M. (2007) The roles of Tyr(CD1) and Trp(G8) in Mycobacterium tuberculosis truncated hemoglobin O in ligand binding and on the heme distal site architecture. Biochemistry 46, 11440−11450. (54) Ilari, A., Kjelgaard, P., von Wachenfeldt, C., Catacchio, B., Chiancone, E., and Boffi, A. (2007) Crystal structure and ligand binding properties of the truncated hemoglobin from Geobacillus stearothermophilus. Arch. Biochem. Biophys. 457, 85−94. (55) Ouellet, Y., Milani, M., Couture, M., Bolognesi, M., and Guertin, M. (2006) Ligand interactions in the distal heme pocket of Mycobacterium tuberculosis truncated hemoglobin N: roles of TyrB10 and GlnE11 residues. Biochemistry 45, 8770−8781. (56) Jamil, F., Teh, A. H., Schadich, E., Saito, J. A., Najimudin, N., and Alam, M. (2014) Crystal structure of truncated haemoglobin from an extremely thermophilic and acidophilic bacterium. J. Biochem. 156, 97−106. (57) Ciaccio, C., Pesce, A., Tundo, G. R., Tilleman, L., Bertolacci, L., Dewilde, S., Moens, L., Ascenzi, P., Bolognesi, M., Nardini, M., and Coletta, M. (2013) Functional and structural roles of the N-terminal extension in Methanosarcina acetivorans protoglobin. Biochim. Biophys. Acta, Proteins Proteomics 1834, 1813−1823. (58) Milani, M., Pesce, A., Ouellet, Y., Ascenzi, P., Guertin, M., and Bolognesi, M. (2001) Mycobacterium tuberculosis hemoglobin N displays a protein tunnel suited for O2 diffusion to the heme. EMBO J. 20, 3902−3909. (59) Boechi, L., Marti, M. A., Milani, M., Bolognesi, M., Luque, F. J., and Estrin, D. A. (2008) Structural determinants of ligand migration in Mycobacterium tuberculosis truncated hemoglobin O. Proteins: Struct., Funct., Genet. 73, 372−379. (60) Pesce, A., Milani, M., Nardini, M., and Bolognesi, M. (2008) Mapping heme-ligand tunnels in group I truncated(2/2) hemoglobins. Methods Enzymol. 436, 303−315. (61) Krissinel, E., and Henrick, K. (2007) Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774−797. (62) Fadouloglou, V. E., Kokkinidis, M., and Glykos, N. M. (2008) Determination of protein oligomerization state: two approaches based on glutaraldehyde crosslinking. Anal. Biochem. 373, 404−406. (63) Oliveira, A., Singh, S., Bidon-Chanal, A., Forti, F., Marti, M. A., Boechi, L., Estrin, D. A., Dikshit, K. L., and Luque, F. J. (2012) Role of PheE15 gate in ligand entry and nitric oxide detoxification function of mycobacterium tuberculosis truncated hemoglobin N. PLoS One 7, e49291. (64) Seregelyes, C., and Dudits, D. (2003) Phytoglobins and nitric oxide: new partners in an old signalling system in plants. Acta Biol. Hung. 54, 15−25. (65) Couture, M., Yeh, S. R., Wittenberg, B. A., Wittenberg, J. B., Ouellet, Y., Rousseau, D. L., and Guertin, M. (1999) A cooperative oxygen-binding hemoglobin from Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. U. S. A. 96, 11223−11228. (66) Ouellet, H., Ouellet, Y., Richard, C., Labarre, M., Wittenberg, B., Wittenberg, J., and Guertin, M. (2002) Truncated hemoglobin HbN protects Mycobacterium bovis from nitric oxide. Proc. Natl. Acad. Sci. U. S. A. 99, 5902−5907. (67) Freitas, T. A., Saito, J. A., Hou, S., and Alam, M. (2005) Globincoupled sensors, protoglobins, and the last universal common ancestor. J. Inorg. Biochem. 99, 23−33. (68) Saito, J. A., Wan, X., Lee, K. S., Hou, S., and Alam, M. (2008) Globin-coupled sensors and protoglobins share a common signaling mechanism. FEBS Lett. 582, 1840−1846. (69) Lama, A., Pawaria, S., Bidon-Chanal, A., Anand, A., Gelpi, J. L., Arya, S., Marti, M., Estrin, D. A., Luque, F. J., and Dikshit, K. L. (2009) Role of Pre-A motif in nitric oxide scavenging by truncated hemoglobin, HbN, of Mycobacterium tuberculosis. J. Biol. Chem. 284, 14457−14468.

(70) Oldfield, C. J., and Dunker, A. K. (2014) Intrinsically disordered proteins and intrinsically disordered protein regions. Annu. Rev. Biochem. 83, 553−584. (71) van der Lee, R., Buljan, M., Lang, B., Weatheritt, R. J., Daughdrill, G. W., Dunker, A. K., Fuxreiter, M., Gough, J., Gsponer, J., Jones, D. T., Kim, P. M., Kriwacki, R. W., Oldfield, C. J., Pappu, R. V., Tompa, P., Uversky, V. N., Wright, P. E., and Babu, M. M. (2014) Classification of intrinsically disordered regions and proteins. Chem. Rev. 114, 6589−6631. (72) Kurotani, A., Yamada, Y., Shinozaki, K., Kuroda, Y., and Sakurai, T. (2015) Plant-PrAS: a database of physicochemical and structural properties and novel functional regions in plant proteomes. Plant Cell Physiol. 56, e11. (73) Johnson, E. A., Rice, S. L., Preimesberger, M. R., Nye, D. B., Gilevicius, L., Wenke, B. B., Brown, J. M., Witman, G. B., and Lecomte, J. T. (2014) Characterization of THB1, a Chlamydomonas reinhardtii truncated hemoglobin: linkage to nitrogen metabolism and identification of lysine as the distal heme ligand. Biochemistry 53, 4573− 4589. (74) Boffi, A., and Chiancone, E. (2004) Evaluating cooperativity in dimeric hemoglobins. Methods Enzymol. 379, 55−64. (75) Hargrove, M. S., Barry, J. K., Brucker, E. A., Berry, M. B., Phillips, G. N., Jr., Olson, J. S., Arredondo-Peter, R., Dean, J. M., Klucas, R. V., and Sarath, G. (1997) Characterization of recombinant soybean leghemoglobin a and apolar distal histidine mutants. J. Mol. Biol. 266, 1032−1042. (76) Arredondo-Peter, R., Hargrove, M. S., Sarath, G., Moran, J. F., Lohrman, J., Olson, J. S., and Klucas, R. V. (1997) Rice hemoglobins. Gene cloning, analysis, and O2-binding kinetics of a recombinant protein synthesized in Escherichia coli. Plant Physiol 115, 1259−1266.

Q

DOI: 10.1021/acs.biochem.5b01013 Biochemistry XXXX, XXX, XXX−XXX