- Email: [email protected]
Redox-dependent conformational transition of catalytic domain of protein disulfide isomerase indicated by crystal structure-based molecular dynamics simulation
Accepted Manuscript Title: Redox-dependent conformational transition of catalytic domain of protein disulfide isomerase indicated by crystal structure-based molecular dynamics simulation Author: Koya Inagaki Tadashi Satoh Satoru G. Itoh Hisashi Okumura Koichi Kato PII: DOI: Reference:
S0009-2614(14)00958-0 http://dx.doi.org/doi:10.1016/j.cplett.2014.11.017 CPLETT 32623
To appear in: Received date: Revised date: Accepted date:
23-9-2014 8-11-2014 10-11-2014
Please cite this article as: K. Inagaki, T. Satoh, S.G. Itoh, H. Okumura, K. Kato, Redoxdependent conformational transition of catalytic domain of protein disulfide isomerase indicated by crystal structure-based molecular dynamics simulation, Chem. Phys. Lett. (2014), http://dx.doi.org/10.1016/j.cplett.2014.11.017 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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*Graphical Abstract (pictogram) (for review)
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Highlights ・ Crystal structures of PDI b′–a′ region in its redox states were both determined. ・ MD simulation of a′ domain indicated its redox-dependent conformational transition.
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・ Insight into the redox-dependent domain rearrangement mechanism was provided.
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Redox-dependent conformational transition of catalytic domain of protein disulfide isomerase indicated by crystal structure-based molecular dynamics simulation
Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori,
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Koya Inagaki1,2, Tadashi Satoh1,3, Satoru G. Itoh4,5, Hisashi Okumura4,5, and Koichi Kato1,2,6,7,*
2
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Mizuho-ku, Nagoya 467-8603, Japan;
Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, 5-1
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Higashiyama, Myodaiji, Okazaki, Aichi 444-8787, Japan;
JST, PRESTO, 3-1 Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan;
4
Department of Theoretical and Computational Molecular Science, Institute for Molecular Science,
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3
National Institutes of Natural Sciences, 38 Nishigo-Naka, Myodaiji, Okazaki, Aichi 444-8585, Japan; Department of Structural Molecular Science, The Graduate University for Advanced Studies,
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Okazaki, Aichi 444-8585, Japan;
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5
Division of Biomolecular Science, Institute for Molecular Science, National Institutes of Natural
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Sciences, 5-1 Higashiyama, Myodaiji, Okazaki, Aichi 444-8787, Japan 7
Department of Functional Molecular Science, The Graduate University for Advanced Studies,
Okazaki, Aichi 444-8585, Japan
*
Addresses for correspondence: Koichi Kato, Ph.D., Okazaki Institute for Integrative Bioscience
and Institute for Molecular Science, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki, Aichi 444-8787, Japan, Tel. +81-564-59-5225, Fax: +81-564-59-5224, e-mail: [email protected]
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Abstract Protein disulfide isomerase is a multidomain protein operating as an essential folding catalyst.
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The b′ and a′ domains of this enzyme exhibit a domain rearrangement depending on the redox states of the a′ domain, which is coupled with an open–closed conformational change of substrate-binding
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hydrophobic surface. Here we performed crystallographic analysis along with molecular dynamics
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simulations to study the structural mechanisms underlying this domain rearrangement. Based on our data, we propose that oxidization of the a′ active site induces conformational changes in its
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b′-interacting segments, which is concealed by crystal packing, resulting in segregation of these two
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domains.
Keywords
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Protein disulfide isomerase
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Crystal structure, Domain rearrangement, Molecular dynamics simulation, Multidomain protein,
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1. Introduction Sophisticated biofunctions exerted by proteins are coupled with their dynamic
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conformational transition, as exemplified by allosteric enzymes composed of multiple domains
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and/or subunits [1]. In the present study, we characterized conformational change of protein disulfide isomerase (PDI), which serves as a folding catalyst for disulfide-containing proteins in
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the endoplasmic reticulum (ER) [2]. This enzyme consists of four thioredoxin-like domains, a, b, b′, and a′, and an acidic C-terminal extension [3-5]. The a and a′ domains possess a cysteine
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pair in a WCGHCK active-site motif that is directly involved in thiol/disulfide exchange
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reactions [6], whereas b and b′ domains contain no active site motif. Furthermore, substrate binding is primarily mediated by b′ and a′ domains, which are connected through a flexible
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linker [7,8]. By the combined use of small-angle X-ray scattering and spectroscopic techniques,
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we have previously demonstrated that these two domains exhibited an intramolecular domain
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rearrangement, depending on the redox state of the active site cysteine pair in the a′ domain, which was coupled with an open–closed conformational change of the substrate-binding hydrophobic surface spanning across these domains [8,9]. Based on these findings, we propose a functional mechanism by which the unfolded substrate is connected to the exposed hydrophobic surface of the oxidized, open form of the PDI b′–a′ domains, which transfer the disulfide bridge to the substrate and consequently become the reduced, closed form releasing the folded substrate with the disulfide bond. The 3D structures of full-length and truncated forms of PDI from human, yeast, and thermophilic fungus have been determined thus far [6,8,10-14]. However, the structural mechanisms underlying the redox-dependent rearrangement of the substrate-binding domains of 4
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PDI remain unexplored at the atomic level. In this study, we attempted to address this issue by combined use of crystallographic analysis and molecular dynamics (MD) simulations to
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characterize potential conformational change in thermophilic fungal PDI. Our data indicated
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that redox-dependent conformational transition occurs in the PDI a′ domain at its interface with the b′ domain, thereby providing structural insight into the molecular mechanism underlying the
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2. Materials and Methods
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functional domain rearrangement of this protein.
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2.1. Crystallization, X-ray data collection, and structure determination The purification of reduced and oxidized b′–a′ domains (residues 208−449) from
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Humicola insolens was performed as previously described [8,15]. For crystallization, the
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reduced and oxidized proteins, dissolved in 10 mM Tris-HCl (pH 8.0) containing 10 mM
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dithiothreitol (DTT) or 0.1 mM oxidized glutathione, respectively, were concentrated to 15 mg/mL. Crystals of the reduced and oxidized forms of this protein were grown in a buffer containing 36% PEG2000 monomethyl ether, 100 mM sodium acetate (pH 4.6), and 200 mM ammonium sulfate or 30% PEG4000, 100 mM Tris-HCl (pH 8.5), and 200 mM magnesium chloride, respectively, on incubation at 20°C. All the diffraction images were processed using HKL2000 software [16]. The crystal parameters are shown in Table 1. The crystal structure of the reduced b′–a′ domains was solved by the molecular replacement method, using the program MOLREP [17] with the NMR structures of separated b′ and a′ domains (PDB codes: 2KP2 and 2KP1, respectively) [8] as search models. In addition, the crystal structure of the oxidized b′–a′ domains was solved by molecular replacement using 5
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the truncated b′ and a′ domains generated from our determined crystal structure of the reduced b′–a′ domains as search models. Further model building and refinement were performed using
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COOT [18] and REFMAC5 [19]. The stereochemical quality of the refined coordinates was
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assessed using RAMPAGE [20]. The refinement statistics are summarized in Table 1. The
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molecular graphics were prepared using PyMOL (http://www.pymol.org/).
2.2. Molecular dynamics simulation
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The 3D models of H. insolens PDI a′ domain (residues 334−449) derived from the crystal
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structures of oxidized and reduced b′–a′ domains were used as initial structures as follows: for the reduced state, the X-ray structure of the a′ domain in the reduced form of the b′–a′ segments
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was isolated and then energy-minimized by the conjugate gradient method in a vacuum after
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complementing the missing hydrogen atoms. We also utilized the X-ray structure of the
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oxidized b′–a′ domains for MD simulation of the oxidized structure of the a′ domain. However, in the crystal structure, the electron density of Ala449 was ambiguous. Hence, the fragment composed of Ala448 and Ala449 was grafted from the crystal structure of the reduced form to that of the oxidized form by superimposing Ala448 through rigid translations and rotations. Subsequently, the hydrogen atoms were positioned onto the models, which were then energy-minimized in a vacuum. The energy-minimized structures were put into explicit water solvent. The system, under oxidized and reduced conditions comprised 4,094 and 4,093 water molecules, respectively, and one molecule of each corresponding form of the PDI a′ domain, along with two sodium ions Na+ as counter ions. The GEMB (Generalized-Ensemble Molecular Biophysics) program [21], developed by 6
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one of the authors (H.O.) was used to perform the MD simulations. The AMBER parm99SB force field [22] was used for the PDI a′ domain, and the TIP3P rigid-body model [23] was used
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for the water molecules. Temperature was controlled at 303 K by the Nosé-Hoover thermostat
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[24-26]. Pressure was controlled at 0.1 MPa by the Andersen barostat [27]. The symplectic
quaternion scheme was used for the rigid-body water molecules [28,29]. A cubic unit cell was
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used with periodic boundary conditions. The electrostatic potential was calculated by the particle mesh Ewald method [30]. Cutoff distance was 12 Å for the Lennard–Jones potential.
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Reversible multiple time scale molecular dynamics techniques [31] were applied for time
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development. The time step was considered to be Δt = 0.5 fs for the bonding interactions of the protein atoms and Δt = 2.0 fs for the non-bonding interactions of the protein atoms and that
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between the protein atoms and solvent molecules, and Δt = 4.0 fs for the interaction between the
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solute molecules. We first performed MD simulations for 10 ns for each structure as an
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equilibration process. We then performed 140 ns simulations for data collection to calculate physical quantities. The
root-mean-square
RMSF i
q
i COG
i qCOG
fluctuation
2
(RMSF)
of
residue
i
is
defined
by
i represents time average and qCOG is the
, where
i
coordinate vector of the center of geometry for residue i. To calculate qCOG , the coordinates of i i backbone C, O, N, and Cα atoms were employed, and qCOG is given by qCOG
1 Ni i qj . N i j 1
Here, Ni is the number of the employed atoms in residue i. The vector qij is the coordinate vector of the employed atom j in residue i and is obtained by superposition between the simulation conformation and the initial conformation. 7
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3. Results and Discussion
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3.1. Crystal structures of reduced and oxidized forms of PDI b′–a′ domains
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To provide structural insights into the molecular mechanisms of the redox-dependent
domain rearrangement of PDI, we first attempted to determine the crystal structures of the b′–a′
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domains (composed of residues 208−449) of H. insolens PDI in its reduced and oxidized states. The final model of the reduced form of the b′–a′ domains, refined to a resolution of 1.85 Å, had
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an Rwork of 23.8% and Rfree of 27.8% (Table 1). The crystal belonged to space group P1, with four
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molecules per asymmetric unit. The structures of molecules A−D were highly similar to each other, with a RMSD value of 0.25−0.47 Å for superimposed 242 Cα atoms. In contrast, the
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oxidized form of the b′–a′ domains belonged to space groups P31, and diffracted up to 3.30-Å
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resolution. In the crystals, three molecules were contained per asymmetric unit. The final model
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of the oxidized form had an Rwork of 22.1% and Rfree of 31.3% (Table 1). The structures of molecules A−C can be closely superimposed with RMSD of 0.11−0.13 Å for 242 Cα atoms. Molecules A in the reduced and oxidized crystal structures, which have lower average B values (Table 1), were used for the comparative analysis and are described below. The crystal structures of the oxidized and reduced b′–a′ domains showed that each
domain adopts a typical thioredoxin fold, as expected (Figure 1). The b′ and a′ domains are composed of 1-α1-2-α2-3-α3-4-5-α4 and 1-α1-2-α2-3-4-5-α3 arrangements, respectively. Our crystal (reduced form, chain A) and previously reported NMR structures of b′ and a′ domains (PDB codes: 2KP2 and 2KP1, respectively) [8] are essentially identical with the RMSD of 1.14 and 1.26 Å for 108 and 111 Cα atoms, respectively. The structural variation is 8
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mainly observed in loop segments. Crystal structures of the b′ and a′ domains from fungal (reduced form, chain A) and human (reduced form, PDB code: 4EKZ) [10] PDIs can be closely
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superimposed with RMSD of 1.89 and 1.03 Å for 89 and 104 Cα and atoms, respectively,
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indicating their common thioredoxin fold.
The spatial arrangement of b′ and a′ domains was compared between the reduced and
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oxidized states. Although the domain arrangement between b′–a′ domains was profoundly affected by crystal packing, the two domains, connected through the flexible linker, exhibited
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“open conformation” in the oxidized state (Figure. 1). In contrast, unlike the crystal structure
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reported for human PDI in its reduced state [10,11], the present crystal structure of the fungal PDI b′–a′ domains continued to adopt an open conformation, even in the reduced state (Figure.
d
1). In the crystal lattice, the potential domain–domain interactions could be hindered by the
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crystallographically neighboring molecule (Supporting Information Figure S1). Moreover,
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different crystal packing results in different domain–domain arrangements. Consequently, the b′–a′ interaction, bridged by an active-site tryptophan side-chain observed in the closed conformation of human PDI [10,11], could not be found in our crystal structures. To identify key residues in the catalytic a′ domain responsible for the redox-dependent
domain rearrangement observed in solution, we compared the crystal structures of the catalytic a′ domain between the reduced and oxidized states. However, both structures of a′ domain were essentially identical, with an RMSD value of 0.23 Å for 112 Cα atoms (Figure 2a). Even in the active site, no significant structural change was observed between them. Unlike the crystal structure of human reduced PDI (Figure 2b) [10,11], tryptophan flipping at the active site was not observed in the thermophilic fungal PDI b′–a′ crystal structures (Figure 2a). These 9
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crystallographic results underscore the importance of structural characterization of this protein
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3.2. Molecular dynamics of catalytic a′ domain of PDI in its redox states
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in the solution environment.
To study the structural mechanisms underlying the conformational transition of PDI in
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solution, we focused on the early stage of this process, triggered by the redox change of the a′ domain active site. Hence, we performed MD simulation analysis on the basis of the 3D
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structure frameworks of PDI a′ domain provided by our crystallographic analyses.
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Conformational fluctuations during the MD simulations were compared between the reduced and oxidized forms (Figure 3 and Supporting Information Figures S2–S5). Upon
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disulfide bond formation in the a′ active site, the surrounding residues exhibited smaller
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fluctuations. The flipping out of the Trp side-chain at the active site (Trp364), which was
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observed in the crystal structure of human PDI in its reduced state [10,11], was not pronounced in the present MD simulation. Notably, the oxidized form of the a′ domain exhibited remarkable fluctuations in the 3-4 loop and its spatial proximity, including the 1 helix, in comparison with the reduced form. These regions are involved in the crystal packing or the interaction with the b′ domain observed in the crystal structure of human PDI [10,11]. These results indicated that conformations of these a′ segments are intrinsically different between the oxidized and reduced states but become similar, as observed in the previously reported (human) [10,11] and currently determined (thermophilic fungal) crystal structures of the b′–a′ domains because of their intermolecular or interdomain interactions.
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Therefore, we propose a mechanistic model of the redox-dependent conformational change of the substrate-binding domains of PDI: the oxidization of the active site of the a′
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domain induces conformational changes in its b′-interacting segments comprising the 3-4
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loop and the 1 helix, resulting in the segregation of these two domains. To validate this model,
it is necessary to perform experimental characterization of isolated a′-domain conformation; this
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study is currently underway in our groups.
The molecular processes involving functional conformational changes are often characterized
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based on crystallographic snapshots of the proteins before and after the conformational
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transitions. However, crystal structures of flexible multidomain proteins can be considerably influenced by crystal packing. On the basis of crystallographic data, this study focuses on the
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crystal packing.
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utility of MD simulation for its ability to characterize protein conformation in the absence of
Acknowledgements
We thank Drs Takeshi Hiromoto (Nagoya City University, Japan), Tsunehiro Mizushima
(University of Hyogo, Japan), and Masato Kawasaki (Photon Factory, KEK, Japan) for the help with X-ray data collection, and Drs. Osamu Asami and Tsumoto Kajino of Toyota Central Research and Development Laboratory for providing the cDNA of the fungal PDI. We are grateful to the beamline staff of Osaka University BL44XU at SPring-8 (Harima, Japan) and BL13B1 at NSRRC (Hsinchu, Taiwan) for providing the data collection facilities and for their support. We also acknowledge the Research Center for Computational Science, National Institutes of Natural Sciences (Okazaki, Japan) for providing the computational resources for 11
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the MD simulation. This work was supported in part by JSPS KAKENHI (Grant Numbers
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24770102, 25121730 to T.S., and 25102008 to K.K.) and by the Okazaki ORION project.
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Accession numbers. The coordinates and structural factors of the crystal structures of the
reduced and oxidized b′–a′ domains of H. insolens PDI have been deposited in the Protein Data
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Bank under accession numbers (3WT1) and (3WT2), respectively.
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References
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cr
(2012) 646. [2] B. Wilkinson, H.F. Gilbert, Biochimica et biophysica acta 1699 (2004) 35.
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[3] J.C. Edman, L. Ellis, R.W. Blacher, R.A. Roth, W.J. Rutter, Nature 317 (1985) 267. [4] R.B. Freedman, P. Klappa, L.W. Ruddock, EMBO reports 3 (2002) 136.
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[7] P. Klappa, L.W. Ruddock, N.J. Darby, R.B. Freedman, The EMBO journal 17 (1998) 927. [8] O. Serve, Y. Kamiya, A. Maeno, M. Nakano, C. Murakami, H. Sasakawa, Y. Yamaguchi, T.
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Harada, E. Kurimoto, M. Yagi-Utsumi, T. Iguchi, K. Inaba, J. Kikuchi, O. Asami, T. Kajino, T. Oka, M. Nakasako, K. Kato, Journal of molecular biology 396 (2010) 361.
te
[9] M. Nakasako, A. Maeno, E. Kurimoto, T. Harada, Y. Yamaguchi, T. Oka, Y. Takayama, A. Iwata, K. Kato, Biochemistry 49 (2010) 6953. C. Wang, W. Li, J. Ren, J. Fang, H. Ke, W. Gong, W. Feng, C.C. Wang, Antioxidants
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& redox signaling 19 (2013) 36.
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chemistry 287 (2012) 1139.
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Asami, T. Kajino, K. Kato, Journal of biomolecular NMR 36 Suppl 1 (2006) 44. [16]
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A. Vagin, A. Teplyakov, J Appl Crystallogr 30 (1997) 1022.
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P. Emsley, B. Lohkamp, W.G. Scott, K. Cowtan, Acta Crystallogr D Biol Crystallogr
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G.N. Murshudov, A.A. Vagin, E.J. Dodson, Acta Crystallogr D Biol Crystallogr 53
S.C. Lovell, I.W. Davis, W.B. Arendall, 3rd, P.I. de Bakker, J.M. Word, M.G. Prisant,
J.S. Richardson, D.C. Richardson, Proteins 50 (2003) 437.
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H. Okumura, Proteins 80 (2012) 2397.
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V. Hornak, R. Abel, A. Okur, B. Strockbine, A. Roitberg, C. Simmerling, Proteins 65
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Chem Phys 116 (2002) 8649.
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Figure legends
Figure 1. Overall structures of oxidized and reduced b′–a′ domains of PDI. Ribbon models of the oxidized and reduced b′–a′ domains of PDI from H. insolens (a and b) and Homo sapiens (c and d). The secondary structure numbers and respective domains are indicated. The active-site cysteine residues are shown in sphere models.
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Figure 2. Comparison of active site of the a′ domain in the redox states. Close-up views of
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the active sites of PDI a′ domain from (a) H. insolens (oxidized; marine blue, and reduced; red)
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and (b) H. sapiens (oxidized; cyan, and reduced; magenta), colored as in Figure 1. Active-site cysteine and adjoining tryptophan residues are shown in stick models. The proposed residue
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(Arg300) involving domain–domain interaction in human PDI b′ domain [10,11], and the structurally corresponding residue (Ala269) in fungal PDI b′ domain are also represented in
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stick models.
Figure 3. MD simulation of the a′ domain in the redox states. Plots of root-mean-square
d
fluctuation (RMSF) during MD simulation of PDI a′ domain in oxidized and reduced states are
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shown in (a) and (b), respectively. The secondary structure elements are indicated above the
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plots. In the right panels, mapping of residues showing higher RMSF values on the PDI a′ crystal structures during the MD simulation are shown. The residues are colored in red with color gradients indicating the degree of the fluctuation. The unanalyzed residues from the b′ domain and flexible linker are shown in gray.
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Page 16 of 20
Table
Table 1. Data collection and refinement statistics for reduced and oxidized b′-a′ domain of PDI b′-a′ domain of PDI (oxidized)
Crystallographic data P1
P31
Unit cell
65.8/67.8/70.3
96.3/96.3/69.5
105.9/113.1/96.6
90.0/90.0/120.0
Beam line
NSRRC 13B1
SPring-8 BL44XU
Wavelength (Å)
1.00000
0.90000
Resolution (Å)
50-1.85 (1.88-1.85)
50-3.30 (3.36-3.30)
Total/unique reflections
173,477/88,599
62,727/10,825
Completeness (%)
92.3 (81.7)
Rmerge (%)
5.5 (37.8)
I / (I)
26.2 (1.5)
a/b/c (Å) α/β/γ (°)
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Space group
Rwork / Rfree (%)
23.8/27.8
an 8.8 (36.8)
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20.0-1.85
99.9 (100)
d
Resolution (Å)
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Data processing statistics
Refinement statistics
Ac ce pt e
R.m.s. deviations from ideal
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b′-a′ domain of PDI (reduced)
32.0 (6.9)
20.0-3.30 22.1/31.3
Bond lengths (Å)
0.015
0.012
Bond angles (°)
1.67
1.51
Favored
98.6
92.7
Allowed
1.4
7.3
Protein atoms (A/B/C/D)
1,901/1,878/1,883/1,883
1,879/1,879/1,879
Water molecules
262
-
24
-
Protein atoms (A/B/C/D)
38.2/44.9/41.2/41.8
74.6/88.8/93.1
Water molecules
39.0
-
Glycerol molecules
42.8
-
Ramachandran plot (%)
Number of atoms
Glycerol molecules 2
Average B-values (Å )
Page 17 of 20
(b)
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(a) α2 α1
β3
β5
β1 β5
α3
α1
ed
α4
Ac
α3
β2
β5
β4
β3
α4
β5
α4
a′
β1
β5
a′
α1 α2
β3
α2
α3 β2 β4
β2
β5
α4
β4
β3
α3 β5
β1
b′
α1
β5
α1
α3
β1
β4
α2
α3
(d)
α2
α1
β4
b′
ce pt
(c)
β3
β2
a′
b′
β2
β2 β4
α3
β3
α2
α2
β3
M an
β2 β4
β3 β2 β4
β1
b′
α2
α1
β1
α3
β1
cr
i
Figure 1
α1
a′ Page 18 of 20
M an
us
cr
i
Figure 2
(a)
(b)
b′
a′
ed
α2
A269
ce pt
α3
C365
C368
b′
R300
W396
α2
a′
C397 C400
Ac
W364
α3
Page 19 of 20
β1 α1
β2
α2
β3
β4
β5
cr
(a)
i
Figure 3 α3
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6
α2
α1
β1
M an
RMSF (Å)
5 4 3
α3 β3 β2 β4
0
β5 α4
RMSF (Å)
5 4 3
β2
α2
β3
β4
β5
2
RMSF (Å) 3.5 3.0 2.5 2.0 1.5 0.0
α2
α1 β1 β3 α2
β2
α3
β3
β2 α4
1
α1
a′
α3
Ac
β1 α1
ce pt
(b)
β5
b′
330 340 350 360 370 380 390 400 410 420 430 440 450 Residue number
α3
β1
ed
1
β4
β3
α2
2
6
β2
β4
β4
α3 β5
β1
β5 α1
0 330 340 350 360 370 380 390 400 410 420 430 440 450 Residue number
b′
a′ Page 20 of 20
S0009-2614(14)00958-0 http://dx.doi.org/doi:10.1016/j.cplett.2014.11.017 CPLETT 32623
To appear in: Received date: Revised date: Accepted date:
23-9-2014 8-11-2014 10-11-2014
Please cite this article as: K. Inagaki, T. Satoh, S.G. Itoh, H. Okumura, K. Kato, Redoxdependent conformational transition of catalytic domain of protein disulfide isomerase indicated by crystal structure-based molecular dynamics simulation, Chem. Phys. Lett. (2014), http://dx.doi.org/10.1016/j.cplett.2014.11.017 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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*Graphical Abstract (pictogram) (for review)
Page 1 of 20
Highlights ・ Crystal structures of PDI b′–a′ region in its redox states were both determined. ・ MD simulation of a′ domain indicated its redox-dependent conformational transition.
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・ Insight into the redox-dependent domain rearrangement mechanism was provided.
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Page 2 of 20
Redox-dependent conformational transition of catalytic domain of protein disulfide isomerase indicated by crystal structure-based molecular dynamics simulation
Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori,
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1
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Koya Inagaki1,2, Tadashi Satoh1,3, Satoru G. Itoh4,5, Hisashi Okumura4,5, and Koichi Kato1,2,6,7,*
2
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Mizuho-ku, Nagoya 467-8603, Japan;
Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, 5-1
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Higashiyama, Myodaiji, Okazaki, Aichi 444-8787, Japan;
JST, PRESTO, 3-1 Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan;
4
Department of Theoretical and Computational Molecular Science, Institute for Molecular Science,
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3
National Institutes of Natural Sciences, 38 Nishigo-Naka, Myodaiji, Okazaki, Aichi 444-8585, Japan; Department of Structural Molecular Science, The Graduate University for Advanced Studies,
6
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Okazaki, Aichi 444-8585, Japan;
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5
Division of Biomolecular Science, Institute for Molecular Science, National Institutes of Natural
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Sciences, 5-1 Higashiyama, Myodaiji, Okazaki, Aichi 444-8787, Japan 7
Department of Functional Molecular Science, The Graduate University for Advanced Studies,
Okazaki, Aichi 444-8585, Japan
*
Addresses for correspondence: Koichi Kato, Ph.D., Okazaki Institute for Integrative Bioscience
and Institute for Molecular Science, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki, Aichi 444-8787, Japan, Tel. +81-564-59-5225, Fax: +81-564-59-5224, e-mail: [email protected]
2
Page 3 of 20
Abstract Protein disulfide isomerase is a multidomain protein operating as an essential folding catalyst.
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The b′ and a′ domains of this enzyme exhibit a domain rearrangement depending on the redox states of the a′ domain, which is coupled with an open–closed conformational change of substrate-binding
cr
hydrophobic surface. Here we performed crystallographic analysis along with molecular dynamics
us
simulations to study the structural mechanisms underlying this domain rearrangement. Based on our data, we propose that oxidization of the a′ active site induces conformational changes in its
an
b′-interacting segments, which is concealed by crystal packing, resulting in segregation of these two
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domains.
Keywords
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Protein disulfide isomerase
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Crystal structure, Domain rearrangement, Molecular dynamics simulation, Multidomain protein,
3
Page 4 of 20
1. Introduction Sophisticated biofunctions exerted by proteins are coupled with their dynamic
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conformational transition, as exemplified by allosteric enzymes composed of multiple domains
cr
and/or subunits [1]. In the present study, we characterized conformational change of protein disulfide isomerase (PDI), which serves as a folding catalyst for disulfide-containing proteins in
us
the endoplasmic reticulum (ER) [2]. This enzyme consists of four thioredoxin-like domains, a, b, b′, and a′, and an acidic C-terminal extension [3-5]. The a and a′ domains possess a cysteine
an
pair in a WCGHCK active-site motif that is directly involved in thiol/disulfide exchange
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reactions [6], whereas b and b′ domains contain no active site motif. Furthermore, substrate binding is primarily mediated by b′ and a′ domains, which are connected through a flexible
d
linker [7,8]. By the combined use of small-angle X-ray scattering and spectroscopic techniques,
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we have previously demonstrated that these two domains exhibited an intramolecular domain
Ac ce p
rearrangement, depending on the redox state of the active site cysteine pair in the a′ domain, which was coupled with an open–closed conformational change of the substrate-binding hydrophobic surface spanning across these domains [8,9]. Based on these findings, we propose a functional mechanism by which the unfolded substrate is connected to the exposed hydrophobic surface of the oxidized, open form of the PDI b′–a′ domains, which transfer the disulfide bridge to the substrate and consequently become the reduced, closed form releasing the folded substrate with the disulfide bond. The 3D structures of full-length and truncated forms of PDI from human, yeast, and thermophilic fungus have been determined thus far [6,8,10-14]. However, the structural mechanisms underlying the redox-dependent rearrangement of the substrate-binding domains of 4
Page 5 of 20
PDI remain unexplored at the atomic level. In this study, we attempted to address this issue by combined use of crystallographic analysis and molecular dynamics (MD) simulations to
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characterize potential conformational change in thermophilic fungal PDI. Our data indicated
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that redox-dependent conformational transition occurs in the PDI a′ domain at its interface with the b′ domain, thereby providing structural insight into the molecular mechanism underlying the
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2. Materials and Methods
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functional domain rearrangement of this protein.
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2.1. Crystallization, X-ray data collection, and structure determination The purification of reduced and oxidized b′–a′ domains (residues 208−449) from
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Humicola insolens was performed as previously described [8,15]. For crystallization, the
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reduced and oxidized proteins, dissolved in 10 mM Tris-HCl (pH 8.0) containing 10 mM
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dithiothreitol (DTT) or 0.1 mM oxidized glutathione, respectively, were concentrated to 15 mg/mL. Crystals of the reduced and oxidized forms of this protein were grown in a buffer containing 36% PEG2000 monomethyl ether, 100 mM sodium acetate (pH 4.6), and 200 mM ammonium sulfate or 30% PEG4000, 100 mM Tris-HCl (pH 8.5), and 200 mM magnesium chloride, respectively, on incubation at 20°C. All the diffraction images were processed using HKL2000 software [16]. The crystal parameters are shown in Table 1. The crystal structure of the reduced b′–a′ domains was solved by the molecular replacement method, using the program MOLREP [17] with the NMR structures of separated b′ and a′ domains (PDB codes: 2KP2 and 2KP1, respectively) [8] as search models. In addition, the crystal structure of the oxidized b′–a′ domains was solved by molecular replacement using 5
Page 6 of 20
the truncated b′ and a′ domains generated from our determined crystal structure of the reduced b′–a′ domains as search models. Further model building and refinement were performed using
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COOT [18] and REFMAC5 [19]. The stereochemical quality of the refined coordinates was
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assessed using RAMPAGE [20]. The refinement statistics are summarized in Table 1. The
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molecular graphics were prepared using PyMOL (http://www.pymol.org/).
2.2. Molecular dynamics simulation
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The 3D models of H. insolens PDI a′ domain (residues 334−449) derived from the crystal
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structures of oxidized and reduced b′–a′ domains were used as initial structures as follows: for the reduced state, the X-ray structure of the a′ domain in the reduced form of the b′–a′ segments
d
was isolated and then energy-minimized by the conjugate gradient method in a vacuum after
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complementing the missing hydrogen atoms. We also utilized the X-ray structure of the
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oxidized b′–a′ domains for MD simulation of the oxidized structure of the a′ domain. However, in the crystal structure, the electron density of Ala449 was ambiguous. Hence, the fragment composed of Ala448 and Ala449 was grafted from the crystal structure of the reduced form to that of the oxidized form by superimposing Ala448 through rigid translations and rotations. Subsequently, the hydrogen atoms were positioned onto the models, which were then energy-minimized in a vacuum. The energy-minimized structures were put into explicit water solvent. The system, under oxidized and reduced conditions comprised 4,094 and 4,093 water molecules, respectively, and one molecule of each corresponding form of the PDI a′ domain, along with two sodium ions Na+ as counter ions. The GEMB (Generalized-Ensemble Molecular Biophysics) program [21], developed by 6
Page 7 of 20
one of the authors (H.O.) was used to perform the MD simulations. The AMBER parm99SB force field [22] was used for the PDI a′ domain, and the TIP3P rigid-body model [23] was used
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for the water molecules. Temperature was controlled at 303 K by the Nosé-Hoover thermostat
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[24-26]. Pressure was controlled at 0.1 MPa by the Andersen barostat [27]. The symplectic
quaternion scheme was used for the rigid-body water molecules [28,29]. A cubic unit cell was
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used with periodic boundary conditions. The electrostatic potential was calculated by the particle mesh Ewald method [30]. Cutoff distance was 12 Å for the Lennard–Jones potential.
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Reversible multiple time scale molecular dynamics techniques [31] were applied for time
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development. The time step was considered to be Δt = 0.5 fs for the bonding interactions of the protein atoms and Δt = 2.0 fs for the non-bonding interactions of the protein atoms and that
d
between the protein atoms and solvent molecules, and Δt = 4.0 fs for the interaction between the
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solute molecules. We first performed MD simulations for 10 ns for each structure as an
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equilibration process. We then performed 140 ns simulations for data collection to calculate physical quantities. The
root-mean-square
RMSF i
q
i COG
i qCOG
fluctuation
2
(RMSF)
of
residue
i
is
defined
by
i represents time average and qCOG is the
, where
i
coordinate vector of the center of geometry for residue i. To calculate qCOG , the coordinates of i i backbone C, O, N, and Cα atoms were employed, and qCOG is given by qCOG
1 Ni i qj . N i j 1
Here, Ni is the number of the employed atoms in residue i. The vector qij is the coordinate vector of the employed atom j in residue i and is obtained by superposition between the simulation conformation and the initial conformation. 7
Page 8 of 20
3. Results and Discussion
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3.1. Crystal structures of reduced and oxidized forms of PDI b′–a′ domains
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To provide structural insights into the molecular mechanisms of the redox-dependent
domain rearrangement of PDI, we first attempted to determine the crystal structures of the b′–a′
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domains (composed of residues 208−449) of H. insolens PDI in its reduced and oxidized states. The final model of the reduced form of the b′–a′ domains, refined to a resolution of 1.85 Å, had
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an Rwork of 23.8% and Rfree of 27.8% (Table 1). The crystal belonged to space group P1, with four
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molecules per asymmetric unit. The structures of molecules A−D were highly similar to each other, with a RMSD value of 0.25−0.47 Å for superimposed 242 Cα atoms. In contrast, the
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oxidized form of the b′–a′ domains belonged to space groups P31, and diffracted up to 3.30-Å
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resolution. In the crystals, three molecules were contained per asymmetric unit. The final model
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of the oxidized form had an Rwork of 22.1% and Rfree of 31.3% (Table 1). The structures of molecules A−C can be closely superimposed with RMSD of 0.11−0.13 Å for 242 Cα atoms. Molecules A in the reduced and oxidized crystal structures, which have lower average B values (Table 1), were used for the comparative analysis and are described below. The crystal structures of the oxidized and reduced b′–a′ domains showed that each
domain adopts a typical thioredoxin fold, as expected (Figure 1). The b′ and a′ domains are composed of 1-α1-2-α2-3-α3-4-5-α4 and 1-α1-2-α2-3-4-5-α3 arrangements, respectively. Our crystal (reduced form, chain A) and previously reported NMR structures of b′ and a′ domains (PDB codes: 2KP2 and 2KP1, respectively) [8] are essentially identical with the RMSD of 1.14 and 1.26 Å for 108 and 111 Cα atoms, respectively. The structural variation is 8
Page 9 of 20
mainly observed in loop segments. Crystal structures of the b′ and a′ domains from fungal (reduced form, chain A) and human (reduced form, PDB code: 4EKZ) [10] PDIs can be closely
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superimposed with RMSD of 1.89 and 1.03 Å for 89 and 104 Cα and atoms, respectively,
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indicating their common thioredoxin fold.
The spatial arrangement of b′ and a′ domains was compared between the reduced and
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oxidized states. Although the domain arrangement between b′–a′ domains was profoundly affected by crystal packing, the two domains, connected through the flexible linker, exhibited
an
“open conformation” in the oxidized state (Figure. 1). In contrast, unlike the crystal structure
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reported for human PDI in its reduced state [10,11], the present crystal structure of the fungal PDI b′–a′ domains continued to adopt an open conformation, even in the reduced state (Figure.
d
1). In the crystal lattice, the potential domain–domain interactions could be hindered by the
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crystallographically neighboring molecule (Supporting Information Figure S1). Moreover,
Ac ce p
different crystal packing results in different domain–domain arrangements. Consequently, the b′–a′ interaction, bridged by an active-site tryptophan side-chain observed in the closed conformation of human PDI [10,11], could not be found in our crystal structures. To identify key residues in the catalytic a′ domain responsible for the redox-dependent
domain rearrangement observed in solution, we compared the crystal structures of the catalytic a′ domain between the reduced and oxidized states. However, both structures of a′ domain were essentially identical, with an RMSD value of 0.23 Å for 112 Cα atoms (Figure 2a). Even in the active site, no significant structural change was observed between them. Unlike the crystal structure of human reduced PDI (Figure 2b) [10,11], tryptophan flipping at the active site was not observed in the thermophilic fungal PDI b′–a′ crystal structures (Figure 2a). These 9
Page 10 of 20
crystallographic results underscore the importance of structural characterization of this protein
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3.2. Molecular dynamics of catalytic a′ domain of PDI in its redox states
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in the solution environment.
To study the structural mechanisms underlying the conformational transition of PDI in
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solution, we focused on the early stage of this process, triggered by the redox change of the a′ domain active site. Hence, we performed MD simulation analysis on the basis of the 3D
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structure frameworks of PDI a′ domain provided by our crystallographic analyses.
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Conformational fluctuations during the MD simulations were compared between the reduced and oxidized forms (Figure 3 and Supporting Information Figures S2–S5). Upon
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disulfide bond formation in the a′ active site, the surrounding residues exhibited smaller
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fluctuations. The flipping out of the Trp side-chain at the active site (Trp364), which was
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observed in the crystal structure of human PDI in its reduced state [10,11], was not pronounced in the present MD simulation. Notably, the oxidized form of the a′ domain exhibited remarkable fluctuations in the 3-4 loop and its spatial proximity, including the 1 helix, in comparison with the reduced form. These regions are involved in the crystal packing or the interaction with the b′ domain observed in the crystal structure of human PDI [10,11]. These results indicated that conformations of these a′ segments are intrinsically different between the oxidized and reduced states but become similar, as observed in the previously reported (human) [10,11] and currently determined (thermophilic fungal) crystal structures of the b′–a′ domains because of their intermolecular or interdomain interactions.
10
Page 11 of 20
Therefore, we propose a mechanistic model of the redox-dependent conformational change of the substrate-binding domains of PDI: the oxidization of the active site of the a′
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domain induces conformational changes in its b′-interacting segments comprising the 3-4
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loop and the 1 helix, resulting in the segregation of these two domains. To validate this model,
it is necessary to perform experimental characterization of isolated a′-domain conformation; this
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study is currently underway in our groups.
The molecular processes involving functional conformational changes are often characterized
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based on crystallographic snapshots of the proteins before and after the conformational
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transitions. However, crystal structures of flexible multidomain proteins can be considerably influenced by crystal packing. On the basis of crystallographic data, this study focuses on the
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crystal packing.
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utility of MD simulation for its ability to characterize protein conformation in the absence of
Acknowledgements
We thank Drs Takeshi Hiromoto (Nagoya City University, Japan), Tsunehiro Mizushima
(University of Hyogo, Japan), and Masato Kawasaki (Photon Factory, KEK, Japan) for the help with X-ray data collection, and Drs. Osamu Asami and Tsumoto Kajino of Toyota Central Research and Development Laboratory for providing the cDNA of the fungal PDI. We are grateful to the beamline staff of Osaka University BL44XU at SPring-8 (Harima, Japan) and BL13B1 at NSRRC (Hsinchu, Taiwan) for providing the data collection facilities and for their support. We also acknowledge the Research Center for Computational Science, National Institutes of Natural Sciences (Okazaki, Japan) for providing the computational resources for 11
Page 12 of 20
the MD simulation. This work was supported in part by JSPS KAKENHI (Grant Numbers
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24770102, 25121730 to T.S., and 25102008 to K.K.) and by the Okazaki ORION project.
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Accession numbers. The coordinates and structural factors of the crystal structures of the
reduced and oxidized b′–a′ domains of H. insolens PDI have been deposited in the Protein Data
Ac ce p
te
d
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an
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Bank under accession numbers (3WT1) and (3WT2), respectively.
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Page 13 of 20
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References
[1] S.S. Taylor, R. Ilouz, P. Zhang, A.P. Kornev, Nature reviews. Molecular cell biology 13
cr
(2012) 646. [2] B. Wilkinson, H.F. Gilbert, Biochimica et biophysica acta 1699 (2004) 35.
us
[3] J.C. Edman, L. Ellis, R.W. Blacher, R.A. Roth, W.J. Rutter, Nature 317 (1985) 267. [4] R.B. Freedman, P. Klappa, L.W. Ruddock, EMBO reports 3 (2002) 136.
[5] O. Serve, Y. Kamiya, K. Kato, Protein Folding (E.C.Walters ed.), NOVA Science Publishers
an
(New York) (2011) 489.
[6] J. Kemmink, N.J. Darby, K. Dijkstra, M. Nilges, T.E. Creighton, Biochemistry 35 (1996) 7684.
M
[7] P. Klappa, L.W. Ruddock, N.J. Darby, R.B. Freedman, The EMBO journal 17 (1998) 927. [8] O. Serve, Y. Kamiya, A. Maeno, M. Nakano, C. Murakami, H. Sasakawa, Y. Yamaguchi, T.
d
Harada, E. Kurimoto, M. Yagi-Utsumi, T. Iguchi, K. Inaba, J. Kikuchi, O. Asami, T. Kajino, T. Oka, M. Nakasako, K. Kato, Journal of molecular biology 396 (2010) 361.
te
[9] M. Nakasako, A. Maeno, E. Kurimoto, T. Harada, Y. Yamaguchi, T. Oka, Y. Takayama, A. Iwata, K. Kato, Biochemistry 49 (2010) 6953. C. Wang, W. Li, J. Ren, J. Fang, H. Ke, W. Gong, W. Feng, C.C. Wang, Antioxidants
Ac ce p
[10]
& redox signaling 19 (2013) 36.
[11]
C. Wang, J. Yu, L. Huo, L. Wang, W. Feng, C.C. Wang, The Journal of biological
chemistry 287 (2012) 1139.
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G. Tian, S. Xiang, R. Noiva, W.J. Lennarz, H. Schindelin, Cell 124 (2006) 61.
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V.D. Nguyen, K. Wallis, M.J. Howard, A.M. Haapalainen, K.E. Salo, M.J. Saaranen,
A. Sidhu, R.K. Wierenga, R.B. Freedman, L.W. Ruddock, R.A. Williamson, Journal of molecular biology 383 (2008) 1144.
[14]
A.Y. Denisov, P. Maattanen, C. Dabrowski, G. Kozlov, D.Y. Thomas, K. Gehring,
The FEBS journal 276 (2009) 1440. [15]
M. Nakano, C. Murakami, Y. Yamaguchi, H. Sasakawa, T. Harada, E. Kurimoto, O.
Asami, T. Kajino, K. Kato, Journal of biomolecular NMR 36 Suppl 1 (2006) 44. [16]
Z. Otwinowski, W. Minor, Methods in Enzymology 276 (1997) 307.
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[17]
A. Vagin, A. Teplyakov, J Appl Crystallogr 30 (1997) 1022.
[18]
P. Emsley, B. Lohkamp, W.G. Scott, K. Cowtan, Acta Crystallogr D Biol Crystallogr
66 (2010) 486.
(1997) 240. [20]
ip t
G.N. Murshudov, A.A. Vagin, E.J. Dodson, Acta Crystallogr D Biol Crystallogr 53
S.C. Lovell, I.W. Davis, W.B. Arendall, 3rd, P.I. de Bakker, J.M. Word, M.G. Prisant,
J.S. Richardson, D.C. Richardson, Proteins 50 (2003) 437.
cr
[19]
H. Okumura, Proteins 80 (2012) 2397.
[22]
V. Hornak, R. Abel, A. Okur, B. Strockbine, A. Roitberg, C. Simmerling, Proteins 65
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(2006) 712.
W.L. Jorgensen, J. Chandrasekhar, J.D. Madura, R.W. Impey, M.L. Klein, J Chem
Phys 79 (1983) 926.
an
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W.G. Hoover, Physical review. A 31 (1985) 1695.
[25]
S. Nosé, Mol Phys 52 (1984) 255.
[26]
S. Nosé, J Chem Phys 81 (1984) 511.
[27]
H.C. Andersen, J Chem Phys 72 (1980) 2384.
[28]
T.F. Miller, M. Eleftheriou, P. Pattnaik, A. Ndirango, D. Newns, G.J. Martyna, J
te
Chem Phys 116 (2002) 8649.
d
M
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H. Okumura, S.G. Itoh, Y. Okamoto, J Chem Phys 126 (2007) 084103.
[30]
U. Essmann, L. Perera, M.L. Berkowitz, T. Darden, H. Lee, L.G. Pedersen, J Chem
Ac ce p
[29]
Phys 103 (1995) 8577.
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Figure legends
Figure 1. Overall structures of oxidized and reduced b′–a′ domains of PDI. Ribbon models of the oxidized and reduced b′–a′ domains of PDI from H. insolens (a and b) and Homo sapiens (c and d). The secondary structure numbers and respective domains are indicated. The active-site cysteine residues are shown in sphere models.
14
Page 15 of 20
Figure 2. Comparison of active site of the a′ domain in the redox states. Close-up views of
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the active sites of PDI a′ domain from (a) H. insolens (oxidized; marine blue, and reduced; red)
cr
and (b) H. sapiens (oxidized; cyan, and reduced; magenta), colored as in Figure 1. Active-site cysteine and adjoining tryptophan residues are shown in stick models. The proposed residue
us
(Arg300) involving domain–domain interaction in human PDI b′ domain [10,11], and the structurally corresponding residue (Ala269) in fungal PDI b′ domain are also represented in
M
an
stick models.
Figure 3. MD simulation of the a′ domain in the redox states. Plots of root-mean-square
d
fluctuation (RMSF) during MD simulation of PDI a′ domain in oxidized and reduced states are
te
shown in (a) and (b), respectively. The secondary structure elements are indicated above the
Ac ce p
plots. In the right panels, mapping of residues showing higher RMSF values on the PDI a′ crystal structures during the MD simulation are shown. The residues are colored in red with color gradients indicating the degree of the fluctuation. The unanalyzed residues from the b′ domain and flexible linker are shown in gray.
15
Page 16 of 20
Table
Table 1. Data collection and refinement statistics for reduced and oxidized b′-a′ domain of PDI b′-a′ domain of PDI (oxidized)
Crystallographic data P1
P31
Unit cell
65.8/67.8/70.3
96.3/96.3/69.5
105.9/113.1/96.6
90.0/90.0/120.0
Beam line
NSRRC 13B1
SPring-8 BL44XU
Wavelength (Å)
1.00000
0.90000
Resolution (Å)
50-1.85 (1.88-1.85)
50-3.30 (3.36-3.30)
Total/unique reflections
173,477/88,599
62,727/10,825
Completeness (%)
92.3 (81.7)
Rmerge (%)
5.5 (37.8)
I / (I)
26.2 (1.5)
a/b/c (Å) α/β/γ (°)
cr
Space group
Rwork / Rfree (%)
23.8/27.8
an 8.8 (36.8)
M
20.0-1.85
99.9 (100)
d
Resolution (Å)
us
Data processing statistics
Refinement statistics
Ac ce pt e
R.m.s. deviations from ideal
ip t
b′-a′ domain of PDI (reduced)
32.0 (6.9)
20.0-3.30 22.1/31.3
Bond lengths (Å)
0.015
0.012
Bond angles (°)
1.67
1.51
Favored
98.6
92.7
Allowed
1.4
7.3
Protein atoms (A/B/C/D)
1,901/1,878/1,883/1,883
1,879/1,879/1,879
Water molecules
262
-
24
-
Protein atoms (A/B/C/D)
38.2/44.9/41.2/41.8
74.6/88.8/93.1
Water molecules
39.0
-
Glycerol molecules
42.8
-
Ramachandran plot (%)
Number of atoms
Glycerol molecules 2
Average B-values (Å )
Page 17 of 20
(b)
us
(a) α2 α1
β3
β5
β1 β5
α3
α1
ed
α4
Ac
α3
β2
β5
β4
β3
α4
β5
α4
a′
β1
β5
a′
α1 α2
β3
α2
α3 β2 β4
β2
β5
α4
β4
β3
α3 β5
β1
b′
α1
β5
α1
α3
β1
β4
α2
α3
(d)
α2
α1
β4
b′
ce pt
(c)
β3
β2
a′
b′
β2
β2 β4
α3
β3
α2
α2
β3
M an
β2 β4
β3 β2 β4
β1
b′
α2
α1
β1
α3
β1
cr
i
Figure 1
α1
a′ Page 18 of 20
M an
us
cr
i
Figure 2
(a)
(b)
b′
a′
ed
α2
A269
ce pt
α3
C365
C368
b′
R300
W396
α2
a′
C397 C400
Ac
W364
α3
Page 19 of 20
β1 α1
β2
α2
β3
β4
β5
cr
(a)
i
Figure 3 α3
us
6
α2
α1
β1
M an
RMSF (Å)
5 4 3
α3 β3 β2 β4
0
β5 α4
RMSF (Å)
5 4 3
β2
α2
β3
β4
β5
2
RMSF (Å) 3.5 3.0 2.5 2.0 1.5 0.0
α2
α1 β1 β3 α2
β2
α3
β3
β2 α4
1
α1
a′
α3
Ac
β1 α1
ce pt
(b)
β5
b′
330 340 350 360 370 380 390 400 410 420 430 440 450 Residue number
α3
β1
ed
1
β4
β3
α2
2
6
β2
β4
β4
α3 β5
β1
β5 α1
0 330 340 350 360 370 380 390 400 410 420 430 440 450 Residue number
b′
a′ Page 20 of 20