DHODH Microdomain-Membrane Interactions are Influenced by the

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HsDHODH Microdomain-Membrane Interactions are Influenced by the Lipid Composition Eduardo Festozo Vicente, Indra Dev Sahu, Edson Crusca Jr., Luis Guilherme Mansor Basso, Claudia Elisabeth Munte, Antonio Jose Costa-Filho, Gary A Lorigan, and Eduardo Maffud Maffud Cilli J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b09642 • Publication Date (Web): 17 Nov 2017 Downloaded from http://pubs.acs.org on November 19, 2017

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HsDHODH Microdomain-Membrane Interactions are Influenced by the Lipid Composition

Eduardo F. Vicentea, Indra D. Sahub, Edson Crusca Jrc,e, Luis G. M. Basso d, Claudia E. Muntee, Antonio J. Costa-Filhod, Gary A. Loriganb and Eduardo M. Cillic *

a

School of Science and Engineering, São Paulo State University (UNESP), 17602-496,

Tupã, SP, Brazil. b

Department of Chemistry and Biochemistry, Miami University, 45056, Oxford, OH,

USA. c

Institute of Chemistry, São Paulo State University (UNESP), 14800-900, Araraquara,

SP, Brazil. d

Laboratório de Biofísica Molecular, Departamento de Física, Faculdade de Filosofia,

Ciências e Letras de Ribeirão Preto, Universidade de São Paulo (USP), 14040-901, Ribeirão Preto, SP, Brazil. e

Instituto de Física de São Carlos, Universidade de São Paulo (USP), 13566-590 - São

Carlos, SP, Brazil.

* Corresponding author: [email protected] +55 16 33019678

ACS Paragon Plus Environment


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Abstract

Human dihydroorotate dehydrogenase (HsDHODH) enzyme has been studied as selective target for inhibitors to block the enzyme activity, intending to prevent proliferative diseases. The N-terminal microdomain seems to play an important role in the enzyme function. However, the molecular mechanism of action and dynamics of this region are not totally understood yet. This study analyzes the interaction and conformation in model membranes of HsDHODH microdomain using peptide analogues containing the paramagnetic amino acid TOAC at strategic positions. In buffer solution, the analogues presented a disordered conformation, but acquired a high content of α-helical structure in membrane mimetics, which was found to be lipid dependent. The microdomain peptide structure in micelles showed a very different peptide conformation when compared to the reported crystal structure, displaying a conformational flexibility of its helices, promoted by the connecting loop, which might be functionally relevant. Electron spin resonance in membrane compositions containing POPC, POPE and cardiolipin showed that interaction of the analogues was enhanced by the presence of cardiolipin, indicating that the microdomain preferentially interacts with cardiolipin-containing membranes. Therefore, the great flexibility of the microdomain and the cardiolipin affinity should be considered in further studies aimed at finding new inhibitory compounds to fight proliferative diseases.


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Introduction

The inhibition of metabolic pathways of nucleotide biosynthesis has been widely employed as a strategy for new drug development.1 Since this pathway is essential for virtually all organisms, the detailed study of the route components is fundamental in the search of treatment optimization for several diseases, such as cancer, autoimmune diseases, rheumatoid arthritis, multiple myeloma, among other serious proliferative diseases.2-3 Dihydroorotate dehydrogenase (DHODH), a flavin-dependent enzyme which is involved in the chemical catalysis of dihydroorotate to orotate, using the cofactor ubiquinone as the electron acceptor to regenerate the flavin mononucleotide (FMN), is crucial for the biosynthesis of new pyrimidine molecules and is directly involved in the respiratory complex.4 DHODH catalyzes the fourth reaction in the pyrimidine de novo pathway and the only step that uses a redox reaction, representing the rate limiting step in pyrimidine biosynthesis.5 DHODHs can be classified in two classes, 1 and 2, regarding the amino acid sequences, localization in the cell and types of substrate/cofactors used in catalysis. The human DHODH (HsDHODH - E. C. 1.3.5.2 and PDB ID: 1D3H) is a class 2 member, which are monomeric catalytic proteins found in higher eukaryotes and that use quinones as electron acceptors. Also, it has a special characteristic of being membrane-associated, making HsDHODH the only protein of the pyrimidine de novo pathway attached to the membrane, while the others are located within the cytosol. Because of its important metabolic role, HsDHODH enzyme has been studied as effective target for many inhibitors designed to bind and block the enzyme activity.6-7 Structurally, HsDHODH comprises two domains: a large C-terminal domain (Met78 - Arg396), very similar to class 1 families, located in the intermembrane space of mitochondria, and a short domain, which interacts with the inner mitochondrial mem-



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brane by the N-terminal region.8 This portion contains a microdomain (Met30 - Leu68), named here as N-t(DH) peptide, which comprises two α-helices (α1 and α2), connected by a four-residue loop, in a highly conserved domain found in almost all class 2 DHODH enzymes.9 Few studies have reported on the role and possible function of the N-terminal portion. Evidences have shown that this N-terminal region, which is buried into the membrane, could potentially control the entry of quinones by hydrophobic interactions, driving them to the active site, thus acting as a regulatory microdomain of HsDHODH.10-11 Although Couto et al. proposed a putative model for the participation of the N-terminal microdomain in the enzyme functional cycle,11-12 the complete mechanism and dynamics of this N-terminal microdomain, at a molecular level, are not fully understood yet. To gather more information on how the HsDHODH N-terminal microdomain acts in contact with different types of lipids and its specific dynamics, this work evaluates conformational changes in this microdomain, using N-t(DH) peptide analogues, in the presence of membrane mimetics by electron spin resonance (ESR), nuclear magnetic resonance (NMR), and circular dichroism (CD). To investigate the mobility of the peptide

backbone,

we

used

the

synthetic

paramagnetic

amino

acid

2,2,6,6-

tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC), which has been extensively used as a powerful ESR probe for peptides.13-15 Incorporation of TOAC into the peptide sequence allowed a precise evaluation of the local ordering, rotational dynamics and conformational properties of specific regions of the peptide.16 In addition, we employed vesicles of different lipid compositions to evaluate the interaction between the peptide containing the microdomain and membrane mimetics and to obtain more realistic and detailed information on the N-t(DH) peptide mechanism of action.


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Experimental Methods

Peptide Synthesis The peptide analogues were manually synthesized by Solid Phase Peptide Synthesis following the standard Nα-Fmoc protecting group strategy.17 The Rink Amide resin (purchased from Synpep®) was employed as the synthesis solid support, containing 0.6 mmol g−1 of substitution degree. Deprotection of the α-amino group of the resin and amino acids was made with 20% piperidine in dimethylformamide (DMF) to remove the base-labile Fmoc protecting group. The amino acid coupling reaction was performed using diisopropylcarbodiimide (DIC)/N-hydroxybenzotriazole (HOBt) in methylene chloride (DCM)/DMF approximately 1:1 (v/v) for two hours stirring, with three-fold excess for all coupling reagents and amino acids. If necessary, O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium

hexafluorophosphate

(HBTU)/

diisopropylethylamine (DIEA) in DCM/N-methylpyrrolidone (NMP) 1:1 (v/v) were used to improve the coupling reaction, in cases of negative coupling. The resin was washed with three or four cycles of DCM and DMF to remove excess reagents and byproducts. TOAC incorporation in the analogues [TOAC0]N-t(DH), [TOAC12]N-t(DH) and [TOAC20]N-t(DH) was performed with 1.2 molar equivalent excess fold, using as acylating

reagents

1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo-[4,5-

b]pyridiniumhexafluorophosphate-3-oxide] (HATU)/DIEA with 3.0 and 4.0 molar equivalent excess, respectively, in DCM/NMP 1:1 (v:v) solvents over the amino component in the resin. An acetyl group capping was added to the N-terminus after TOAC coupling and, due to the resin functionalization, the peptides had an amidated Cterminus. The next amino acid coupling in the analogues [TOAC12]N-t(DH) and [TOAC20]N-t(DH) was performed with six consecutives coupling steps, using the



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Fmoc-amino acid (Met for [TOAC12]N-t(DH) and Asp for [TOAC20]N-t(DH)) with five-fold excess, using HATU/DIEA as coupling agents at high temperatures (around 55ºC) for approximately 4h each step.13 After this amino acid incorporation, the resins were acetylated to avoid byproducts. Finally, after the last amino acid of each analogue, an acetylation reaction was performed to mimic the native protein sequence structure. The cleavage was performed using trifluoroacetic acid (TFA), triisopropylsilane (TIS) and water (95:2.5:2.5, v:v:v, respectively). The sample was treated with cold diethyl ether and centrifuged three times. The precipitate was resuspended in aqueous solution, obtaining the crude peptides, which were lyophilized. The extracted spin-labeled analogue was treated with ammonium hydroxide for complete N−O deprotonation, step monitored by analytical HPLC. After that, purification of the peptides were performed by semi-preparative HPLC Beckman System Gold (Brea, CA, USA) with a reverse phase C-18 column in a linear gradient, flow rate 5 mL min−1, using aqueous 0.02 mol L−1 ammonium acetate (pH 5.0) and 90% acetonitrile in ammonium acetate solution as solvents A and B, respectively.13 The purity of the peptides were checked by analytical HPLC Varian (Santa Clara, CA, USA), flow rate of 1.0 mL min−1, UV detection at 220 nm, using solvents A (0.045% TFA:H2O) and B (0.036% TFA:ACN) with a linear gradient of 5–95% (v/v) of solvent B for 30 min. The confirmation of the peptide analogues obtained was performed by Electrospray Mass Spectrometry, on ZMD Micromass model equipment (Milford, MA, USA) (Table 1). All HPLC profiles and Mass Spectra of the synthesized peptide analogues are shown in the Supporting information (Figure S1).

Circular Dichroism


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CD measurements were performed at 25°C on a Jasco Products Company, Inc. J-715 (Oklahoma City, OK, USA) spectropolarimeter, using a 1 mm path-length quartz cell. Spectra were acquired every 0.2 nm from 250 to 190 nm at a scan speed of 50 nm min−1, with 2 nm bandwidth and a response time of 3 s. Each spectrum represents an average of 8 successive scans and is expressed as molar ellipticity [θ] (deg. cm2. dmol−1). Samples were prepared using 30 µmol L−1 of peptide analogues at the following conditions: buffer solution (Tris-HCl 0.01 mol L−1, NaCl 0.01 mol L−1, pH 7.4); 60% of 2,2,2-Trifluoroethanol (TFE) in buffer solution (v/v); 1-palmitoyl-2-hydroxysn-glycero-3-phosphocholine (LPC) at a concentration of 10 mmol L−1; sodium dodecyl sulfate (SDS) at a concentration of 20 mmol L−1. Both LPC and SDS detergents were previously weighted from a powder lipid (Avanti, Alabaster, AL). For the CD measurements in liposomes, Multilamellar vesicles (MLV) were prepared using 1-palmitoyl2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid (Avanti, Alabaster, AL) and added into a flask tube. After the film preparation, the peptide was added to the film resuspension in Tris buffer solution, reaching a concentration of 30 µmol L−1 in a final molar peptide/lipid ratio of 1:250, and final volume of 300 µL. This peptide-to-lipid molar ratio was obtained by ESR and presented, among the samples studied (1:100, 1:250 and 1:400), the best signal-to-noise results (data not shown). The solution was equilibrated by repeating at least 10 freeze-thaw-sonication cycles until the sample became clear.

NMR spectroscopy and structure calculations All 1H NMR spectra were recorded on a Bruker DRX 600 spectrometer, equipped with a cryoprobe TXI, using the presaturated procedure for water suppression. Two-dimensional TOCSY (total correlation spectroscopy) and NOESY (nuclear Overhauser effect spectroscopy) spectra of 0.7 mmol L−1 N-t(DH) peptide in 100 mmol L−1



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DPC-d38 micelles were acquired in 10 mmol L−1 Tris buffer solution containing 10% D2O at pH 7.4 and using mixing times of 65 ms and 200 ms, respectively. All 2D spectra were acquired with 4,096 complex points and 256 τ1 increments. The measurements were performed at 35ºC using 80 scans with DSS (2,2-dimethyl-2-silapentane 5sulfonate sodium salt) as an internal standard. Additionally, natural abundance 13C and 15

N HSQC experiments were carried out and the chemical shifts obtained were used to

perform a secondary structure prediction as well as applied as input restraints for structure calculation. NMR data processing and analyses were carried out using Topspin (Bruker) and NMRViewJ programs, respectively.18 1H chemical shifts were assigned according to standard procedures.19 Briefly, to build an ensemble of 3D structures NOESY crosspeaks were characterized based on total volumes and upper bounds for the NOE constraints, calibrated using the internal calibration utility of NMRViewJ known 1H (β)—1H(β’) distances, in three classes: strong (≤ 2.7 Å), medium (≤ 3.5 Å) and weak (≤ 5.0 Å). The lower distance limits were taken as the sum of the van der Waals radii of two hydrogen atoms (1.8 Å). The set of 442 manually assigned distance restraints was used to calculate 1,500 structures with the program CYANA 2.1 by simulated annealing (SA) algorithm.20 The structure calculation started with an extended model, with 18,000 steps at high temperature, and 9,000 steps of cooling. An ensemble of the 20 lowest energy structures was chosen to represent the peptide 3D structure and minimized by GROMACS 4.5 package and AMBER force field.21-22 Quantitative analyses of the structures were carried out with PROCHECK-NMR and Pymol programs.2324

The NMR structure was deposited at Protein Data Bank (www.rcsb.org) and assigned

with the PDB code 5TCE.

Electronic Spin Resonance


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Continuous-wave (CW) ESR measurements in micelles were performed on a Varian (Santa Clara, CA, USA) E-109 X-band (9.5 GHz) CW-EPR spectrometer (located in the Laboratório de Biofísica Molecular at Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto - Universidade de São Paulo). ESR measurements in MLV liposomes were carried out at 22ºC on a X-band (9.5 GHz) Bruker EMX spectrometer (Ohio Advanced EPR Laboratory at Miami University) using thin quartz microtubes. The experimental conditions for all experiments were: central field, 3,362 G; sweep width, 100 G; modulation amplitude, 0.5 G; modulation frequency, 100 kHz; microwave power, 10 mW; time constant, 128 ms, and acquisition time of 150 s. The previous CD sample conditions were also used for ESR assays. The peptide concentration was 30 µmol L−1. Regarding the ESR studies with liposomes, MLVs were made with the

phospholipids

POPC,

1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine

(POPE), and cardiolipin (CL), all purchased from Avanti Polar Lipids. The following MLV lipid compositions were used: pure POPC, POPC:POPE (2.15:1), POPC:CL (4.67:1), and POPC:POPE:CL (4.67:2.17:1). The concentration of the TOAC-bearing peptide analogues was 50 µmol L−1. The composition of the ternary POPC:POPE:CL lipid mixture is very similar to the real inner mitochondrial lipid composition,25 to which the HsDHODH microdomain is anchored,8 while the other MLVs were used to gain a better understanding of the individual contribution of each lipid in the membrane interaction with the N-t(DH) analogues.

Nonlinear-least-squares (NLLS) simulations of the ESR spectra NLLS simulations of the single- and multicomponent ESR spectra of the TOACbearing peptides were performed using the Multicomponent software developed by Dr. Christian Altenbach (University of California, Los Angeles, California)26-27. The rota-



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tional diffusion rates, rotational correlation times, and order parameters were obtained as described by Vicente et al.13 The MOMD (microscopic order, macroscopic disorder) model28 was used to account for the tendency of TOAC to become partially ordered in the randomly oriented lipid micelles and model membranes. Seed values of the g-tensor and hyperfine-tensor components of TOAC in the peptide analogues were taken from Nesmelov et al,29 but were allowed to vary during the fitting process. Further details of the NLLS simulations can be found elsewhere.13, 30

Results and Discussion

Peptide Analogues Design and Features We have successfully used SPPS to obtain the TOAC-labeled N-t(DH) analogues. In this study, the spin label TOAC was inserted at strategic positions in the three N-t(DH) peptide derivatives to understand in detail the peptide conformational changes as monitored by the dynamics of the probe in the presence of model membranes of different lipid compositions. To perform the spin-labeling of the analogues, residues of proline were strategically substituted by the TOAC spin-label. Also, TOAC can induce turns or curvatures and stabilize helices, which can increase the peptide helicity.16 In the named [TOAC0]N-t(DH) analogue, TOAC was added to the N-terminus, which is a region known to play an important role in several protein/peptide interactions.13,

31-33

For the analogue [TOAC20]N-t(DH), the spin label was attached to the α1 helix, replacing a proline residue, whereas [TOAC12]N-t(DH) carries TOAC in the end of the microdomain loop, i.e., the beginning of the α2 helix (Table 1). In general, the peptide analogues have a hydrophobic ratio of approximately 38%, calculated according to Wang et al.34 and the total net charge at pH 7 is − 1.8 on average, for each analogue.35


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Table 1. Amino acid sequences and molecular weights of N-t(DH) peptide and its TOAC-bearing analogues. The highlighted letter “O” in yellow represents the TOAC paramagnetic amino acid. Peptide

MW (g mol-1)

Sequence

N-t(DH) 0

[TOAC ]N-t(DH)

GDERFYAEHLMPTLQGLLDPESAHRLAVRFTSLG

3,869.3

OGDERFYAEHLMPTLQGLLDPESAHRLAVRFTSLG

4,066.6

12

GDERFYAEHLMOTLQGLLDPESAHRLAVRFTSLG

3,969.5

20

GDERFYAEHLMPTLQGLLDOESAHRLAVRFTSLG

3,969.5

[TOAC ]N-t(DH) [TOAC ]N-t(DH)

Peptide conformation in micelle model membranes To analyze the structure of the peptides, firstly far-UV CD spectroscopy measurements were performed to verify the secondary structure of the peptide analogues in buffer, in 60% TFE/buffer solution (v/v), in micelles of LPC and SDS, and in POPC lipid vesicles. Figure 1 shows the CD spectra of the peptide analogues.

Figure 1. CD spectra of the synthetic peptide analogues (A) [TOAC0]N-t(DH), (B) [TOAC12]N-t(DH), and (C) [TOAC20]N-t(DH). The spectra were obtained in buffer solution (black lines), TFE 60% (v/v) (green lines), LPC 10 mmol L−1 (red lines), SDS 20 mmol L−1 (dark blue lines) and POPC vesicles (light blue lines), using a peptide-tolipid ratio of 1:250. The peptide concentration was 30 µmol L−1. 25

25

30

A

B [θ] x 10 (deg . cm . dmol )

-1

-1

[θ] x 10 (deg . cm . dmol )

-1

15

10

5

0

0

-5

-5

-10

-10 190

200

210

220

λ (nm)

230

240

250

15 10 5

3

3

5

20

2

2

10

3

2

15

C

25

20

20

[θ] x 10 (deg . cm . dmol )

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

0 -5 -10 -15

190

200

210

220

230

240

λ (nm)


250

190

200

210

220

λ (nm)

230

240

250


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The negative bands at 222 and 208 nm and the positive band at 195 nm in the CD spectrum of [TOAC12]N-t(DH) in buffer are characteristics of α-helical conformation. The α-helical structure of [TOAC12]N-t(DH) in aqueous solution can be attributed to the insertion of TOAC at the α1 helix, which induced a helicity increase of this molecule. This fact was observed in other peptide analogues containing TOAC, such as Ctx[Ile21]-Ha antimicrobial peptide13 and trichogin A IV,36 since TOAC is a constrained nitroxide side chain spin label, in which the nitroxide ring is rigidly attached to the peptide backbone. On the other hand, [TOAC0]N-t(DH) and [TOAC20]Nt(DH) peptide analogues presented predominantly a disordered conformation in buffer solution, as evidenced by the negative band around 202 nm. It is worth mentioning that structural changes induced by TOAC placed at different positions along the peptide sequence in solution are not relevant in this study, since we are mainly interested in the interactions between the peptides and membrane mimetics. TFE was used in the CD studies to stabilize secondary structures and, in our case, to induce a helical structure, as evidenced by the CD spectra.37 In this environment, it was observed approximately the same amount of α-helical content in the three analogues. These data show that all peptides have a similar structure, and TOAC incorporation does not promote large structural changes or destabilization in the peptide analogues. In zwitterionic LPC and the negatively charged SDS micelles, the peptides mostly presented an α-helical structure. These results are in accordance with Langmuir monolayer and Polarization Modulation-Infrared Reflection-Adsorption spectroscopy (PM-IRRAS) studies of the N-t(DH) microdomain peptide, which have shown that the peptide acquires a disordered structure in aqueous subphase, but assumes an α-helical conformation when in contact with monolayer.5 The phosphocholine lipid head group


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could increase the amount of α-helix content of the N-t(DH) peptide, as verified with dipalmitoyl phosphatidylcholine (DPPC) monolayers.5 In POPC vesicles, the spin-labeled peptides showed different intensities of conformations. The [TOAC0]N-t(DH) presented the lowest amount of α-helix when compared to the other analogues, which can be due to TOAC position in the peptide analogue sequence. The [TOAC0]N-t(DH) CD spectrum clearly presents a negative band around 203-204 nm and a weak and positive band at 195 nm, which can be interpreted as a mixture of two populations in different conformations: membrane bound (α-helix) and free in solution (disordered). The same effect occurred, but less pronounced, for [TOAC20]N-t(DH). The helicity of [TOAC12]N-t(DH) exhibited less variation of intensity when compared with [TOAC20]N-t(DH) and [TOAC0]N-t(DH). The reduced amount of α-helix found for [TOAC20]N-t(DH) in POPC as compared to [TOAC12]Nt(DH) is probably due to the proximity of the spin label to the loop, which may disturb the normal mobility of this region. In general, the insertion of TOAC in the peptide sequence did not affect the original N-t(DH)-membrane interaction properties, showing approximately the same pattern of conformational changes.5 Overall, the helical content of N-t(DH) analogues were found to be dependent upon the environment: larger helix amount in micelles, likely due to their decreased molecular packing and natural high positive curvature, and little lower content in liposomes, possibly due to the more packed hydrophobic core of lipid bilayers and to the mobility featured by the microdomain.10 It has been suggested that the loop plays an important role in the microdomain structure by helping to select the most suitable conformation in order to allow the enzymatic reaction.11 In order to evaluate the peptide structure in micelles and understand the peptide changes in terms of its conformation compared to the crystal structure, NMR studies were carried out.



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NMR derived structure of N-t(DH) bound to DPC micelles The complete sequential assignments of N-t(DH) have been made for the peptide bound to DPC micelles and the three-dimensional structure was determined based on 442 distance constraints resulting in an ensemble of the 20 lowest-energy NMR structures. The secondary Hα chemical shifts of N-t(DH) (Figure 2) predominantly showed two α-helical segments, suggesting that the secondary structure of N-t(DH) is, in general, almost similar to those reported in the HsDHODH crystal structures.9 The fingerprint (NH-Hα and NH-NH) regions display many strong interresidue (i, i+1), NH/NH and medium range (i, i+1), and (i, i+3) NH-Hα cross-peaks, indicating a helical structure (Figure S5). The DPC-induced N-t(DH) conformation displays an αhelix at the N-terminus from residues Glu3 – Gly16 and another helix at the C-terminus from residues Ser22 – Ser32 connected by a bend consisted of residues Leu17 to Glu21 (Figure 3). Overall, the refined structure is well defined with average backbone and heavy atoms (N, Cα and C’) root mean square deviations of 0.8 and 1.4 Å, respectively, compared to the mean structure (Table S2). The final structural statistics are reported in Table S2 and S3 of the Supporting Information. A bundle of the 20 lowest energy structures is shown in Figure 3 (PDB code ID 5TCE). The crystal structure of the microdomain, reported by Liu et al. (Figure 4), has both helices very close to each other, with the detergent molecule N,Ndimethyldecylamine-N-oxide (DDAO) tightly bound to the center of the hydrophobic patch between the helices. Interestingly, the NMR structure here reported and acquired in DPC micelles showed a different conformation for the N-t(DH) peptide, mainly with the α1 helix twisted at about 180º relative to the α2 helix (Figure 4). This motif shifting


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occurs mainly because of the flexibility of the connecting loop, which thus promotes a significant alteration in the microdomain peptide conformation.

Figure 2. Deviation of Hα (A), NH (B) and Cα (C) chemical shifts of N-t(DH) from random coil values for 0.7 mmol L-1 N-t(DH) peptide in 100 mmol L-1 DPC-d38 solution, pH 7.4 at 35ºC.



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Figure 3. Superposition of backbone atoms (N, Cα and C’) of 20 lowest energy structures of N-t(DH) bound to DPC micelles obtained from CYANA 2.1 (top). Cartoon representation of the lowest energy structure determined by NMR spectroscopy (bottom).

Figure 4. Comparison between the crystal structure obtained by Liu et al. (green) and the lowest energy structure determined by NMR spectroscopy (blue) by superposition of α1 helix backbone atoms (N, Cα and C’); highlighted in red are the proline residues that were replaced by the TOAC spin label, and in orange arginine residues as reference.



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The NMR structure showed a twist conformation of the residues, which can be crucial for the interaction of the microdomain with different types of membrane mimetics. Moreover, it can be inferred that the microdomain could adopt different conformations depending on the local membrane composition. Differences regarding crystallographic and NMR structures of different proteins were systematically compared by Sikic et al.38 and they noted some important patterns, such as: beta strands match better between NMR and crystal structures than α-helices and loops. Beyond that, conformational differences between loops are independent of crystal packing interactions, and rarely, side chains buried in the protein interior demonstrated to acquire different orientations in the crystal and in solution.38 Although these results were found for proteins, the latter statement might be applied in our peptide-micelle system, since the detergent molecule was found to be tightly bound to the microdomain in the crystal structure. In this way, our studies showed that this region can adopt a different conformation in DPC micelles than that found in crystal structure. Also, the presented flexibility in the peptide structure is in accordance with previous Double Electron-Electron Resonance (DEER) studies,10 which showed different distances between the spin-labeled peptide ends de-


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pending on the membrane mimetic analyzed. Moreover, for the N-t(DH) NMR structure, analyzing the theoretical distance between both α-carbons from Gly1 (N-terminus) to Gly34 (C-terminus) using the PyMOL software, it was observed a value of 39.4 Å (data not shown). This result is close to the distance found at same conditions (i.e. DPC micelles) in DEER experiments, which was observed a distance value of 32 Å ± 4 Å between the spin probes attached at the N and C-termini, in a doubly-labeled N-t(DH) peptide analogue.10 The distance between the α-carbon from Gly1 (N-terminus) and Gly34 (C-terminus) obtained from the N-t(DH) NMR structure in DPC micelles (39.4 Å) is more consistent with the DEER experiments than the distance between the same atoms found in the crystal structure in solution (13.5 Å). These results provide strong support for the more open conformation of N-t(DH) peptide in micelles found in our NMR studies. Also, this information suggests that the NMR structure of the N-t(DH) peptide agrees with the conformation that this molecule acquires in micelles, evidenced previously by DEER spectroscopy, even considering the high dynamics of the spin probes studied. Finally, the loop connecting the two helices of the microdomain provides great conformational flexibility and dynamics to the helices, which could play a fundamental role in the catalysis of the HsDHODH enzyme. To obtain more information about this molecular mechanism and the peptide/membrane interaction, ESR experiments were also performed with vesicles of different lipid compositions.

Peptide dynamics in different vesicle compositions ESR is a very powerful tool to unraveling the dynamics of specific regions of the peptide backbone. The ESR spectra acquired from TOAC-labeled molecules report on local ordering, mobility, accessibility to polar and nonpolar paramagnetic compounds and the polarity of the surrounding microenvironment of the spin label.39 The line shape



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analysis of the ESR data for TOAC-labeled proteins or peptides can probe the structural model of the molecule with spatial resolution at the backbone level.40-41 Initially, ESR spectra of the N-t(DH) peptide analogues were obtained in aqueous solution and in micelles to analyze the rotational dynamics of TOAC spin label attached to different positions of the peptide backbone. Figure 5 shows the ESR spectra acquired in buffer and TFE 60% solutions and in the presence of LPC and SDS micelles, along with the best fits obtained from nonlinear least-squares simulations. Table 2 displays the rotational diffusion rates (R), correlation times (τ), and order parameters (S0) obtained from the best NLLS fits and the experimental peak-to-peak width of the central line (W0) and maximum hyperfine splitting (2Amax) of each spectrum. The gand A-tensor magnetic components calculated from the spectra are shown in Table S1 of the Supporting Information.

Figure 5. Experimental (black) and best-fit (red) ESR spectra of (A) [TOAC0]N-t(DH), (B) [TOAC12]N-t(DH), and (C) [TOAC20]N-t(DH) in buffer and 60% (v/v) TFE/water solutions, and in 10 mmol L-1 LPC and 20 mmol L-1 SDS micelles. The ESR spectrum of [TOAC0]N-t(DH) in LPC presents two components, represented in gray and blue. Scan width: 160 G.


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Table 2. Best-fit rotational diffusion rates (R), rotational correlation times (τ), and order parameters (S0) obtained from NLLS simulations along with the experimental peak-topeak width of the central line (W0) and the maximum hyperfine splitting (2Amax) of the ESR spectra of N-t(DH) peptide analogues in different conditions.

System

Comp / Pop

R (×107 s-1)

τ (ns)

S0

W0 (G)

2Amax (G)

1 1 1 (17 %) 2 (83 %) 1

43.6 14.1 13.5 4.8 13.2

0.9 1.2 1.2 3.5 1.5

̶

1.7 2.3 2.6 ̶ 2.3

̶

̶

1 1 1 1

10.0 2.1 1.6 3.4

1. 7 8.0 10.5 4.9

̶ 0.5 0.2

3.0 5.0 5.5 4.3

̶ 55 64 58

1 1 1 1

11.5 4.6 1.7 4.7

1.4 3.6 10.1 3.6

̶ 0.3 0.5 0.1

2.6 4.4 5.0 3.8

̶ 59 60 51

[TOAC0]N-t(DH) Buffer TFE LPC SDS

̶ ̶ 0.2 ̶

̶ ̶ ̶


̶


Estimated uncertainties: R (4%); τ (4%); S0 (± 0.04); 2Amax (± 1 G).

In buffer, the mobility of the TOAC analogues was much higher than in the other environments, indicating a high degree of spin label freedom, as evidenced by the narrow lines of the ESR spectra (Figure 5) and the low W0 values (Table 2). This result is in accordance with the CD assays that show, for all peptides, higher content of disordered structure or just freely tumbling in solution. NLLS simulations showed that TOAC presents the higher mobility at the N-terminus ([TOAC0]N-t(DH) - R = 4.36 × 108 s-1, W0 = 1.7 G), and an intermediate spin label freedom at position n = 20 (R = 1.15 × 108 s-1, W0 = 2.6 G), as shown for [TOAC20]N-t(DH), where the probe can be slightly



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sterically hindered by the backbone itself. The [TOAC12]N-t(DH) shows the slowest mobility of TOAC (R = 1.0 ×108 s-1), which is likely due to the position of the nitroxide spin label in the peptide sequence (n = 12) and the improved helicity that TOAC imposes to the first helix of the peptide, as previously discussed. These reduced dynamics are also reflected in the line shape broadening (W0 = 3.0 G). Only slight changes in the line shape of the ESR spectrum of [TOAC0]N-t(DH) in TFE can be observed (Figure 5A), which is likely related to the α-helical secondary structure acquired by this analogue. Despite that, a remarkable three-fold decrease in the rotational dynamics of the backbone took place (R = 1.41 × 108 s-1). For [TOAC12]Nt(DH) and [TOAC20]N-t(DH) derivatives, the line shapes changed considerably and became broader (W0 = 5.0 G and 4.4 G, respectively) in TFE. The rotational dynamics of the spin label in the [TOAC12]N-t(DH) (R = 2.1 ×107 s-1) is more than two-fold slower than that in the [TOAC20]N-t(DH) derivative (R = 4.6 ×107 s-1). This result is in agreement with the TOAC position in the peptide sequence, located in the middle of the helix, while the spin label at n = 20 is in the turn. Our NLLS simulations also showed that the nitroxide probe in the [TOAC20]N-t(DH) derivative experiences an orienting potential (S0 = 0.29), likely imposed by the peptide conformation, that restricts the reorientational motion of the spin probe. This ordering effect is further evidenced by the larger 2Amax parameter (Table 2). The rotational dynamics of the peptide analogues is faster in SDS than in LPC (Figure 5 and W0 values in Table 2). This is primarily due to a faster tumbling of SDS micelles in solution than that evidenced in LPC, which averages the magnetic interactions and decreases the spectral anisotropy.42-43 Furthermore, the reorientational motion of the spin probe in all paramagnetic peptide derivatives are more restricted in LPC than in SDS micelles, as evidenced by the order parameters (S0, Table 2).


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Second, there is clearly a second component in the ESR spectrum of [TOAC0]Nt(DH) in LPC micelles, as judged by the appearance of a shoulder in the low field peak (Figure 5A). NLLS simulations showed that both spin populations presented very different mobilities as compared to the dynamics of the peptide analogue in buffer. While the rotational diffusion rate of the less populated component (17%) decreased 3-fold (R = 1.35 × 108 s-1), a remarkable 9-fold decrease of mobility (R = 4.79 × 107 s-1) along with an order parameter of 0.17 was found for the less mobile, more populated site (83%). These results indicate that both peptide populations are due to LPC-bound peptide analogues and thus may correspond to different conformational states of the [TOAC0]N-t(DH) N-terminus. This same effect was previously observed for a TOAClabeled analogue of Ctx(Ile21)-Ha peptide from Hypsiboas albopunctatus, where the spin probe was attached to the N-terminus.13 Third, the mobility of the peptide derivatives in LPC and SDS micelles follows the same order as in buffer, i.e. [TOAC12]N-t(DH) < [TOAC20]N-t(DH) < [TOAC0]Nt(DH). However, the effects of both micelles were more pronounced in the rotational diffusion rate of [TOAC0]N-t(DH) than in the other TOAC-containing peptides (Table 2). In order to investigate the mobility of the TOAC-labeled peptides in model membranes that better mimic the natural lipid environment of the HsDHODH microdomain, we performed ESR experiments in the presence of the following membrane mimetics: POPC, POPC:POPE, POPC:CL, and POPC:POPE:CL. The latter lipid mixture was chosen due to its similarity with the lipid composition of the inner mitochondrial membrane system.25,

44

Different peptide-to-lipid (P/L) molar ratios were tested

(data not shown) and we show herein the results with P/L = 1/250 due to the best signalto-noise ratio achieved, which allowed for performing NLLS simulations with all pep-



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tides. Figure 6 shows the experimental ESR spectra (top panel) of the peptide analogues in different membrane mimetics along with some representative best-fit spectra at selected conditions (middle panel). The parameters obtained from the NLLS simulations are illustrated in Table 3 and all simulated spectra are shown in Figures S2-S4 of Supporting Information.

Figure 6. Peptide analogues partition into lipid membranes. Experimental and best-fit ESR spectra of (A) [TOAC0]N-t(DH), (B) [TOAC12]N-t(DH), and (C) [TOAC20]Nt(DH) in multilamellar vesicles of different lipid compositions at a peptide-to-lipid molar ratio of 1:250. Top panel: Experimental ESR spectra of the peptides in POPC (black), POPC:POPE (red), POPC:CL (gray), and POPC:POPE:CL (blue). Middle panel: Best NLLS fit (red) to the experimental spectra (black) of the peptides at selected conditions. The ESR spectra of the free (gray) and membrane-bound (blue) components are also shown. Bottom panel: Populations of the membrane-bound peptide analogues in different model membranes. Scan width: 100 G.


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Table 3. Best-fit rotational diffusion rate (R), rotational correlation time (τ), and order parameter (S0) obtained from NLLS simulations along with the maximum hyperfine splitting (2Amax) of the ESR spectra of N-t(DH) peptide analogues in lipid model membranes.

System

Comp / Pop

R (×107 s-1)

τ (ns)

S0

2Amax

1 (63 %) 2 (37 %) 1 (single) 1 (58 %) 2 (42 %) 1 (single) 1 (29 %) 2 (71 %) 1 (19 %) 2 (81 %)

43.6 5.4 44.7 43.6 5.0 42.7 43.6 6.2 43.6 5.5

0.4 3.1 0.4 0.4 3.3 0.4 0.4 2.7 0.4 3.1

̶

̶

1 (47 %) 2 (53 %) 1 (18 %) 2 (82 %) 1 1

10.0 1.7 10.0 1.9 2.3 2.2

1 (38 %) 2 (62 %) 1 (47 %) 2 (53 %) 1 (24 %) 2 (76 % ) 1

11.5 1.2 11.5 1.2 11.5 1.7 1.6

[TOAC0]N-t(DH) POPC

POPC:POPE

POPC:CL POPC:POPE:CL

̶

̶ ̶

̶

̶

̶ ̶

̶ ̶

̶

̶

̶ ̶

̶

̶ ̶

̶

̶

1.7 9.6 1.7 8.8 7.3 7.6

̶ 0.6 ̶ 0.7 0.5 0.5

̶ 66 ̶ 66 66 65

1.4 13.8 1.4 13.8 1.4 9.5 10.3

̶ 0.6 ̶ 0.6 ̶ 0.6 0.6

̶ 60 ̶ 60 ̶ 60 61

[TOAC12]N-t(DH) POPC POPC:POPE POPC:CL POPC:POPE:CL

[TOAC20]N-t(DH) POPC POPC:POPE POPC:CL POPC:POPE:CL

• •

In the ESR spectra of [TOAC12]N-t(DH), it was found a very small content of free TOAC in POPC (0.8 %) and in POPC:POPE (0.5 %). Estimated uncertainties: o site 1: R (4%); τ (4%); S0 (± 0.04); 2Amax (± 1 G). o site 2: R (5%); τ (5%); S0 (± 0.06); 2Amax (± 1 G).


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For [TOAC0]N-t(DH), the line shape of the ESR spectrum in buffer is almost the same as in the presence of POPC and POPC:POPE (Figure 6A). However, the appearance of a shoulder in the low field peak upon addition of POPC:CL and POPC:POPE:CL is indicative of a second, membrane-bound component possibly with different ordering and dynamics. The use of a single component in the simulations of [TOAC0]N-t(DH) ESR signals in POPC and POPC:POPE led to well fitted spectra (Figure S2) with parameters very close to those found for the peptide free in solution. This means that either the peptide does not bind to the membranes or it does so but the TOAC at the N-terminus displays the same conformational freedom as in solution, despite great part of peptide backbone likely being in contact with the liposome. As discussed before, the CD spectrum of [TOAC0]N-t(DH) in POPC results from a mixture of membrane-bound and unbound peptide conformations (Figures 1A). Thus, if a population of peptides does bind to POPC and acquires helical structure, we can test this possibility by including a second component in our NLLS simulations and keeping the parameters of the first component fixed at those of [TOAC0]N-t(DH) in solution. Figures S2A and S2B show that a slightly better fitting was achieved upon addition of a second spectral population, which accounted for 37% (42%) in the presence of POPC (POPC:POPE) (Table 3). The shoulders surrounding the resonance lines were better fitted. The 8-fold decrease of the rotational mobility of the second component compared to R obtained in solution (site 1) suggests that this spin population likely corresponds to membrane-bound [TOAC0]Nt(DH) peptides. The presence of cardiolipin in the POPC:CL and POPC:POPE:CL model membranes led to the appearance of an evident and well populated second membrane-bound component of [TOAC0]N-t(DH) (Figures 6A and S2C). Differences in the rotational



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diffusion rates among the four membrane mimetics may reflect different peptide-lipid interactions (Table 3). Incorporation of cardiolipin seems to increase the affinity of peptide analogues, which is likely related to its unique features such as the four acyl chains and two negative charges, although forming a relatively small anionic head group.45 In fact, since the concentration of CL per mol of vesicle in POPC:CL is higher than in POPC:POPE:CL and the membrane-bound population follows an opposite order (71% in POPC:CL and 81% in POPC:POPE:CL), both electrostatic and hydrophobic forces of CL with [TOAC0]N-t(DH) might contribute to the higher affinity of the peptide to CLcontaining membranes. The ESR spectra of the peptide analogues [TOAC12]N-t(DH) and [TOAC20]Nt(DH) in the liposomes presented either one or two components depending on the lipid composition of the membrane. Irrespective of the membrane mimetic, an immobilized, broad component is observed in all conditions, which is more intense in the CLcontaining membranes (Figures 6B and 6C). NLLS simulations of the ESR spectra (Figures S3 and S4) indicate that the parameters of the narrow, mobile component of peptide/membrane systems are the same as those of the peptides in solution. Conversely, the rotational diffusion rates and order parameters found for the immobilized spin population are consistent with membrane-bound peptides (R ~ 1.2−2.3×107 s-1; S0 ~ 0.53−0.68; Table 3). The high values of S0 indicate a very restricted reorientational motion of the spin label in [TOAC12]N-t(DH) and [TOAC20]N-t(DH) analogues bound to the membranes. Therefore, the two spectral components correspond to peptide partitioning between membrane and water phases, whose populations are in slow-exchange in the ESR time scale. Notably, the peptide-membrane interactions evidenced in this study were found to be modulated by the lipid composition of the membranes. This is in agreement with


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Afonin et al.,46 which showed that the peptide orientation, membrane insertion, and peptide–lipid interactions are largely governed by physicochemical properties of the constituent lipids of the membranes. Moreover, cardiolipin plays an important role in the peptide interaction with the membrane. The natural peptide microdomain environment, i.e. the inner mitochondrial membrane, contains a high percentage of cardiolipin (~ 20%) as compared to other lipid membranes in human cells. Unlike other lipids, cardiolipin presents two phosphatidyl moieties linked by a glycerol group, which, as mentioned above, results in a relatively small negative head group and a large hydrophobic tail formed by four acyl chains. Thus, because of the restricted movement resulting from its size, cardiolipin is also not able to form hydrogen bonds that can stabilize the negative charge of the head group and, therefore, the interaction with other lipids reduces the effective size of the polar head, promoting the formation of nonlamellar structures (i.e. mitochondrial cristae). In addition, the binary mixtures of CL with PC tend to form stable mixtures, resulting in only lamellar phases without any domains or structures.45 Interestingly, the electrostatic surface of the peptide presented in Figure S6 (Supporting Information) shows that negative and positive charges along the peptide sequence are roughly in opposite sides of the peptide. This charge arrangement is assumed to be formed after the membrane binding, when the peptides acquire the helical conformation, since in aqueous solution they presented a predominant disordered structure. Although, the peptide also has considerable hydrophobic surface (38%), which can assist the membrane anchoring and affinity. Our ESR assays clearly show a preferential binding to CL-membranes, especially for [TOAC12]N-t(DH) and [TOAC20]N-t(DH) in the ternary POPC-POPE-CL membrane composition. In this case, even with a peptide net charge at pH 7 around – 2 and a negatively charged membrane surface density, the hydrophobic forces in this pep-



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tide/lipid system are strong enough to favor virtually all peptide binding to the membrane. It is well known from the literature that membrane-protein interactions are classically described as a first electrostatic contact followed by the insertion of hydrophobic groups into the lipid bilayer.47 As proposed by Couto et al.11 and Vicente et al.,10 the microdomain peptide presented flexibility between the two helices, which is promoted by the connecting loop (see NMR section). Such flexibility seems to play a very important role in the affinity of the peptide for the membrane, triggering and driving the forces that lead to higher binding for some of the membrane mimetics studied. Also, it indicates that microdomain motions increases the affinity for specific membrane lipid domains, especially those containing cardiolipin.

Conclusions Our results demonstrate the molecular interactions of the microdomain from HsDHODH with membrane mimetics. The connecting loop provides an important conformational flexibility to both microdomain helices, helping the molecule to bind to different membrane mimetics. Particularly, cardiolipin seems to be essential for HsDHODH microdomain membrane interactions, since it promotes a complete peptide partitioning into CL-containing membranes. Therefore, to the best of our knowledge, this study evidences for the first time, using peptide analogues models to analyze the affinity and dynamics of the microdomain, that CL could play an important role in the HsDHODH microdomain function and enzyme activity, thus might play an essential role for the complete catalysis. Moreover, the great flexibility demonstrated by the Nt(DH) microdomain should be considered in further studies aimed at the research and development of new HsDHODH enzyme inhibitory compounds to fight proliferative diseases.


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Acknowledgments This work was generously supported and funded by CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) with PDSE/BEX scholarship Grant Number 6834/12-4 (to E.F.V.), CNPq (Conselho Nacional de Desenvolvimento Científíco e Tecnológico), FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) Grant Number 2010/06526-6 (to E.M.C.) and 2015/18390-5 (to A.J.C.F). LGMB thanks FAPESP for the fellowship (Grant Number 2014/00206-0). E.C.J thanks FAPESP for the fellowship (Grant Number 2010/12953-4).

Supporting Information. Chromatograms and Mass Spectra of the synthesized peptide analogues. Experimental and best-fit ESR spectra NLLS simulations of analogues in liposomes, g- and hyperfine-tensor components of TOAC in NLLS simulations. Summary of NMR structure parameters, NOE statistics, charges distribution and chemical shifts of N-t(DH) peptide.



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References (1) Lane, A. N.; Fan, T. W. Regulation of Mammalian Nucleotide Metabolism and Biosynthesis. Nucleic Acids Res. 2015, 43, 2466-2485. (2) Herrmann, M. L.; Schleyerbach, R.; Kirschbaum, B. J. Leflunomide: an Immunomodulatory Drug for the Treatment of Rheumatoid Arthritis and other Autoimmune Diseases. Immunopharmacology 2000, 47, 273-289. (3) Baumann, P.; Mandl-Weber, S.; Volkl, A.; Adam, C.; Bumeder, I.; Oduncu, F.; Schmidmaier, R. Dihydroorotate Dehydrogenase Inhibitor A771726 (Leflunomide) Induces Apoptosis and Diminishes Proliferation of Multiple Myeloma Cells. Mol. Cancer Ther. 2009, 8, 366-375. (4) Fang, J. X.; Uchiumi, T.; Yagi, M.; Matsumoto, S.; Amamoto, R.; Takazaki, S.; Yamaza, H.; Nonaka, K.; Kang, D. C. Dihydroorotate Dehydrogenase is Physically Associated with the Respiratory Complex and Its Loss Leads to Mitochondrial Dysfunction. Biosci. Rep. 2013, 33, 217-227. (5) Vicente, E. F.; Nobre, T. M.; Pavinatto, F. J.; Oliveira, O. N.; Costa-Filho, A. J.; Cilli, E. M. N-Terminal Microdomain Peptide from Human Dihydroorotate Dehydrogenase: Structure and Model Membrane Interactions. Protein Pept. Lett. 2014, 22, 119-129. (6) Li, S. L.; Luan, G. Q.; Ren, X. L.; Song, W. L.; Xu, L. X.; Xu, M. H.; Zhu, J. S.; Dong, D.; Diao, Y. Y.; Liu, X. F. et al. Rational Design of BenzylidenehydrazinylSubstituted Thiazole Derivatives as Potent Inhibitors of Human Dihydroorotate Dehydrogenase with In Vivo Anti-Arthritic Activity. Sci. Rep. 2015, 5, 14836. (7) Munier-Lehmann, H.; Lucas-Hourani, M.; Guillou, S.; Helynck, O.; Zanghi, G.; Noel, A.; Tangy, F.; Vidalain, P. O.; Janin, Y. L. Original 2-(3-Alkoxy-1H-pyrazol-1yl)pyrimidine Derivatives as Inhibitors of Human Dihydroorotate Dehydrogenase (DHODH). J. Med. Chem. 2015, 58, 860-877. (8) Rawls, J.; Knecht, W.; Diekert, K.; Lill, R.; Loffler, M. Requirements for the Mitochondrial Import and Localization of Dihydroorotate Dehydrogenase. Eur. J. Biochem. 2000, 267, 2079-2087. (9) Liu, S. P.; Neidhardt, E. A.; Grossman, T. H.; Ocain, T.; Clardy, J. Structures of Human Dihydroorotate Dehydrogenase in Complex with Antiproliferative Agents. Structure Fold. Des. 2000, 8, 25-33. (10) Vicente, E. F.; Sahu, I. D.; Costa-Filho, A. J.; Cilli, E. M.; Lorigan, G. A. Conformational Changes of the HsDHODH N-terminal Microdomain via DEER Spectroscopy. J. Phys. Chem. B 2015, 119, 8693-8697. (11) Couto, S. G.; Nonato, M. C.; Costa-Filho, A. J. Site Directed Spin Labeling Studies of Escherichia coli Dihydroorotate Dehydrogenase N-terminal Extension. Biochem. Biophys. Res. Commun. 2011, 414, 487-492. (12) Couto, S. G.; Nonato, M. C.; Costa-Filho, A. J. Defects in Vesicle Core Induced by Escherichia coli Dihydroorotate Dehydrogenase. Biophys. J. 2008, 94, 1746-1753. (13) Vicente, E. F.; Basso, L. G.; Cespedes, G. F.; Lorenzon, E. N.; Castro, M. S.; Mendes-Giannini, M. J.; Costa-Filho, A. J.; Cilli, E. M. Dynamics and Conformational Studies of TOAC Spin Labeled Analogues of Ctx(Ile21)-Ha Peptide from Hypsiboas albopunctatus. PLoS One 2013, 8, e60818. (14) Cilli, E. M.; Vicente, E. F.; Crusca Jr, E.; Nakaie, C. R. EPR Investigation of the Influence of Side Chain Protecting Groups on Peptide-Resin Solvation of the Asx and Glx Model Containing Peptides. Tetrahedron Lett. 2007, 48, 5521-5524. (15) Teixeira, L. G.; Malavolta, L.; Bersanetti, P. A.; Schreier, S.; Carmona, A. K.; Nakaie, C. R. Conformational Properties of Seven Toac-Labeled Angiotensin I


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DHODH Microdomain-Membrane Interactions are Influenced by the

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