Subscriber access provided by - Access paid by the | UCSB Libraries
Article
Structure-Function Relationships in the Oligomeric NADPH-Dependent Assimilatory Sulfite Reductase Isabel Askenasy, Daniel Murray, Rachel M. Andrews, Vladimir N. Uversky, Huan He, and M. Elizabeth Stroupe Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00446 • Publication Date (Web): 22 May 2018 Downloaded from http://pubs.acs.org on May 29, 2018
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 41 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
Biochemistry
Structure-Function Relationships in the Oligomeric NADPH-Dependent Assimilatory Sulfite Reductase Isabel Askenasy#^, Daniel T. Murray#, Rachel M. Andrews#%, Vladimir N. Uversky&, Huan He@+, and M. Elizabeth Stroupe#@* #
Department of Biological Science and @Institute of Molecular Biophysics, Florida State
University, 91 Chieftain Way Tallahassee, FL 32306 ^Current address: University of WisconsinMadison, Department of Biomolecular Chemistry, 440 Henry Mall, Biochemical Sciences Building, Room 4206C, Madison, WI 53706, USA %Current address: Department of Microbiology, University of Alabama, Bevill Biomedical Research Building, Suite 276/11, 1720 2nd Avenue South, Birmingham, AL 35294-2170 and &Department of Molecular Medicine and USF Health Byrd Alzheimer's Research Institute, Morsani College of Medicine, University of South Florida, Tampa, Florida 33612, USA; Institute for Biological Instrumentation of the Russian Academy of Sciences, Institutskaya str., 7, Pushchino, Moscow region, 142290 Russia +
Translational Science Laboratory, College of Medicine, Florida State University, Tallahassee, FL 32306
*To whom correspondences should be addressed: e-mail:
[email protected] phone: 850-644-1751
ACS Paragon Plus Environment
1
Biochemistry 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
Page 2 of 41
KEYWORDS: NADPH-dependent assimilatory sulfite reductase (SiR); SiR flavoprotein; SiR hemoprotein; cytochrome p450 reductase; electron transfer; intrinsically disordered protein (IDP); intrinsically disordered protein region (IDPR); isothermal titration calorimetry (ITC); protein-protein interactions
ABSTRACT: The central step in the assimilation of sulfur is a six-electron reduction of sulfite to sulfide, catalyzed by the oxidoreductase NADPH-dependent assimilatory sulfite reductase (SiR). SiR is composed of two subunits. One is a multi-domain flavin-binding reductase (SiRFP) and the other an iron-containing oxidase (SiRHP). Both enzymes are primarily globular, as expected from their functions as redox enzymes. Consequently, we know a fair amount about their structures but not how they assemble. Curiously, both structures have conspicuous regions that are structurally undefined, leaving questions about their functions and raising the possibility that they are critical in forming the larger complex. Here, we used UV - Visible and CD spectroscopy, isothermal titration calorimetry, proteolytic sensitivity tests, electrospray ionization mass spectrometry, and activity assays to explore the effect of altering specific amino acids in SiRFP on their function in the holoenzyme complex. Additionally, we used computational analysis to predict the propensity for intrinsic disorder within both subunits and found that SiRHP’s N-terminus is predicted to have properties associated with intrinsic disorder. Both proteins also contained internal regions with properties indicative of intrinsic disorder. We showed that SiRHP’s N-terminal disordered region is critical for complex formation. Together with our analysis of SiRFP amino acid variants, we show how molecular interactions outside the core of each SiR globular enzyme drive complex assembly of this prototypical oxidoreductase.
ACS Paragon Plus Environment
2
Page 3 of 41 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
Biochemistry
INTRODUCTION: Reduced sulfur is essential because it is a building block of sulfur containing biomolecules. The central step in the bio-geo cycling of sulfur is a six-electron reduction that converts sulfite (SO32-) to sulfide (S2-), catalyzed by the oxidoreductase sulfite reductase (Figure 1A) (1). Sulfur reduction is a universal biochemical process as the basis for sulfur-based anaerobic respiration in a wide range of microorganisms (dissimilatory sulfur reduction) or to prepare sulfur for incorporation into sulfur-containing biomolecules (assimilatory sulfur reduction). In γ-proteobacteria like Escherichia coli, NADPH-dependent assimilatory sulfite reductase (SiR, E. C. 1.8.1.2) is an oligomeric, 800 kDa complex composed of eight copies of a flavin-binding reductase called the flavoprotein (SiRFP) and four copies of a metalloenzyme oxidase called the hemoprotein (SiRHP) (2, 3). SiRFP is a cytochrome p450 reductase (CPR) homolog that channels electrons from NADPH through tightly-bound FAD and FMN cofactors and on to SiRHP (4). SiRHP uses a Fe4S4 cluster covalently bound through one of its cysteine ligands to the iron of an iron-containing tetrapyrrole called siroheme (2). Siroheme-dependent SiRHP is the most commonly found enzyme that performs sulfur reduction in the dissimilatory and assimilatory pathways and the E. coli homolog is the model for understanding how SiR participated in the transition from a sulfur- to an oxygen-based environment (5). CPR and its homologs are multidomain reductases that deliver electrons to their oxidase partners through a closed-to-open transition whose importance has been inferred from X-ray crystal structures of CPR (Figure 1B and C) (6-9). When CPR is closed, the FMN binding domain, which is homologous to flavodoxin (Fld), sits adjacent to the FAD/NADPH binding domain, which is homologous to ferredoxin/NADP+ reductase (FNR) (6, 8, 10). When CPR is open, the Fld domain swings away to deliver electrons to its oxidase partner (7, 9). The exact
ACS Paragon Plus Environment
3
Biochemistry 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
Page 4 of 41
Figure 1. SiRFP is a multi-domain protein. A. Schematic of the chemistry performed by SiR. B. Domain architecture, nomenclature, and position of altered amino acids studied in this report. The octamerization domain is yellow and the SiRFP Fld domain (Fld1) is light green. Together, they make an octomer of Fld domains (Fld8). The linker is white, the FAD-binding domain that comprises the ferredoxin motif of SiRFP’s FNR is blue, the connection domain is red, and the
ACS Paragon Plus Environment
4
Page 5 of 41 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
Biochemistry
NADPH-binding domain that comprises the NADP+ reductase motif of SiRFP’s FNR motif is green. The later three domains comprise the monomeric FNR1 domain. SiRFP-60 is a monomeric form of Fld1 and FNR1, without the octamerization domain. C. Model of SiRFP based on an NMR structure of Fld1 (11) and an X-ray crystal structure of FNR1 (12), superimposed on the closed conformation of CPR (10). The domains are colored in a corresponding way to A but neither the N-terminus, which is responsible for SiRFP octamerization, nor the flexible linker joining the domains are pictured.
ACS Paragon Plus Environment
5
Biochemistry 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
Page 6 of 41
position of the domains in either conformation depends, at least in part, on the crystal context (6, 10) or subunit modification (7, 8) but the conformational change has also been measured in solution (13). CPR interacts with its cytochrome P450 oxidase (CYP) transiently through both charge-charge and hydrophobic interactions (7, 14-16). In contrast, SiRFP and SiRHP form a stable α8β4 holoenzyme, the nature of which has not been fully assessed because of three properties that are specific to SiR. First, SiRFP’s N-terminal 51 amino acids mediate formation of an octamer that is unique to this homolog (17). With the N-terminus, the Fld domain is also octameric (Fld8) but without it is monomeric (Fld1) (Figure 1B). Truncation of those amino acids results in a 60 kDa monomer (SiRFP-60) that binds and mediates electron transfer to SiRHP as a minimal, functional dimer (18). Second, SiRHP binds the FNR domain of SiRFP (3), which is monomeric (FNR1, Figure 1B), but the electrons pass from the Fld domain to SiRHP’s siroheme/Fe4-S4 cluster (19). Third, SiRHP’s 60 N-terminal amino acids must be removed to grow crystals of the enzyme so little is known about their structure despite their importance for complex formation (3, 20). Here, we tested the hypothesis that stable binding between SiRFP and SiRHP is driven by hydrophobic interactions between FNR1 and the N-terminus of SiRHP at a structural binding site driven by a disorder-to-order transition that is distinct from the functional, transient interaction that allows for electron transfer between the subunits. We used UV - Visible and CD spectroscopy, isothermal titration calorimetry, proteolytic sensitivity tests, electrospray ionization mass spectrometry (ESI-MS), and activity assays to measure the binding affinity between SiRFP-60 and SiRHP and to identify two amino acids in SiRFP, F496 and V500, that play an important role in subunit-subunit binding.
EXPERIMENTAL METHODS:
ACS Paragon Plus Environment
6
Page 7 of 41 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
Biochemistry
Protein expression and purification – The N-terminal six-histidine tagged SiRFP-60 and FNR1 expression constructs were generated by subcloning 5’ truncated forms from pJWY613 (21) and inserting then independently into the pBAD/myc/hisA plasmid (Life Technologies, Inc., Carlsbad, CA, USA). Each SiRFP-60 amino acid variant was generated by Q5 site-directed mutagenesis (New England BioLabs, Ipswich, MA, US) or a standard QuikChange protocol (22) with the appropriate set of primers (SI Table 1). SiRFP-60 variants and SiRHP/FNR1 dimer were expressed in LMG194 E. coli cells grown in LB media to O.D.600 = 0.6, induced with 0.05% arabinose and incubated for 4 hours at 25ºC. Cells were harvested, resuspended, and lysed in 1 X SPG buffer (12.5 mM Succinic Acid, pH 6.8, 50 mM NaH2PO4 and 37.5 mM Glycine), 200 mM NaCl. FNR1-expresssing cells were mixed with SiRHP-expressing cells (3) before lysis. For each preparation, cell lysate was clarified by centrifugation at 16,000 X g for 35 min in a 5810R Eppendorf AG centrifuge (Hamburg, Germany) equipped with an F-34-6-38 fixed angle rotor. All steps were performed at 4ºC. The clarified cell lysate was stirred with 0.1% polyethyleneimine for 20 min and then centrifuged at 10,000 X g. The supernatant was loaded onto a 5-ml HisTrap HP NiNTA column (GE Healthcare, Little Chalfont, UK). Protein was eluted with a gradient of 5 to 500 mM imidazole in 1 X SPG buffer, 200 mM NaCl. SiRFP-60or SiRHP/FNR1-containing fractions were dialyzed overnight in 0.2 X SPG buffer, 1 mM EDTA. The dialyzed sample was then loaded onto a 5-ml HiTrap Q-Sepharose Fast Flow column (GE Healthcare, Little Chalfont, UK) buffer and eluted with a gradient of KCl from 10 to 1,000 mM in 0.2 X SPG, 1 mM EDTA. Finally, the fractions containing SiRFP-60 or SiRHP/FNR1 were concentrated and loaded onto a HiPrep 26/60 Sephacryl S300 HR (GE Healthcare, Little Chalfont, UK) for SiRFP-60 or a Superose 6 (GE Healthcare, Little Chalfont, UK) for SiRHP/FNR1 size exclusion chromatography (SEC) column. SEC columns were
ACS Paragon Plus Environment
7
Biochemistry 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
Page 8 of 41
equilibrated in 1 X SPG buffer, 1 mM EDTA. Protein purity was assessed by SDS-PAGE. Importantly, between each individual protein preparation and the next, columns were washed with 0.2 M NaOH to ensure the integrity of every preparation used in this study. Concentrations for each protein variant were determined using variant-specific extinction coefficients obtained from individual bicinchoninic acid assays (Life Technology, Inc., Carlsbad, CA, USA) following the manufacturer’s protocol. Cofactor occupancy was monitored by UVVis spectroscopy on an Agilent 8453 spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). ITC measurementsITC experiments were run on a VP-ITC instrument (Malvern-MicroCal, Malvern, UK) at 25º C. All samples were dialyzed overnight in 1 X SPG buffer, 1 mM EDTA, and degassed for 30 min. SiRHP or NADP+ (Sigma-Aldrich, St. Louis, MO, USA) was injected into SiRFP-60 (WT or variants) in a total of 40 injections of 6 µl at 200 sec intervals per titration, with agitation upon addition of each injection. In the protein-protein titrations, concentrations were adjusted to 5 µM SiRFP-60 (WT and variants) and 50 µM SiRHP. In the SiRFP-60/NADP+ titrations, concentrations were adjusted to 9.0 µM SiRFP-60 and 1 mM NADP+. The baseline was determined by titrating SiRHP or NADP+ into buffer alone. We used SiRFP-60 to simplify the ITC analysis, and NADP+ rather than NADPH to avoid complications from partial oxidation of the reductant. There is a known 1:1 stoichiometry between SiRFP-60 and SiRHP (18). In contrast to its tight binding to SiRHP, SiRFP-60 does not bind NADP+ with high affinity. Consequently, in our experimental setup for measuring NADP+ binding the Wiseman “c” parameter, which is the product of the Ka, the total molecular concentration in the reaction cell, and the number of interaction sites, was < 1. Although this c parameter was lower than the “experimental window” for which unconstrained fitting of the
ACS Paragon Plus Environment
8
Page 9 of 41 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
Biochemistry
isotherm is optimal (23), we know with certainty that there is a single NADP+ binding site on SiRFP-60 because it binds the nucleotide with a Rossmann fold (12, 24). Therefore, all data was analyzed with AffiniMeter (affinimeter.com), using its global fitting algorithm and the simple 1:1 model to determine the Kd, ∆H, and T∆S for each isotherm. Each binding assay was performed in triplicate. Disorder prediction in SiRFP and SiRHPIntrinsic disorder propensities of SiRFP and SiRHP were analyzed using several established per-residue disorder predictors each having a unique strength, including PONDR® VLXT (25), PONDR® VSL2B (26), PONDR® VL3 (27), IUPred (28), and PONDR® FIT (29). For each test, scores above 0.5 correspond to the disordered residues/regions. We also calculated the mean disorder propensity by averaging disorder profiles of individual predictors because empirical observations report that use of consensus for evaluating intrinsic disorder can increase the predictive performance over the use of a single predictor (30, 31). We also used ANCHOR (32, 33) to look for predictable sites that undergo disorder-to-order transitions when interacting with specific partners in SiRFP and SiRHP. Activity assaysThe ability of SiRFP-60 and variants to transfer electrons from NADPH to cytochrome c was measured under anaerobic conditions. All solutions were degassed through three freeze-thaw cycles followed by a 30 min purge step under vacuum. Reactions were set up in triplicate in a Unilab anaerobic chamber (mBRAUN, Stratham, NH, USA) under a nitrogen environment and each was sealed in a 1 mL gas-tight quartz cuvette with a 1 cm pathlength. The reaction mixture contained 50 mM Tris, pH 7.5, 0.2 mM NADPH (Sigma-Aldrich, St. Louis, MO, USA), 0.05 mM oxidized cytochrome c from equine heart (Sigma-Aldrich, St. Louis, MO, USA), 10 units of glucose oxidase (Sigma-Aldrich, St. Louis, MO, USA), and 10 mM glucose. The reactions were initiated with injection of 5 µl of 10 nM SiRFP-60 WT or variants. Reduction
ACS Paragon Plus Environment
9
Biochemistry 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
Page 10 of 41
of cytochrome c was monitored at 550 nm as previously described (34) on an 8453 UV-Vis spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). We also measured the specific activity of electron transfer from SiRFP to SiRHP by mixing
SiRFP-60
variants
and
SiRHP
at
a
2:1
ratio.
Activity
was
monitored
spectrophotometrically at 340 nm to measure SO32--dependent NADPH oxidation (19). Reactions contained 1 mM potassium phosphate pH 7.7, 0.2 mM NADPH (Sigma-Aldrich, St. Louis, MO, USA), 10 units of glucose oxidase (Sigma-Aldrich, St. Louis, MO, USA), 0.5 mM NaSO3 and 10 mM glucose, and were initiated by injection of 5 µl of the enzyme to anaerobic cuvettes. All enzymatic activities were background corrected for non-specific activity due to oxygen introduced during protein injection. For specific activity measurements monitoring reduction of cytochrome c, the control reaction did not include NADPH. For specific activity measurements monitoring SO32--dependent NADPH oxidation, the control reaction did not include SO32-. CD spectroscopySamples of SiRFP-60 WT or variants were dialyzed overnight into 50 mM potassium phosphate buffer, pH 7.7, 1 mM EDTA. The protein concentrations were adjusted to 0.08 mg/ml. CD experiments were carried out in an AVIV spectrometer, model 410 (AVIV Biomedical, Lakewood, NJ) attached to a CFT-33 circulating chiller (NESLAB, Portsmouth, NH). CD spectra were collected between 260 and 190 nm using a 1 mm path quartz cuvette with a 1 nm bandwidth and 3 sec averaging time. Three scans were collected for each sample. Data was background-corrected, smoothed and converted to mean residue ellipticity. Proteolytic sensitivitySiRFP-60, SiRHP, and SiRHP/FNR1 were dialyzed into thermolysin buffer containing 50 mM Tris buffer, pH 7.8, 150 mM KCl, and 5 mM CaCl2. Proteins were diluted to 0.5 mg/mL and thermolysin was added to a final concentration of 1 mg/mL. For
ACS Paragon Plus Environment
10
Page 11 of 41 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
Biochemistry
SiRFP-60, time points were taken immediately upon thermolysin addition as well as at 1 and 2 minutes. For SiRHP or SiRHP/FNR1, time points were taken immediately upon thermolysin addition as well as at 5 and 10 minutes. All samples were quenched with 10 mM ETDA before being prepared for SDS-PAGE analysis. The resulting protein fragments were identified with Nterminal Edman degradation on a PPSQ-53A protein sequencer (Shimadzu, Kyoto, Japan) or tryptic digestion mass spectrometry. SiRHP/FNR1 was used rather than SiRHP/SiRFP-60 to avoid complications from the extreme sensitivity of Fld1 and because SiRHP/FNR1 dimer can be purified stoichiometrically. Nano-liquid chromatography nLC/MS/MS In-gel protein trypsin digestion was performed with a ProteoExtract All-in-One Trypsin Digestion Kit (EMD Millipore, Billerica, MA, USA). Gel bands were excised and de-stained with wash buffer; cut into ~ 1 mm gel pieces; and dried by incubation with acetonitrile followed by vacuum treatment. Dried gel pieces were rehydrated with 50 µL digestion buffer. 1 uL 0.25 µg/µL Trypsin was added and the mixture was incubated at 37oC overnight. 50 µL of 6% formic acid aqueous solution was added to quench the digestion and the supernatant was collected. The gel pieces were again incubated with 75 µL acetonitrile before the supernatant was collected and dried in a SpeedVac (Thermo Scientific, Sunnyvale, CA, USA). The dried peptide mixture was re-dissolved in 0.5% formic acid and separated by nLC with an Easy Nano LC II system (Thermo Scientific) with a 100 µm x 2 cm trap column (Thermo Scientific) and a 75 µm x 10 cm C18AQ analytical column (Thermo Scientific). Mobile phases were A (99.9% H2O and 0.1% formic acid) and B (99.9% acetonitrile and 0.1% formic acid). A linear gradient from 0% to 30% B over 1 hr was performed with a flow rate of 300 nL/min. Eluate was on-line ionized by nano-electrospray ionization (nESI) and detected by a
ACS Paragon Plus Environment
11
Biochemistry 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
Page 12 of 41
Velos LTQ-Orbitrap Mass Spectrometer (Thermo Scientific). The precursor ions were detected with a mass resolution of 60 K (at m/z of 800 Da) in the Orbitrap. Data-dependent MS2 was carried out on the top 10 most abundant precursor ions with collision-induced dissociation in the LTQ. Acquired Xcalibur .raw files were analyzed by Proteome Discoverer 1.4 with Sequest HT (Thermo Scientific) against a database containing only SiRFP-60, SiRHP and thermolysin. The result data were then combined for further manual analysis. For peptide abundance comparison and to account for the difference of protein loading amount, the acquired area under curve (AUC) of each peptide was normalized to the most abundant peptide of the corresponding protein with good reproducibility. RESULTS Design and generation of SiRFP variantsWe designed a series of rational amino acid variants in each SiRFP-60 domain. First, we targeted a conserved, hydrophobic amino acid within the Fld domain, Y101 (Figure 1B and C). The tyrosine’s bulky sidechain was predicted to play a role in electron transfer so we altered it to an alanine (Y101A). Next, we targeted a polar amino acid within the Fld domain, Q191, which we altered to a positively charged lysine. The analogous amino acid in the closed CPR conformation makes charge-charge interactions with the opposing domain (Figure 1B and C) but Q191’s sidechain interactions are not defined because there is no structure of full-length SiRFP-60 (12, 35). We also targeted two hydrophobic amino acids within the FNR domain, F496 and V500 (Figure 1B and C), and altered each to an aspartic acid to disrupt any potential hydrophobic interactions they might mediate (F496D and V500D). We then synthesized point mutations in expression constructs for SiRFP-60.
ACS Paragon Plus Environment
12
Page 13 of 41 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
Biochemistry
Figure 2. SiRFP-60 variants. A. SDS-PAGE analysis of purified monomeric SiRFP-60 variants shows each can be purified to homogeneity. B. UV-Vis spectroscopy of the SiRFP-60 variants shows that flavin co-factor occupancy is not altered by the mutagenesis, indicating proper folding of the enzymes.
ACS Paragon Plus Environment
13
Biochemistry 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
SiRFP-60 variant WT Y101A Q191K F496D V500D Y101A-Q191K Y101A-V500D Q191K-V500D Y/A-Q/K-V/D
∆H T∆S A455:A280 (K*cal/mol) (cal/mol) ratio 2.7 ± 0.4 -16,000 ± 1000 -4,90 ± 1,000 0.13 2.8 ± 0.7 -16,000 ± 100 -5,500 ± 3,000 0.14 2.9 ± 0.4 -16,000 ± 3000 -4,200 ± 2,000 0.13 N. M. N. M. N. M. 0.14 160 ± 20 -14,000 ± 200 -4,500 ± 2,000 0.12 2.2 ± 0.6 -17,000 ± 200 -4,900 ± 2,000 0.16 180 ± 70 -16,000 ± 100 -5,000 ± 1,000 0.16 240 ± 80 -15,000 ± 600 -5,900 ± 500 0.17 250 ± 50 -15,000 ± 600 -5,90 ± 700 0.15 N. M. - not measurable N. D. - not determined K d (nM)
Page 14 of 41
spec. act. to cyt c -
(e /min flavin) 2500 ± 200 1800 ± 200 2400 ± 200 570 ± 70 2400 ± 300 N. D. N. D. N. D. N. D.
spec. act. to SiRHP -
(e /min siroheme) 120 ± 10 70 ± 20 99 ± 20 4±2 46 ± 5 110 ± 40 41 ± 3 42 ± 8 44 ± 10
Table 1. Biochemical parameters from ITC, UV-Vis, and activity assays. Standard deviations were calculated from averaging three independent runs.
ACS Paragon Plus Environment
14
Page 15 of 41 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
Biochemistry
We expressed and purified to homogeneity each of the single variants (Y101A, Q191K, F496D, and V500D) and the double (Y101A-Q191K, Y101A-V500D, Q191K-V500D) or triple (Y101AQ191K-V500D) variants. Our ability to generate >99% pure protein sample was not affected by alteration of the amino acid sequence (Figure 2A). SiRFP activity depends on integral flavin cofactor binding. The ratio of the UV-Visible (UV-Vis) absorbance at 280 nm due to tryptophan and tyrosine versus UV-Vis absorbance at 455 nm due to the flavin cofactors is an excellent measure of proper folding because misfolded proteins are not able to bind the cofactor, resulting in a low A455:A280 ratio. Similarly, the presence of non-flavin-containing proteins or flavincontaining proteins with different extinction coefficients would alter the A455:A280 ratio. By these measures, no single amino acid variant or combination thereof abolished SiRFP-60’s ability to bind FAD or FMN (Figure 2B and Table 1). Together, SDS-PAGE and UV-Vis analysis showed our protein reagents were of high quality. SiRFP-60 and SiRHP bind with nM affinityWe first set out to characterize the tight subunit binding that is unique to the SiR oxidoreductase by use of isothermal titration calorimetry (ITC; Table 1). We used monomeric SiRFP-60 in these experiments to simplify the analysis because there is a single stable SiRHP binding site on SiRFP-60. Titrating SiRHP into SiRFP-60 resulted in a curve with a profile characteristic of strong, low-nanomolar binding with a steep transition from unbound to bound (Table 1 and SI Figure 1A). Lowering the protein concentration or altering the injection volume resulted in isotherms with low signal-to-noise ratios, an observation that is also characteristic of tight binding – and one that has been made by others who were unsuccessful at making these measurements by ITC (18). Variation of the amino acids predicted to play a role in electron transfer (Y101) or conformational change (Q191) did not significantly affect the steep titration profile or strength of binding (SI Figures 1B and C). In contrast, the
ACS Paragon Plus Environment
15
Biochemistry 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
Page 16 of 41
Figure 3. Intrinsic disorder propensity of SiR subunits. A. SiRFP and B. SiRHP were evaluated by the members of PONDR family of disorder predictors, PONDR® VLXT (25), PONDR® VSL2 (26), PONDR® VL3 (27), IUPred_short and IUPred_long (28), and PONDR® FIT (29). Mean disorder propensity (blue) was calculated by averaging disorder profiles of individual predictors. The standard deviation across predictors is shown in cyan.
ACS Paragon Plus Environment
16
Page 17 of 41 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
Biochemistry
single variant F496D did not show measurable binding in the ITC isotherm (SI Figure 1D). V500D resulted in a shallow transition from unbound to bound and ~70-fold weaker binding (SI Figure 1E). The double variant Y101A-Q191K bound to SiRHP in a manner similar to the single variants Y101A and Q191K (SI Figure 1F) whereas all the multiple variants that include V500D (Y101A-V500D, Q191K-V500D, and Y101A-Q191K-V500D) bound with reduced affinity (SI Figures 1G, H, and I). Intrinsically disordered regions in SiR are pervasiveSiRFP exhibits significant conformational heterogeneity that limited analysis by X-ray crystallography (12). In SiRHP, the N-terminal 60 amino acids are proteolytically cleaved and the next 20 amino acids are not ordered in its X-ray crystal structure (20). Based on these observations, we hypothesized that some regions of each protein might exhibit characteristics of intrinsic disorder, perhaps playing a role in subunit-subunit assembly via a disorder-based binding site. To test this hypothesis, we analyzed their primary structure by use of per-residue disorder predictors (Figure 3). Not surprisingly, the whole SiRFP polypeptide had a mean-mean disorder prediction of 0.29 ± 0.2 and the whole SiRHP polypeptide had a mean-mean disorder prediction of 0.27 ± 0.1, indicating mainly globular enzyme subunits. However, both SiRHP and SiRFP have several predicted intrinsically disordered protein regions (IDPRs) in their least well structurally characterized regions (Figure 3). The N-terminus of SiRFP was predicted to be highly disordered and expected to contain a disorder-based binding site from residues 35 to 42 (Figure 3A). Additionally, disorder was predicted within the linker between the SiRFP Fld and FNR domains, residues 200 - 280 (Figure 3A). Similarly, the N-terminus of SiRHP was predicted to be mostly disordered and to house a disorder-based binding site between amino acids 48 and 55 (Figure 3B). A second
ACS Paragon Plus Environment
17
Biochemistry 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
Page 18 of 41
SiRFP variant
Kd (mM)
∆H (cal mol-1)
T∆S (K*cal mol-1)
WT F496D V500D
0.12 ± 0.03 N. D. 0.15 ± 0.02
-7,400 ± 1,000 N. D. -16,000 ± 1,000
-1,200 ± 2,000 N. D. -11,000 ± 800
Table 2: NAD+ binding to SiRFP-60 and its F496D and V500D variants measured by ITC.
Figure 4: CD analysis of SiRFP-60 and its F496D and V500D variants.
ACS Paragon Plus Environment
18
Page 19 of 41 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
Biochemistry
predicted region of disorder in SiRHP was identified between amino acids 300 and 350 and a third at its C-terminus (Figure 3B). NADP+ cofactor binding and activity assaysSiRFP uses a dissociable NADP(H) cofactor as an electron source that initiates electron transfer (36). The F496D and V500D variants altered amino acids close to the NADP(H) binding site, although those amino acids do not directly bind the cofactor (Figure 1). We tested whether those variants disrupted NADP+ binding by use of ITC. SiRFP-60 binding to NADP+ resulted in a hyperbolic curve (SI Figure 2A), characteristic of weak binding with a low c parameter in the experimental setup (23). F496D binding of NADP+ was not measurable by ITC (SI Figure 2B and Table 2) whereas V500D bound NADP+ with similar affinity as WT SiRFP-60 (SI Figure 2C and Table 2). As expected from integral flavin binding by both the F496D and V500D variants, neither enzyme showed secondary structure altered from that of the WT enzyme (Figures 2B and 4). We then used a cytochrome c-based activity assay to test the variants’ ability to move electrons from NADPH to a non-physiological electron acceptor of SiRFP-60 that depends on the presence of a functional reductase but not on tight binding of the electron acceptor (34, 37). No single amino acid variant abolished SiRFP-60’s ability to reduce cytochrome c (Table 1), although the Y101A and F496D variants each showed reduced activity compared to that of WT enzyme. Similarly, the Y101A variant showed reduced activity in reducing its natural partner, SiRHP, whereas the Q191K variant was unaffected. In contrast, the V500D variant was reduced by about 60% in its ability to transfer electrons to SiRHP but the F496D variant was inactive (Table 1). Proteolytic sensitivity of SiRFP-60 and SiRHPProteolytic sensitivity is one marker of intrinsic disorder (38) so we tested the effect of thermolysin on SiRFP-60 and SiRHP. Both are
ACS Paragon Plus Environment
19
Biochemistry 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
Page 20 of 41
Figure 5: SiRFP-60 and SiRHP are highly modular proteins. A. Thermolysin (TL) proteolysis of SiRFP-60 shows rapid cleavage at the linker between the Fld and FNR domains. In = input; 0’ = timepoint after TL addition; 1’ = 1 minute timepoint; 2’ = 2 minute timepoint; T = TL. All fragments, including the TL fragment (TL frag) were identified by N-terminal Edman degradation and/or proteolytic mass spectrometry. Marker masses in kDa. Text at bottom: Ntermini of fragments, per Edman degradation. B. TL proteolysis of SiRHP and SiRHP/FNR1 show the SiRHP N-terminus is protected upon binding FNR1. In = input; 0’ = timepoint after TL addition; 5’ = 5 minute timepoint; 10’ = 10 minute timepoint; T = TL. Fragment 3 and 4 were identified with N-terminal Edman degradation and proteolytic mass spectrometry. HisFNR1 and FNR1 (*) were confirmed with Western blotting (SI Figure 3B). Marker masses in kDa. Text at bottom: N-termni of fragments, per Edman degradation.
ACS Paragon Plus Environment
20
Page 21 of 41 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
Biochemistry
highly modular enzymes and this was reflected in their sensitivity to thermolysin degradation (Figure 5). Thermolysin treatment of SiRFP-60 resulted in two fragments of ~40 kDa (band 1 in Figure 5A) and ~20 kDa (band 2 in Figure 5A). Over the remaining two minutes those fragments persisted. N-terminal sequencing of the larger fragment revealed it began at I226, 14 amino acids away from the start of the 43 kDa FNR domain due to degradation at the linker between the Fld and FNR domains. We predicted, then, that the 20 kDa fragment corresponded to the Fld domain so N-terminal sequencing would not be useful in identifying the site of C-terminal degradation. Therefore, we used ESI-MS to measure the peptide abundance in that sample and showed the presence of peptides corresponding to the N-terminus of SiRFP-60 through amino acid 210 (SI Figure 3A), confirming the degradation of the linker between SiRFP’s globular domains. We also assessed the proteolytic sensitivity of SiRHP’s N-terminus. SiRHP binds stably to FNR1 but not Fld8 (3). Although SiRHP also binds SiRFP-60 through its FNR domain (SI Figure 1 and Table 1), the SiRHP/SiRFP-60 dimer is not sufficiently well separated from free SiRFP-60 by SEC to purify it stoichiometrically. Further, SiRFP-60 is proteolytically sensitive. Therefore, we used SiRHP/FNR1 dimer to focus on the change in proteolytic sensitivity of SiRHP upon binding its partner. Thermolysin treatment of SiRHP results in fragments of ~60 kDa (band 3 in Figure 5B) and ~45 kDa (band 4 in Figure 5B). N-terminal sequencing, confirmed by ESI-MS (SI Figure 3B), showed that the larger fragment began at L75 whereas the shorter fragement began at I206. L75 corresponds to the end of the predicted region of disorder in SiRHP’s N-terminus (Figure 3B) and I206 corresponds to a loop near the active site that is disordered in the X-ray crystal structure (20). In marked contrast, neither of these fragments forms when SiRHP is bound to FNR1 (Figure 5B). Rather, the N-terminal histidine tag on FNR1
ACS Paragon Plus Environment
21
Biochemistry 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
Page 22 of 41
Figure 6. Model for the roles of IDPRs (black dashed lines) in the SiRFP-SiRHP holoenzyme. SiRFP is colored as in Figure 1. Each SiRHP sulfite and nitrite reductase repeat is colored pink or purple. The N-terminus of SiRFP mediates octamerization of SiRFP via a disorder-based binding site. The N-terminus of SiRHP also houses a disordered-based binding site that binds SiRFP at amino acids F496 and V500, which are close to the NADP(H) binding site. Electrons come from SiRFP’s Fld domain (light green) to SiRHP so flexibility in the linker between it and the FNR domain (Green/blue) is essential for productive enzyme activity.
ACS Paragon Plus Environment
22
Page 23 of 41 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
Biochemistry
is degraded leaving intact FNR1, confirmed with Western blotting using antibodies against the 6histidine epitope, SiRFP, or SiRHP but SiRHP persists as the full length protein (SI Figure 3C). DISCUSSION Multiple sites on SiRFP are required for full functionSiRFP, like its homologs CPR, the nitric oxide synthase reductase domain (NOSred), and methionine synthase reductase (MSR), is a multifaceted enzyme. These reductases must 1) bind cofactor 2) retain intra- and inter-molecular electron transfer 3) undergo domain rearrangement and 4) bind their oxidase partner. We altered amino acids that we hypothesized were critical for SiRFP function that, indeed, affected each of these aspects differently (Table 1). For example, the Y101A alteration did not hinder SiRFP’s ability to bind cofactor or SiRHP but reduced its ability to transfer electrons to a transientlybound oxidase or to SiRHP (Figures 2, 4 and Table 1). The Q191K alteration did not have a strong impact on function, suggesting that the closed conformation in SiRFP is different than it is in CPR (Figure 2 and Table 1). The V500D alteration reduced tight subunit-subunit interaction (SI Figure 1 and Table 1), resulting in decreased electron transfer to SiRHP but not a transiently bound oxidase (Table 1). We also identified F496 as an essential element of the interface between SiRFP and SiRHP. F496D bound its flavin cofactors with full occupancy (Figure 2) and had identical secondary structure to the WT enzyme (Figure 4). Nevertheless, this single amino acid alteration reduced both NADP+ and SiRHP binding sufficiently that neither was measurable by ITC (SI Figures 1, 2 and Tables 1, 2). Weak NADP(H) binding explains why the SiRFP-60-F496D had lower activity in reducing a transiently bound electron donor. Weak NADP(H) binding combined with loss of SiRHP binding explains its inability to catalyze SO32- reduction (Table 1). The deleterious effect of the SiRFP F496D and V500D alterations on SiRHP binding strongly supports the idea that the
ACS Paragon Plus Environment
23
Biochemistry 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
Page 24 of 41
SiR subunit-subunit interface was formed primarily by burying hydrophobic “hot-spots” specific amino acids that contribute the bulk of the binding energy, in contrast to many amino acids each contributing a small amount (39). Disorder in SiROthers have proposed that SiRFP and SiRHP interact through charge-charge interactions between SiRFP’s Fld domain and a surface adjacent to the SiRHP active site (35, 40), a prediction based in part on an X-ray crystal structure of SiRHP that lacks its N-terminus (20). We propose an alternative model, where SiRHP binds SiRFP’s FNR domain, far from SiRFP’s Fld domain, the ultimate electron donor (Figures 6). This model leaves open the question of how the Fld domain comes to interact with SiRHP. The answer to this question lies in regions of SiR that are not well characterized structurally (12, 20); shown to be proteolytically sensitive (Figure 5 and SI Figure 3); and predicted to be intrinsically disordered (Figure 3). Intrinsic disorder was historically considered unimportant for catalytic function. Recent analysis shows that IDPRs occur in enzymes at similar lengths and frequencies as in nonenzymes, often found at proteins’ N- or C-termini or as insertions between globular domains (41). These regions impact subunit-subunit interactions and enzyme function (42). We identified terminally- and centrally-located IDPRs in each SiR subunit that are important for holoenzyme structure and enzyme function (Figures 3 and 7). Structurally, we predicted that the N-terminus of SiRHP was disordered (Figures 3B and 6) and showed that it is protected from proteolysis upon binding SiRFP (Figure 5B). Taken with our identification of F496 and V500 as hydrophobic hot spots, we describe for the first time the mechanism underlying tight binding between the SiR subunits: a disorder to order transition when the N-terminus of SiRHP binds SiRFP’s FNR domain around amino acids F496 and V500.
ACS Paragon Plus Environment
24
Page 25 of 41 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
Biochemistry
Functionally, one of the predicted SiRFP IDPRs was within the linker that connects the Fld domain to the rest of the protein (Figures 3A and 6), the structure of which is also unknown (12) but is proteolytically sensitive (Figure 5A). The absence of a strong effect from the Q191K variant on electron transfer to cytochrome c or SiRHP supports the idea that SiRFP is particularly mobile because altering the charge at that position within the Fld domain does not affect electron transfer activity to a generic oxidase (cytochrome c) or its physiological partner. Nevertheless, predicted disorder in this linker likely explains the mechanism by which the Fld electron donor can access SiRHP, bound far from the mobile domain. Stoichiometry in SiR The stoichiometric mismatch between subunit number in SiR has long remained a mystery in the absence of a holoenzyme structure. SiRHP evolved from an ancient siroheme-dependent sulfite reductase that was a homodimer (5). A gene duplication event occurred leading to heterodimeric siroheme-dependent sulfite reductases with two active sites (5, 43-45). A subsequent gene fusion event lead to a pseudo two-fold symmetric monomer with only one active site (20, 46), as in SiRHP, where a long linker joins the halves. In SiRHP, the linker, amino acids 328-347, has no defined secondary structure but structurally mimics the siroheme (20). Interestingly, we predicted that these amino acids are an internal IDPR (Figures 3B and 6). Unlike other regulatory and catalytic functions commonly attributed to IDPRs that depend on the malleable nature of the peptide (47-49), this IDPR has a structural role in filling what would otherwise be an empty cavity. Others have explained the stoichiometric mismatch by positing that in SiRHP’s dimeric precursors, each active site had a dedicated reductase (20). Here, we characterized tight binding between SiRFP and SiRHP that is incompatible with the electron transfer-competent interface, suggesting that the dodecameric holoenzyme is made up of four SiRFP/SiRHP heterodimers and
ACS Paragon Plus Environment
25
Biochemistry 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
Page 26 of 41
four free SiRFP molecules. Six electrons come two at a time from three NADPH cofactors to complete catalysis, which means that SiRHP must interact with SiRFP three times for every SO32- that binds (Figure 1). One consequence of tight subunit binding is an increase of local SiRFP concentration relative to SiRHP in the holoenzyme, ensuring a continued supply of electrons to perform the complete reaction. ConclusionsSiR is an essential enzyme in the global sulfur cycle and shares many commonalities with the biomedically important CYP, NOSred, and MSR. The themes that link these enzymes – domain modularity, conformational dynamics, and transient protein-protein interactions – highlight the shortcomings in our understanding of protein chemistry that has resulted from powerful, but static, structure determination. At the same time, the unique aspects of SiR that set it aside from the other homologous systems – tight oxidase/reductase binding, a disorder-to-order binding event that forms the subunit-subunit interface, and protein-protein interaction driven by burying of otherwise solvent-exposed hydrophobic amino acids – provide new insight into the hierarchy of protein interactions that work together to mediate electron transfer in multisubunit oxidoreductase enzymes. Supporting Information: Supporting Information includes the raw ITC data for SiRFP60/SiRHP and SiRFP-60/NADP+ binding experiments; mass spectrometry data for peptide identification after limited proteolysis; Western blot analysis for protein fragment identification after limited proteolysis; and a table of the plasmids used in this study. Author Contributions: IA, DTM and RMA performed the experiments described in the manuscript and contributed to manuscript preparation. VNU performed the IDP/IDPR calculations and contributed to manuscript preparation. HH performed the mass spectrometry experiments. MES supervised experimentation and wrote the paper.
ACS Paragon Plus Environment
26
Page 27 of 41 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
Biochemistry
Funding Sources: This work was supported by National Science Foundation award MCB1149763 to MES. Acknowledgements: The authors would like to thank Dr. Claudius Mundoma for facilitating experiments on the ITC and CD instruments housed and maintained in the Biophysical Resource Facility in the Institute of Molecular Biophysics at Florida State University. N-terminal Edman degradation sequencing experiments were performed in the Iowa State University of Science and Technology Office of Biotechnology Protein Facility. We also thank Drs. Brian Miller, Scott Stagg, Margaret Seavy and Ángel Piñeiro for helpful conversations and Ms Angela Tavolieri for help with protein variant purifications. Abbreviations: NADPH-dependent assimilatory sulfite reductase (SiR); sulfite reductase flavoprotein (SiRFP); sulfite reductase hemoprotein (SiRHP); cytochrome p450 reductase (CPR); flavodoxin (Fld); ferredoxin-dependent NADP+ reductase (FNR); cytochrome p450 (CYP); 60 kDa monomeric truncation of SiRFP (SiRFP-60); 20-kDa truncation of SiRFP (Fld8); 43 kDa monomeric truncation of SiRFP (FNR1); intrinsically disordered protein regions (IDPR); UV-Visible (UV-Vis); isothermal titration calorimetry (ITC); electrospray ionization mass spectrometry (ESI-MS); nitric oxide synthase reductase domain (NOSred); methionine synthase reductase (MSR); 12.5 mM succinic acid, pH 6.8, 50 mM NaH2PO4 and 37.5 mM glycine buffer (SPG buffer)
ACS Paragon Plus Environment
27
Biochemistry 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
Page 28 of 41
References: [1] Siegel, L. M., Leinweber, F. J., and Monty, K. J. (1965) Characterization of the Sulfite and Hydroxylamine Reducases of Neurospora Crassa, J Biol Chem 240, 2705-2711. [2] Siegel, L. M., Murphy, M. J., and Kamin, H. (1973) Reduced nicotinamide adenine dinucleotide phosphate-sulfite reductase of enterobacteria. I. The Escherichia coli hemoflavoprotein: molecular parameters and prosthetic groups, J Biol Chem 248, 251264. [3] Askenasy, I., Pennington, J. M., Tao, Y., Marshall, A. G., Young, N. L., Shang, W., and Stroupe, M. E. (2015) The N-terminal Domain of Escherichia coli Assimilatory NADPHSulfite Reductase Hemoprotein Is an Oligomerization Domain That Mediates Holoenzyme Assembly, J Biol Chem 290, 19319-19333. [4] Siegel, L. M., and Davis, P. S. (1974) Reduced nicotinamide adenine dinucleotide phosphatesulfite reductase of enterobacteria. IV. The Escherichia coli hemoflavoprotein: subunit structure and dissociation into hemoprotein and flavoprotein components, J Biol Chem 249, 1587-1598. [5] Crane, B. R., and Getzoff, E. D. (1996) The relationship between structure and function for the sulfite reductases, Curr Opin Struct Biol 6, 744-756. [6] Wang, M., Roberts, D. L., Paschke, R., Shea, T. M., Masters, B. S., and Kim, J. J. (1997) Three-dimensional structure of NADPH-cytochrome P450 reductase: prototype for FMNand FAD-containing enzymes, Proc Natl Acad Sci U S A 94, 8411-8416. [7] Hamdane, D., Xia, C., Im, S. C., Zhang, H., Kim, J. J., and Waskell, L. (2009) Structure and function of an NADPH-cytochrome P450 oxidoreductase in an open conformation capable of reducing cytochrome P450, J Biol Chem 284, 11374-11384.
ACS Paragon Plus Environment
28
Page 29 of 41 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
Biochemistry
[8] Xia, C., Hamdane, D., Shen, A. L., Choi, V., Kasper, C. B., Pearl, N. M., Zhang, H., Im, S. C., Waskell, L., and Kim, J. J. (2011) Conformational changes of NADPH-cytochrome P450 oxidoreductase are essential for catalysis and cofactor binding, J Biol Chem 286, 16246-16260. [9] Laursen, T., Jensen, K., and Moller, B. L. (2011) Conformational changes of the NADPHdependent cytochrome P450 reductase in the course of electron transfer to cytochromes P450, Biochim Biophys Acta 1814, 132-138. [10] McCammon, K. M., Panda, S. P., Xia, C., Kim, J. J., Moutinho, D., Kranendonk, M., Auchus, R. J., Lafer, E. M., Ghosh, D., Martasek, P., Kar, R., Masters, B. S., and Roman, L. J. (2016) Instability of the Human Cytochrome P450 Reductase A287P Variant Is the Major Contributor to Its Antley-Bixler Syndrome-like Phenotype, J Biol Chem 291, 20487-20502. [11] Evrard, A., Zeghouf, M., Fontecave, M., Roby, C., and Coves, J. (1999) 31P nuclear magnetic resonance study of the flavoprotein component of the Escherichia coli sulfite reductase, Eur J Biochem 261, 430-437. [12] Gruez, A., Pignol, D., Zeghouf, M., Coves, J., Fontecave, M., Ferrer, J. L., and FontecillaCamps, J. C. (2000) Four crystal structures of the 60 kDa flavoprotein monomer of the sulfite reductase indicate a disordered flavodoxin-like module, J Mol Biol 299, 199-212. [13] Pudney, C. R., Heyes, D. J., Khara, B., Hay, S., Rigby, S. E., and Scrutton, N. S. (2012) Kinetic and spectroscopic probes of motions and catalysis in the cytochrome P450 reductase family of enzymes, FEBS J 279, 1534-1544.
ACS Paragon Plus Environment
29
Biochemistry 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
Page 30 of 41
[14] Kenaan, C., Zhang, H., Shea, E. V., and Hollenberg, P. F. (2011) Uncovering the role of hydrophobic residues in cytochrome P450-cytochrome P450 reductase interactions, Biochemistry 50, 3957-3967. [15] Jang, H. H., Jamakhandi, A. P., Sullivan, S. Z., Yun, C. H., Hollenberg, P. F., and Miller, G. P. (2010) Beta sheet 2-alpha helix C loop of cytochrome P450 reductase serves as a docking site for redox partners, Biochim Biophys Acta 1804, 1285-1293. [16] Shen, A. L., and Kasper, C. B. (1995) Role of acidic residues in the interaction of NADPHcytochrome P450 oxidoreductase with cytochrome P450 and cytochrome c, J Biol Chem 270, 27475-27480. [17] Zeghouf, M., Fontecave, M., Macherel, D., and Coves, J. (1998) The flavoprotein component of the Escherichia coli sulfite reductase: expression, purification, and spectral and catalytic properties of a monomeric form containing both the flavin adenine dinucleotide and the flavin mononucleotide cofactors, Biochemistry 37, 6114-6123. [18] Zeghouf, M., Fontecave, M., and Coves, J. (2000) A simplifed functional version of the Escherichia coli sulfite reductase, J Biol Chem 275, 37651-37656. [19] Siegel, L. M., Davis, P. S., and Kamin, H. (1974) Reduced nicotinamide adenine dinucleotide phosphate-sulfite reductase of enterobacteria. 3. The Escherichia coli hemoflavoprotein: catalytic parameters and the sequence of electron flow, J Biol Chem 249, 1572-1586. [20] Crane, B. R., Siegel, L. M., and Getzoff, E. D. (1995) Sulfite reductase structure at 1.6 A: evolution and catalysis for reduction of inorganic anions, Science 270, 59-67.
ACS Paragon Plus Environment
30
Page 31 of 41 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
Biochemistry
[21] Wu, J. Y., Siegel, L. M., and Kredich, N. M. (1991) High-level expression of Escherichia coli NADPH-sulfite reductase: requirement for a cloned cysG plasmid to overcome limiting siroheme cofactor, J Bacteriol 173, 325-333. [22] Liu, H., and Naismith, J. H. (2008) An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol, BMC Biotechnol 8, 91. [23] Turnbull, W. B., and Daranas, A. H. (2003) On the value of c: can low affinity systems be studied by isothermal titration calorimetry?, J Am Chem Soc 125, 14859-14866. [24] Rossmann, M. G., Moras, D., and Olsen, K. W. (1974) Chemical and biological evolution of nucleotide-binding protein, Nature 250, 194-199. [25] Romero, P., Obradovic, Z., Li, X., Garner, E. C., Brown, C. J., and Dunker, A. K. (2001) Sequence complexity of disordered protein, Proteins 42, 38-48. [26] Obradovic, Z., Peng, K., Vucetic, S., Radivojac, P., and Dunker, A. K. (2005) Exploiting heterogeneous sequence properties improves prediction of protein disorder, Proteins 61 Suppl 7, 176-182. [27] Obradovic, Z., Peng, K., Vucetic, S., Radivojac, P., Brown, C. J., and Dunker, A. K. (2003) Predicting intrinsic disorder from amino acid sequence, Proteins 53 Suppl 6, 566-572. [28] Dosztányi, Z., Csizmok, V., Tompa, P., and Simon, I. (2005) IUPred: web server for the prediction of intrinsically unstructured regions of proteins based on estimated energy content, Bioinformatics 21, 3433-3434. [29] Xue, B., Dunbrack, R. L., Williams, R. W., Dunker, A. K., and Uversky, V. N. (2010) PONDR-FIT: a meta-predictor of intrinsically disordered amino acids, Biochim Biophys Acta 1804, 996-1010.
ACS Paragon Plus Environment
31
Biochemistry 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
Page 32 of 41
[30] Walsh, I., Giollo, M., Di Domenico, T., Ferrari, C., Zimmermann, O., and Tosatto, S. C. (2015) Comprehensive large-scale assessment of intrinsic protein disorder, Bioinformatics 31, 201-208. [31] Peng, Z., and Kurgan, L. (2012) On the complementarity of the consensus-based disorder prediction, Pac Symp Biocomput, 176-187. [32] Mészáros, B., Simon, I., and Dosztányi, Z. (2009) Prediction of protein binding regions in disordered proteins, PLoS Comput Biol 5, e1000376. [33] Dosztányi, Z., Mészáros, B., and Simon, I. (2009) ANCHOR: web server for predicting protein binding regions in disordered proteins, Bioinformatics 25, 2745-2746. [34] Eschenbrenner, M., Coves, J., and Fontecave, M. (1995) The flavin reductase activity of the flavoprotein component of sulfite reductase from Escherichia coli. A new model for the protein structure, J Biol Chem 270, 20550-20555. [35] Sibille, N., Blackledge, M., Brutscher, B., Coves, J., and Bersch, B. (2005) Solution structure of the sulfite reductase flavodoxin-like domain from Escherichia coli, Biochemistry 44, 9086-9095. [36] Siegel, L. M., Faeder, E. J., and Kamin, H. (1972) Flavin interaction in NADPH-sulfite reductase, Z Naturforsch B 27, 1087-1089. [37] Ostrowski, J., Barber, M. J., Rueger, D. C., Miller, B. E., Siegel, L. M., and Kredich, N. M. (1989) Characterization of the flavoprotein moieties of NADPH-sulfite reductase from Salmonella typhimurium and Escherichia coli. Physicochemical and catalytic properties, amino acid sequence deduced from DNA sequence of cysJ, and comparison with NADPH-cytochrome P-450 reductase, J Biol Chem 264, 15796-15808. [38] Tompa, P. (2002) Intrinsically unstructured proteins, Trends Biochem Sci 27, 527-533.
ACS Paragon Plus Environment
32
Page 33 of 41 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
Biochemistry
[39] Moreira, I. S., Fernandes, P. A., and Ramos, M. J. (2007) Hot spots--a review of the proteinprotein interface determinant amino-acid residues, Proteins 68, 803-812. [40] Champier, L., Sibille, N., Bersch, B., Brutscher, B., Blackledge, M., and Coves, J. (2002) Reactivity, secondary structure, and molecular topology of the Escherichia coli sulfite reductase flavodoxin-like domain, Biochemistry 41, 3770-3780. [41] Dunker, A. K., Brown, C. J., Lawson, J. D., Iakoucheva, L. M., and Obradović, Z. (2002) Intrinsic disorder and protein function, Biochemistry 41, 6573-6582. [42] DeForte, S., and Uversky, V. N. (2017) Not an exception to the rule: the functional significance of intrinsically disordered protein regions in enzymes, Mol Biosyst 13, 463469. [43] Hsieh, Y. C., Liu, M. Y., Wang, V. C., Chiang, Y. L., Liu, E. H., Wu, W. G., Chan, S. I., and Chen, C. J. (2010) Structural insights into the enzyme catalysis from comparison of three forms of dissimilatory sulphite reductase from Desulfovibrio gigas, Mol Microbiol 78, 1101-1116. [44] Oliveira, T. F., Vonrhein, C., Matias, P. M., Venceslau, S. S., Pereira, I. A., and Archer, M. (2008) The crystal structure of Desulfovibrio vulgaris dissimilatory sulfite reductase bound to DsrC provides novel insights into the mechanism of sulfate respiration, J Biol Chem 283, 34141-34149. [45] Schiffer, A., Parey, K., Warkentin, E., Diederichs, K., Huber, H., Stetter, K. O., Kroneck, P. M., and Ermler, U. (2008) Structure of the dissimilatory sulfite reductase from the hyperthermophilic archaeon Archaeoglobus fulgidus, J Mol Biol 379, 1063-1074.
ACS Paragon Plus Environment
33
Biochemistry 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
Page 34 of 41
[46] Schnell, R., Sandalova, T., Hellman, U., Lindqvist, Y., and Schneider, G. (2005) Sirohemeand [Fe4-S4]-dependent NirA from Mycobacterium tuberculosis is a sulfite reductase with a covalent Cys-Tyr bond in the active site, J Biol Chem 280, 27319-27328. [47] Dunker, A. K., Lawson, J. D., Brown, C. J., Williams, R. M., Romero, P., Oh, J. S., Oldfield, C. J., Campen, A. M., Ratliff, C. M., Hipps, K. W., Ausio, J., Nissen, M. S., Reeves, R., Kang, C., Kissinger, C. R., Bailey, R. W., Griswold, M. D., Chiu, W., Garner, E. C., and Obradovic, Z. (2001) Intrinsically disordered protein, J Mol Graph Model 19, 26-59. [48] Uversky, V. N. (2015) Functional roles of transiently and intrinsically disordered regions within proteins, FEBS J 282, 1182-1189. [49] Dunker, A. K., Silman, I., Uversky, V. N., and Sussman, J. L. (2008) Function and structure of inherently disordered proteins, Curr Opin Struct Biol 18, 756-764.
ACS Paragon Plus Environment
34
Page 35 of 41 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
Biochemistry
187x186mm (300 x 300 DPI)
ACS Paragon Plus Environment
Biochemistry 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
63x77mm (300 x 300 DPI)
ACS Paragon Plus Environment
Page 36 of 41
Page 37 of 41 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
Biochemistry
84x147mm (300 x 300 DPI)
ACS Paragon Plus Environment
Biochemistry 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
41x29mm (300 x 300 DPI)
ACS Paragon Plus Environment
Page 38 of 41
Page 39 of 41 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
Biochemistry
172x86mm (300 x 300 DPI)
ACS Paragon Plus Environment
Biochemistry 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
104x80mm (300 x 300 DPI)
ACS Paragon Plus Environment
Page 40 of 41
Page 41 of 41 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
Biochemistry
60x44mm (300 x 300 DPI)
ACS Paragon Plus Environment