Biomol NMR Assign DOI 10.1007/s12104-014-9576-9

ARTICLE

Backbone, side chain and heme resonance assignments of the triheme cytochrome PpcD from Geobacter sulfurreducens Joana M. Dantas • Carlos A. Salgueiro Marta Bruix

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Received: 26 May 2014 / Accepted: 3 September 2014 Ó Springer Science+Business Media Dordrecht 2014

Abstract Gene knock-out studies on Geobacter sulfurreducens (Gs) cells showed that the periplasmic triheme cytochrome PpcD is involved in respiratory pathways leading to the extracellular reduction of Fe(III) and U(VI) oxides. More recently, it was also shown that the gene encoding for PpcD has higher transcript abundance when Gs cells utilize graphite electrodes as sole electron donors to reduce fumarate. This sets PpcD as the first multiheme cytochrome to be involved in Gs respiratory pathways that bridge the electron transfer between the cytoplasm and cell exterior in both directions. Nowadays, extracellular electron transfer (EET) processes are explored for several biotechnological applications, which include bioremediation, bioenergy and biofuel production. Therefore, the structural characterization of PpcD is a fundamental step to understand the mechanisms underlying EET. However, compared to non-heme proteins, the presence of numerous proton-containing groups in the redox centers presents additional challenges for protein signal assignment and structure calculation. Here, we report the complete assignment of the heme proton signals together with 1H, 13 C and 15N backbone and side chain assignments of the reduced form of PpcD.

J. M. Dantas
C. A. Salgueiro Requimte-CQFB, Departamento de Quı´mica, Faculdade de Cieˆncias e Tecnologia, Universidade Nova de Lisboa, Campus Caparica, 2829-516 Caparica, Portugal e-mail: [email protected] M. Bruix (&) Departamento de Quı´mica Fı´sica Biolo´gica, Instituto de Quı´mica-Fı´sica ‘‘Rocasolano’’, CSIC, Serrano 119, 28006 Madrid, Spain e-mail: [email protected]

Keywords Geobacter sulfurreducens
PpcD
Multiheme
NMR

Biological context The genome of the bacterium Geobacter sulfurreducens (Gs) encodes for 73 multiheme c-type cytochromes (Methe´ et al. 2003). The presence of such large number of cytochromes in Gs highlights their involvement in a broad range of essential cellular functions and explains the impressive respiratory versatility of this microorganism. Electron transfer towards extracellular terminal acceptors is one the most remarkable features of Gs, by which it can reduce toxic or radioactive metals and convert renewable biomass into electricity. Examples of such applications include degradation of hydrocarbon contaminants in soils, reduction of insoluble Fe(III) and Mn(IV) oxides, precipitation of uranium in contaminated aquifers and electron transfer to electrodes in microbial fuel cells, from which electricity can be harvested (Bond et al. 2002; Lovley et al. 2004). In addition to this, it was recently found that Gs can also transport electrons from extracellular insoluble donors, such as graphite electrodes (Gregory et al. 2004). In this process, the reducing power provided by the electrode can be used to alter the cellular levels of NADH to redirect fermentative pathways or drive respiratory processes, underlying the synthesis of valuable organic compounds and, therefore, opening new perspectives in the field of bioremediation and biofuel production (Rabaey and Rozendal 2010). To assist electron transfer to the cell exterior, or to recruit electrons from outside, the spatial disposition of the redox components in Gs cells differs from those found in the majority of other microorganisms. In fact, in addition to the usual location at the inner membrane,

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several outer-membrane associated multiheme cytochromes were also identified (Mehta et al. 2005). Additionally, an unusual pool of five triheme cytochromes, designated PpcAE was identified in the Gs periplasm (Pokkuluri et al. 2004). To best of our knowledge, the periplasmic PpcD triheme cytochrome is the first multiheme component of Gs electron transfer chains to be clearly involved in extracellular electron transfer in both directions: (1) from cytoplasm to outer membrane components and then to extracellular Fe(III) oxides (Shelobolina et al. 2007) (Aklujkar et al. 2013; Ding et al. 2006; Morgado et al. 2012a, b) and (2) from outer cell surface to the inner membrane toward terminal electron acceptors, such as fumarate (Strycharz et al. 2011). Despite the knowledge about the cellular localization and phenotypes associated with Gs multiheme cytochromes, no structural information is available for any of these proteins in solution, except for the triheme cytochrome PpcA (Morgado et al. 2011, 2012a, b). This relates with the traditional difficulties in obtaining isotopic labeled recombinant multiheme cytochromes with the correct fold and post-translational modification of the heme groups in a cost-effective manner. This bottleneck was recently overcome by the simultaneous use of an expression plasmid with a lac promoter and a second plasmid co-expressing the cytochrome c maturation gene cluster, which allowed the heterologous expression of Gs multiheme cytochromes in Escherichia coli (Fernandes et al. 2008; Londer et al. 2002). Using this expression system, 13 C, 15N-labeled PpcD was obtained, and its high-resolution structure determination by NMR is currently in progress. To our best knowledge, the triheme PpcD is the first multiheme cytochrome uniformly labeled both in 13C and 15N. This protein has a MW of 9.56 kDa and contains three low spin ctype heme groups with bis-histidinyl axial coordination (Pokkuluri et al. 2004). Since the number of structural restraints associated with the heme signals is, on average, four times higher than those involving any protein residue, their assignment is critical to the accomplishment of a highresolution structure. The highly basic PpcD (19 % lysines and an isoelectric point value of 8.96, calculated with the pI/ Mw tool program on the ExPASy Server http://web.expasy. org/compute_pi/) has also high content in glycine residues (15 %). Here we report the complete assignment of the heme proton signals and a near-complete assignment of 1H, 13C and 15N backbone and side chain resonances for PpcD. This information will lay down the foundations to achieve the complete high-resolution structure of PpcD in solution.

Methods and experiments Unlabeled or 13C,15N-labeled PpcD were produced in E. coli and purified as previously described (Fernandes et al. 2008). Briefly, E. coli BL21 (DE3) cells containing

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plasmids pEC86 (encoding for cytochrome c maturation gene cluster ccmABCDEFH and pVA203 (encoding for PpcD) (Pokkuluri et al. 2010) were grown in 2xYT media to an OD600 of *1.5. This culture was then processed as follow: (a) to produce unlabeled PpcD: 10 lM isopropyl b-D-thiogalactoside (IPTG) was added and cultures was grown overnight at 30 °C after which cells were harvested by centrifugation; (b) to produce 15N-PpcD: the cells were collected by centrifugation after reaching an OD600 of 0.6–1.0, washed twice with 250 mL of a salt solution containing 3 g/L KH2PO4, 6 g/L Na2HPO4 and 0.5 g/L NaCl resuspended in minimal media (in a ratio of 250 mL of minimal medium for each liter of 2xYT medium) supplied with 1 g/L 15NH4Cl as nitrogen source, (together with 1 mM of the heme precursor a-aminolevulinic acid, trace amounts of metal salts, biotin and thiamine), grown overnight at 30 °C in the presence of 100 lM IPTG and harvested by centrifugation; (c) to produce 13C, 15N-PpcD: as described in (b) but also supplied with 2 g/L 13C6-glucose as carbon source. In each case, the periplasmic fraction was isolated using lysis buffer (100 mM Tris–HCl, pH 8.0, 0.5 mM EDTA, 20 % sucrose) containing 0.5 mg/mL lysozyme. The proteins were then purified by ion-exchange and gel filtration chromatography. Protein purity was evaluated by SDS gel electrophoresis and Coomassie blue staining. Samples for NMR experiments with 2 mM concentrations were prepared in 92 %H2O/8 %2H2O or in pure 2H2O containing 45 mM sodium phosphate buffer pH 7.1 (with NaCl to 100 mM final ionic strength) and 0.04 % sodium azide. Reduction of the samples was achieved by adding catalytic amounts of the enzyme hydrogenase from Desulfovibrio vulgaris (Hildenborough), as previously described (Morgado et al. 2008). After reduction, the pH values of the samples were confirmed with a glass micro electrode inside an anaerobic glove chamber (MBraun LABstar) with argon circulation to avoid sample reoxidation. The NMR experiments were carried out on a Bruker Avance 800 MHz spectrometer equipped with a z-gradient cryoprobe at 298 K. For backbone and side chain assignments the following spectra were acquired: (1) 2D 1H,15N-HSQC, 3D 1H,15N-TOCSY (45 ms) and 3D 1H,15N-NOESY (80 ms) for 15N-PpcD sample; (2) 2D 1H,13C-HSQC, 3D CBCANH, 3D CBCA(CO)NH, 3D HNCA, 3D HN(CO)CA, 3D HCC(H)-TOCSY and 3D HC(C)H-TOCSY for 13C,15N-PpcD sample and (3) 2D 1H-COSY, 1HTOCSY (60 ms) and 1H-NOESY (50 ms) for the unlabeled PpcD sample, all samples in 92 %H2O/8 %2H2O. For the assignment of the heme signals, 2D 1H-COSY, 1H-TOCSY (45 ms) and 1H-NOESY (100 ms) experiments were acquired for the unlabeled PpcD sample prepared in 2H2O. 1 H chemical shifts were calibrated using the water signal as internal reference and both 13C and 15N chemical shifts

Backbone, side chain and heme resonance assignments Fig. 1 2D 1H,15N-HSQC spectrum of fully reduced PpcD (2 mM, 45 mM sodium phosphate buffer pH 7.1 with NaCl to 100 mM final ionic strength, 298 K, 800 MHz). Orange and blue labels represent 1H-15N connectivities for backbone and side-chains groups (Nd2 of N11 and N13; Ne1 of W47 and Nd1 of axial histidines), respectively

calibrated through indirect referencing (Wishart et al. 1995). All NMR spectra were processed using TOPSPIN software (Bruker Biospin, Karlsruhe, Germany) and analyzed with Sparky (TD Goddard and DG Kneller, Sparky 3, University of California, San Francisco, USA).

Assignments and data deposition All heme proton signals in the reduced protein span the same region of the polypeptide signals and, consequently, NOEs belonging to the heme are obscured by the NOE connectivities involving the amino acid signals. In order to unambiguously assign the heme proton signals, 2D-NMR spectra were acquired for a non-labeled PpcD sample prepared in 2H2O. These spectra were used to assign all the heme proton signals following the strategy described by Turner and co-workers (Turner et al. 1992). After assigning the heme proton signals, the combined analysis of 2D 1 15 H, N-HSQC and the series of 3D NMR spectra (3D CBCANH, 3D CBCA(CO)NH, 3D HNCA, 3D HNCACO) led to the near-complete assignment of backbone 15N (99 %), 1HN (99 %), 13Ca (100 %) and 13Cb (98 %). Figure 1 shows the 2D 1H,15N-HSQC NMR spectrum of labeled PpcD with the backbone nitrogen resonances assigned, as well as those of the side chains of N11, N13, W47 and the six axial histidines (H18, H21, H32, H49, H56 and H70). To extend the assignment to aliphatic protons such as 1Ha, 1Hb, 1Hc, 1Hd and 1He, as well as to the aliphatic carbons 13Cc, 13Cd and 13Ce, we analyzed the 2D 1 13 H, C-HSQC and a series of 3D spectra that included HCC(H)-TOCSY, HC(C)H-TOCSY, 1H,15N-TOCSY and 1 15 H, N-NOESY. These analyses yielded essentially

complete side chain assignments for most residues. The aromatic ring protons were assigned on the basis of 2D 1HCOSY, 1H-TOCSY and 1H-NOESY NMR spectra. Compared to the ring-current effects produced by the amino acid aromatic side chains in non-heme proteins, the ones produced by the three hemes in PpcD are much stronger. Therefore, the chemical shifts of the nuclei located in the proximities of heme groups differ significantly from equivalent residues in non-heme proteins. The more affected nuclei are those of axial histidines. In fact, the ring proton signals (Hd2 and He1) are strongly up-field to the 1.60–0.60 ppm range. This contrasts clearly with the typical positions found for these signals in non-heme proteins (around 7–8 ppm). Also the signals correspondent to Hb of heme axial histidines are typically up-field shifted by at least 2 ppm compared to a non-heme bound histidines. The effect of the ring-current shifts is also extended to the nitrogen atoms (Nd1) of the axial histidine side chains, which appear in very characteristic positions (160–170 ppm) in the 2D 1H,15N-HSQC spectrum (Fig. 1). The chemical shifts of other nuclei located in the neighborhoods of the heme groups are also significantly affected, as it is the case of the N11Hd21 (0.79 ppm), K20HN (5.55 ppm), G37Ha (1.50 and -0.84 ppm), G38 HN (3.77 ppm), K45c (-0.82 and -1.65 ppm); G62Ha (2.51 and 1.16 ppm) and P63Hd (-0.70 ppm). Therefore, as a result of the strong heme ring-current effects, the observed chemical shifts of the nuclei located in the proximities of the heme group(s) are differently affected, depending upon their relative orientation to the closest heme(s). Thus, independently of the structure of the secondary element holding a particular nucleus, the signals can be up- or lowfield shifted, which prevents an accurate prediction of

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the secondary structure for cytochrome PpcD. However, this protein shares 70 % amino acid sequence similarity (60 % amino acid identity) with its homologue PpcA, whose structure was determined in solution (Morgado et al. 2012a, b). Moreover, the heme binding residues are strictly conserved between the two cytochromes, which anticipate a high structural homology between the two proteins. The total extent of the assignment for the 1H, 13C and 15 N, excluding carboxyl, amino and hydroxyl groups, is 91, 99 and 99 %, respectively. The 1H, 13C and 15N chemical shifts have been deposit in the BioMagResBank (http:// www.bmrm.wisc.edu) under BMRB accession number 19985. Acknowledgments This work was supported by the following project grants: PTDC/BBB-BEP/0753/2012 from Fundac¸a˜o para a Cieˆncia e a Tecnologia (FCT), Portugal (to CAS), strategic grant PEst-C/EQB/LA0006/2013 from FCT, Portugal (to REQUIMTE Laborato´rio Associado) and CTQ2011-22514 from the Ministerio De Economia y Competitividad (to MB). JMD is recipient of grant SFRH/BD/89701/2012 from FCT, Portugal. We thank Dr. M. Schiffer for providing the PpcD coding plasmid.

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