Visualization of the Encounter Ensemble of the Transient citocromo c

Visualization of the Encounter Ensemble of the Transient citocromo c

(Parte 1 de 2)

Visualization of the Encounter Ensemble of the Transient

Electron Transfer Complex of Cytochrome c and Cytochrome c Peroxidase

Qamar Bashir,† AlexanderN. Volkov,†,‡ G. MatthiasUllmann,§ and MarcellusUbbink*,†

Leiden Institute of Chemistry, Leiden UniVersity, Gorlaeus Laboratories, P.O. Box 9502, 2300 RA Leiden, The Netherlands, and Structural Biology/Bioinformatics, UniVersity of Bayreuth, UniVersitatsstrasse 30, BGI, 95447 Bayreuth, Germany

Received July 31, 2009; E-mail: m.ubbink@chem.leidenuniv.nl

Abstract: Recent studies have provided experimental evidence for the existence of an encounter complex, a transientintermediatein the formationof proteincomplexes.We use paramagneticrelaxationenhancement NMR spectroscopy in combination with Monte Carlo simulations to characterize and visualize the ensemble of encounter orientations in the short-lived electron transfer complex of yeast cytochrome c (Cc) and cytochrome c peroxidase (CcP). The complete conformational space sampled by the protein molecules duringthedynamicpartof theinteractionwasmappedexperimentally.Theencountercomplexwasdescribed by an electrostatic ensemble of orientations based on Monte Carlo calculations, considering the protein structures in atomic detail. We demonstrate that this visualization of the encounter complex, in combination with the specific complex, is in excellent agreement with the experimental data. Our results indicate that Cc samples only about 15% of the surface area of CcP, surrounding the specific binding interface. The encounter complex is populated for 30% of the time, representing a mere 0.5 kcal/mol difference in the free energies between the two states. This delicate balance is interpreted to be a consequence of the conflicting requirements of fast electron transfer and high turnover of the complex.

Introduction

Electron transfer (ET) protein complexes often require high turnover and thus fast dissociation. A high affinity confers specificity on the interaction but limits the dissociation rate. Therefore, these complexes are on the border of specific and nonspecific as a result of a compromise between tight binding and fast dissociation. Early theoretical work suggested that ET complexes are highly dynamic.1 NMR and kinetic studies, recently reviewed in ref 2, provided evidence for a model of the process of protein complex formation in which proteins approach each other by diffusion and initially associate in an encounter complex, followed by the formation of the specific complex. In the encounter complex, the proteins do not assume a single orientation relative to each other, but rather they rapidly change orientation, thus sampling the surface of the partner. It is thought that the encounter complex accelerates the formation of the specific complex by reduction of the dimensionality of the conformational search. In the encounter complex intermolecular interactions are dominated by electrostatic forces. Longrange charge-charge attraction prolongs the lifetime of the encounter and allows for preorientation of protein molecules, thus limiting the conformational search to a part of the binding surface.2 In the specific complex the proteins assume a single, well-definedorientationthat is stabilizednot only by electrostatic forces but also by short-range interactions like hydrogen bonds and the hydrophobic effect. Some ET complexes appear to be entirelyor mostlynonspecific,with the encountercomplexbeing the dominant form,3-10 whereas in others the specific complex dominates.1-13

Paramagnetic relaxation enhancement (PRE) NMR spectroscopy has proven to be a useful technique for detecting the encountercomplexin protein-protein13,14and protein-DNA15,16 complexes as well as macromolecular self-association17,18 allostery19 and state equilibria.20 Generally, to observe PRE, a

† Leiden University. ‡ Present address: StructuralBiology Brussels, Vrije UniversiteitBrussel, and Department of Molecular and Cellular Interactions, VIB, Pleinlaan 2, 1050 Brussels, Belgium.

(3) Xu, X.; Reinle, W.; Hannemann, F.; Konarev, P. V.; Svergun, D. I.;

Bernhardt, R.; Ubbink, M. J. Am. Chem. Soc. 2008, 130, 6395–6403. (4) Liang, Z.-X.; Nocek, J. M.; Huang, K.; Hayes, R. T.; Kurnikov, I. V.;

Beratan, D. N.; Hoffman, B. M. J. Am. Chem. Soc. 2002, 124, 6849– 6859. (5) Volkov, A. N.; Ferrari, D.; Worrall, J. A. R.; Bonvin, A. M. J. J.;

Ubbink, M. Protein Sci. 2005, 14, 799–811. (6) Liang, Z.-X.; Kurnikov, I. V.; Nocek, J. M.; Mauk, A. G.; Beratan,

D. N.; Hoffman, B. M. J. Am. Chem. Soc. 2004, 126, 2785–2798. (7) Worrall, J. A. R.; Reinle, W.; Bernhardt, R.; Ubbink, M. Biochemistry 2003, 42, 7068–7076. (8) Worrall, J. A. R.; Liu, Y.; Crowley, P. B.; Nocek, J. M.; Hoffman,

B. M.; Ubbink, M. Biochemistry 2002, 41, 11721–11730. (9) Hoffman, B. M.; Celis, L. M.; Cull, D. A.; Patel, A. D.; Seifert, J. L.;

Wheeler, K. E.; Wang, J.; Yao, J.; Kurnikov, I. V.; Nocek, J. M. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 3564–3569. (10) Ubbink, M.; Bendall, D. S. Biochemistry 1997, 36, 6326–6335. (1) Vlasie, M. D.; Fernandez-Busnadiego, R.; Prudencio, M.; Ubbink, M.

J. Mol. Biol. 2008, 375, 1405–1415. (12) Ubbink, M.; Ejdeback, M.; Karlsson, B. G.; Bendall, D. S. Structure 1998, 6, 323–335. (13) Volkov, A. N.; Worrall, J. A. R.; Holtzmann, E.; Ubbink, M. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 18945–18950.

Published on Web 12/04/2009

paramagnetic spin label (SL) is attached to the protein surface via a cysteineresidue.The unpairedelectronon the SL increases the relaxation rate of the nuclei in its proximity due to the magnetic dipolar interactions. The effect depends on the sixth power of the distance between the nucleus and the paramagnetic center, averaged over all positions of the nucleus and the SL (eqs 4 and 5, Materials and Methods). The effect is very strong at short distances,and consequently,even lowly populatedstates in whichthe nucleusis closeto the SL can be detected.Although PRE can be used to map the surface area sampled by a partner protein in the encounter complex, visualization of the orientations of the proteins is impossible solely on the basis of experimental data because the PRE represent an average over time and space of all orientations.Several approacheshave been proposedso far to visualizethe encountercomplexby combining modeling and PRE data, including explicit ensemble refinement,14 Brownian Dynamics simulations,21 and empirical ensemble simulations.3,2

The complex between yeast mitochondrial iso-1-cytochrome c (Cc) and cytochrome c peroxidase (CcP) represents a wellstudied example of ET complexes with a short lifetime. CcP protects the cell against high concentrations of peroxides, receiving electrons from Cc. Here, we report the comprehensive mapping of the Cc-CcP encounter complex and its characterization by PRE NMR and Monte Carlo (MC) simulations. We demonstrate that a combination of the PREs calculated for the simulated ensemble and the specific complex is in excellent agreement with the experimental data. Using this approach we determine the fraction of the complex in the encounter complex to be 30%. The functional reason for the small associated free energy difference between encounter and specific complexes is discussed.

Materials and Methods

Protein Preparation. Single-cysteine CcP mutants were constructed by site-directed mutagenesis using the QuikChange polymerasechainreactionprotocoland CcP (MKT,C128A)plasmid13,23 as a template.The proteinswere expressedand purifiedas described before.23-26 During the purification procedure of CcP mutants on a gel filtration column, dithiothreitol (1 mM) was present in the equilibrationbuffer as well as in the protein sample. Concentrations of ferric Cc and five-coordinated high-spin ferric CcP were determined according to the optical absorbance peaks.27,28 The yields were, respectively, 7-8 mg and 15-25 mg/L of culture of pure Cc (from minimal medium) and CcP mutants (from rich medium).

Conjugation with a paramagnetic label, MTSL [(1-oxyl-2,2,5,5- tetramethyl-3-pyrroline-3-methyl)methanethiosulfonate], or a diamagneticanalogue,MTS [(1-acetyl-2,2,5,5-tetramethyl-3-pyrroline- 3-methyl)methanethiosulfonate], both purchased from Toronto Research Chemicals (Toronto, Canada), was carried out as published.13 The yield of labeling, determined by EPR or a dithiodipyridine assay,29 was more than 90%.

NMR Experiments. NMR samples contained 0.3 mM of a 1:1 15N Cc-CcP-MTS(L) complex in 20 mM sodium phosphate, 0.1

M NaCl, 6% D2O for lock, and 0.1 mM CH3CO15NH2 as internal reference. The pH of the samples was adjusted to 6.0 ( 0.05 with small aliquots of 0.1 M HCl or 0.1 M NaOH. Measurements were performed at 303 K on a Bruker DMX600 spectrometer equipped with a triple-resonance TXI-Z-GRAD probe (Bruker, Karlsruhe, Germany). 2D [15N, 1H] HSQC spectra were obtained with 512 and 128 complex points in the direct and indirect dimensions, respectively, and with spectral widths of 32 (15N) and 16 ppm (1H). All data were processed with the Azara suite of programs (provided by Wayne Boucherand the Departmentof Biochemistry,University of Cambridge, U.K.) and analyzed in ANSIG for Windows.30,31 Assignments of the 15N and 1H nuclei of Cc were taken from previous work.32 Calculation of the PRE. For each observed amide proton of

Cc the intensityratio (Ipara/Idia) of the resonancesin the paramagnetic (CcP-MTSL)and diamagnetic(CcP-MTS)sampleswas determined and Γ2 calculated from eq 133

Ipara

Idia where R2 is the transverse relaxation rate of Cc amide protons in the complex with CcP-MTS, Γ2 is the PRE, and t is the total INEPT evolution time of the HSQC. For the amides of which the resonances disappear in the paramagnetic spectrum, an upper limit for Ipara was estimated from the standard deviation of the noise level of the spectrum. For each peak, R2 was estimated from the width at half-height (∆ν1/2) of a Lorentzian fit in the proton dimension by using R2 ) π∆ν1/2. PRE values were calculated after normalization of the Ipara/Idia ratios. The residues with Ipara/Idia > 0.85 were used for this correction. The upper and lower 10% of Ipara/Idia values were removed, and the average of the remaining values was obtained. All Ipara/Idia values were divided by this average to get the normalized PREs.

Generation of the Encounter Complex Ensemble. A Boltzmann ensemble of encounter complex geometries was generated by a Metropolis Monte Carlo method using a previously described approach34 with small modifications. Cc was moving in the electrostatic potential of CcP, and moves were accepted or rejected according to the Metropolis criterion. An exclusion grid was used to avoid steric overlap between the proteins.1 Protein coordinates were taken from the PDB entry 2PCC.35 The dielectric constants used for the protein and water are 4 and 80, respectively. The electrostaticpotential was calculatedby the MEAD program suite36

(14) Tang, C.; Iwahara, J.; Clore, G. M. Nature 2006, 4, 383–386. (15) Iwahara, J.; Clore, G. M. Nature 2006, 440, 1227–1230. (16) Clore, G. M. Mol. BioSyst. 2008, 4, 1058–1069. (17) Tang, C.; Ghirlando, R.; Clore, G. M. J. Am. Chem. Soc. 2008, 130, 4048–4056. (18) Tang, C.; Louis, J. M.; Aniana, A.; Suh, J.-Y.; Clore, G. M. Nature 2008, 455, 693–696. (19) Tang, C.; Schwieters, C. D.; Clore, G. M. Nature 2007, 449, 1078– 1082. (20) Henzler-Wildman, K. A.; Thai, V.; Lei, M.; Ott, M.; Wolf-Watz, M.;

Fenn, T.; Pozharski, E.; Wilson, M. A.; Petsko, G. A.; Karplus, M.; Hubner, C. G.; Kern, D. Nature 2007, 450, 838–844. (21) Kim, Y. C.; Tang, C.; Clore, G. M.; Hummer, G. Proc. Natl. Acad.

Sci. U.S.A. 2008, 105, 12855–12860. (2) Hulsker, R.; Baranova, M. V.; Bullerjahn, G. S.; Ubbink, M. J. Am.

Chem. Soc. 2008, 130, 1985–1991. (23) Goodin, D. B.; Davidson, M. G.; Roe, J. A.; Mauk, A. G.; Smith, M.

Biochemistry 1991, 30, 4953–4962. (24) Morar, A. S.; Kakouras, D.; Young, G. B.; Boyd, J.; Pielak, G. J.

J. Biol. Inorg. Chem. 1999, 4, 220–2. (25) Pollock, W. B. R.; Rosell, F. I.; Twitchett, M. B.; Dumont, M. E.;

Mauk, A. G. Biochemistry 1998, 37, 6124–6131. (26) Teske, J. G.; Savenkova, M. I.; Mauro, J. M.; Erman, J. E.; Satterlee,

(28) Vitello, L. B.; Huang, M.; Erman, J. E. Biochemistry 1990, 29, 4283– 4288. (29) Riener, C.; Kada, G.; Gruber, H. Anal. Bioanal. Chem. 2002, 373, 266–276. (30) Kraulis, P. J. Magn. Reson. 1989,8 4. (31) Helgstrand, M.; Kraulis, P.; Allard, P.; Hard, T. J. Biomol. NMR 2000, 18, 329–336. (32) Worrall, J. A. R.; Kolczak, U.; Canters, G. W.; Ubbink, M. Biochemistry 2001, 40, 7069–7076. (3) Battiste, J. L.; Wagner, G. Biochemistry 2000, 39, 5355–5365. (34) Ullmann,G. M.; Knapp, E.-W.; Kostic,N. M. J. Am. Chem. Soc. 1997, 119, 42–52. (35) Pelletier, H.; Kraut, J. Science 1992, 258, 1748–1755.

242 J. AM. CHEM. SOC. 9 VOL. 132, NO. 1, 2010

ARTICLES Bashir et al.

using atomic partial charges of the Charmm force field.37 For histidine residues that are not coordinated to the heme iron, average Charmm charges were used, which were calculated as 0.25 × (His_Nε + His_Nδ) + 0.5 × protonated His. The temperature was set to 303 K and the ionic strength to 0.12 M to match the experimental conditions. When the centers of mass of the two molecules had a distance of less than 40 Å, the configuration was saved every 1000 steps. About 1600 configurations were retained for the analysis. This approach implies that configurations with a larger distance contribute negligibly to the free energy change of protein complex formation. These configurations will also be invisible in the PRE analysis.

Ensemble Analysis. The ensemble was analyzed by comparison with the Cc-CcP crystal structure by superposition of either of the proteins.The sphericalcoordinatesθ (elevation)and (azimuth) position the center of mass of a structure in the ensemble relative to the vector between the centers of mass of Cc and CcP in the crystal structure, Figure 3A. For the Cc structures around CcP, the range of θ is small, with 95% of the angles less than 45° and 36% within 10° (Figure 3B, gray areas), whereas is evenly spread (Figure 3C). The inertial ellipsoid of CcP can be described with values 12.2, 9.3, and 9.6 µs-1 for Dz, Dy, and Dx, respectively, with a rotational correlationtime of 16 ns.38 If CcP is approximated as a sphere, the surface area explored by Cc can be calculated. A surface element A of a sphere with radius r is given by eq 2 for Θ ) 45° and Φ ) 360°, A ) 1.84r2, or 14.6% of the total surface (12.57r2). For Θ ) 10° A is 0.76% of the total surface. The fact that the lowest θ bins are most populated implies that in the ensemble Cc is located close to the orientation in the specific complex.

For CcP structures around Cc, the bin of θ ) 35° is the most frequent, indicating that the ensemble orientation is not optimal for forming the specific complex and a rotation of CcP around Cc is required. With 95% of the θ angles between 0° and 90° (Figure 3D) and 90% of the angles between -85° and 95° (Figure 3E), it can be calculatedwith eq 2 that 85% of the CcP structuressamples 25% of the surface area of Cc, again approximating the protein by a sphere (Dz, Dy, and Dx for Cc are 3, 26, and 28 µs-1 , respectively, and the correlation time is 5.7 ns).38 The DRMS metric was defined according to eq 3 (ref 21) where dij is the distance between the CR atoms of two residues i and j from different proteins. N is the total number of pairs (i, j), and dijxray and dijens are the distance matrix elements from the specific and ensemble structures, respectively.

The distances between each Cc backbone amide hydrogen in the ensemble and the oxygen of SL on CcP were measured with XPLOR-NIH39 and averaged using eq 4

(36) Bashford, D. In Scientific Computing in Object-Oriented Parallel

EnVironments; Springer: Berlin, 1997; p 233-240. (37) MacKerell, A. D.; et al. J. Phys. Chem. B 1998, 102, 3586–3616. (38) Garcıa de la Torre, J.; Huertas, M. L.; Carrasco, B. J. Magn. Reson. 2000, 147, 138–146. (39) Schwieters, C. D.; Kuszewski, J. J.; Tjandra, N.; Marius Clore, G. J. Magn. Reson. 2003, 160, 65–73.

Figure 1. CcP spin labeling sites and intermolecular PRE effects on Cc. The ribbon representation of the specific Cc-CcP complex was drawn from PDB entry 2PCC35 with PyMOL.51 Cc and CcP are in pink and gray, with the heme groups shown as cyan sticks. CcP residues that were replaced by cysteines for SL attachment are labeled and shown as red (PRE observed) and blue (no PRE) sticks. For each SL position, the Ipara/Idia plots for the Cc residues are shown. The red circles in the graphs represent the residues for which the resonances disappeared in the spectrum of the paramagnetic sample. The enlarged data graphs with error bars are provided as Supporting Information, Figure S-3.

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ET Complex of Cytochrome c and Cytochrome c Peroxidase ARTICLES

where r is the effective distance for a given proton, m is the number of SL orientations used, and n is the number of structures in the ensemble. To account for the mobility of the SL, the distances to four conformations of the SL were averaged.40 The use of an effective distance is justified only if the rate of interconversion (kex) of the substates is much higher than the PRE (kex . Γ2). From the chemical shift changes observed upon complex formation it can be derived that kex(binding) . 1000 s-1. The interconversion of the substates in the encounter complex and between the encounter and specific complexes is necessarily much faster. Thus, it was assumed that the fast exchange regime can be applied in this case. If the distance between the oxygen of the SL and any Cc CR atom was less than 5 Å, then that SL orientation was not used for that Cc structure because it was assumed that steric collision between the protein and the SL would not allow that SL orientation. The

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