Heteronuclear NMR and Soft Docking:  An Experimental Approach for a Structural Model of the Cytochrome c₅₅₃−Ferredoxin Complex

Abstract

The combination of docking algorithms with NMR data has been developed extensively for the studies of protein-ligand interactions. However, to extend this development for the studies of protein-protein interactions, the intermolecular NOE constraints, which are needed, are more difficult to access. In the present work, we describe a new approach that combines an ab initio docking calculation and the mapping of an interaction site using chemical shift variation analysis. The cytochrome c553-ferredoxin complex is used as a model of numerous electron-transfer complexes. The 15N-labeling of both molecules has been obtained, and the mapping of the interacting site on each partner, respectively, has been done using HSQC experiments. 1H and 15N chemical shift analysis defines the area of both molecules involved in the recognition interface. Models of the complex were generated by an ab initio docking software, the BiGGER program (bimolecular complex generation with global evaluation and ranking). This program generates a population of protein-protein docked geometries ranked by a scoring function, combining relevant stabilization parameters such as geometric complementarity surfaces, electrostatic interactions, desolvation energy, and pairwise affinities of amino acid side chains. We have implemented a new module that includes experimental input (here, NMR mapping of the interacting site) as a filter to select the accurate models. Final structures were energy minimized using the X-PLOR software and then analyzed. The best solution has an interface area (1037.4 A2) falling close to the range of generally observed recognition interfaces, with a distance of 10.0 A between the redox centers.

Direct Electrochemistry of Redox Enzymes as a Tool for Mechanistic Studies

“…This conclusion may hold in ViVo and suggests that electron transport in real biological systems may be faster in situations where the components of a transient complex are not connected together in a single, well defined manner. [347][348][349][350][351][352][353][354][355][356] Hildebrandt and co-workers have also emphasized the similarity between self-assembled monolayers and biological membranes. 357 In the case of bare metal electrodes, the protein-electrode interaction is not expected to mimic the physiological situation.…”

“…This may be reminiscent of a common situation for inter-protein electron transfer. Indeed, it is now admitted that distributions of orientations and electron transfer rates also exist in protein-protein electron transfers, as evidenced in NMR experiments, [347][348][349][350][351][352][353][354] soft-docking, 355 and molecular dynamics simulations, 356 but the heterogeneity of intermolecular ET rate constants is likely to escape detection in traditional kinetic measurements. In contrast, the distribution of interfacial ET rate constants is evident from the shape of the PFV data, and this illustrates an advantage of measuring the complete dependence of rate on driving force rather than a single value of the rate as in homogeneous kinetics.…”

A further investigation of the cytochrome b 5–cytochrome c complex

“…A recently developed computer program [21] allowed us to use the NMR data to provide a model of the interaction. In a similar approach, heteronuclear NMR and docking calculations were used for building structural models of the cytochrome c 553 -ferredoxin complex and a combination of TROSY experiments and docking calculations were used for the study of the [Fe]-hydrogenase-cytochrome c 553 complex [22,23]. Other, more recent, applications of similar strategies include the cytochrome b 5 -myoglobin [24] and the cytochrome ccytochrome f [25] complexes.…”

Structural Model of the Fe-Hydrogenase/Cytochromec 553 Complex Combining Transverse Relaxation-optimized Spectroscopy Experiments and Soft Docking Calculations

“…For protein-protein interactions the use of such algorithms is more difficult, and it is essential that experimental data be used to select the best model. We have recently described an experimental ab initio approach using the software BiGGER in which we have implemented an NMR filter (13). This approach developed for small complexes was carried out using heteronuclear single quantum coherence experiments.…”