A novel approach for assessing macromolecular complexes combining soft-docking calculations with NMR data

Abstract

We present a novel and efficient approach for assessing protein-protein complex formation, which combines ab initio docking calculations performed with the protein docking algorithm BiGGER and chemical shift perturbation data collected with heteronuclear single quantum coherence (HSQC) or TROSY nuclear magnetic resonance (NMR) spectroscopy. This method, termed "restrained soft-docking," is validated for several known protein complexes. These data demonstrate that restrained soft-docking extends the size limitations of NMR spectroscopy and provides an alternative method for investigating macromolecular protein complexes that requires less experimental time, effort, and resources. The potential utility of this novel NMR and simulated docking approach in current structural genomic initiatives is discussed.

Fig. 1.

Validation of the "restrained soft-docking" approach. The atomic coordinates of the free proteins are entered into the protein docking algorithm BiGGER* (Palma et al. 2000Palma et al. 2000), with the larger and smaller proteins of the complex representing the "target" and "probe," respectively. Initial ab initio calculations by BiGGER produce 10⁷–10⁹ binding configurations, which are filtered and scored on the basis of geometric surface complementarity, pairwise amino acid interaction propensities, interaction electrostatic potential, and solvation energy upon complex formation. These alternatively docked geometric configurations are then filtered and scored using NMR chemical shift perturbation analysis data, NOE information, or H-D exchange data to idey the 1000 best theoretical solutions. These solutions are scored and ranked according to their fit to these NMR data. The family of structures which is ranked highest is superimposed with the known complex structure, and the RMS deviation (RMSD) was measured.

Fig. 2.

Docking of the complexes assessed following ab initio calculations and NMR chemical shift perturbation analysis and/or H-D exchange filtering. Panels a, b, and c illustrate the backbone trace (N, C^α, C`) of EIN, Barnase, and Tom20, respectively, superimposed with the 1000 putative docking configurations of their complex partners, which are represented by small solid spheres that denote the center of mass for each docking position, color-coded according to the NMR filtering interaction score (higher scoring solutions, which are scored positive, are represented in red). Panels d, e, and f: the highest ranked cluster of structures obtained from these calculations is shown for each complex (illustrated as red spheres). Panels g, h, and i idey the best solutions for each complex, which are scored from red to white (1–4Å) according to the RMS deviation (calculated by BiGGER) of the calculated backbone trace with the previously determined structure.

Fig. 3.

Superimpostion of the EIN/ HPr complex determined using "restrained soft-docking" (illustrated in blue) with the previously determined average NMR structure (presented in red) (Garrett et al. 1999Garrett et al. 1999). These structures were superimposed and the RMSD determined using the BiGGER software package. The EIN and HPr proteins of this complex are labeled.