A method to trap transient and weak interacting protein complexes for structural studies

Abstract

Several key biological events adopt a "hit-and-run" strategy in their transient interactions between binding partners. In some instances, the disordered nature of one of the binding partners severely hampers the success of co-crystallization, often leading to the crystallization of just one of the partners. Here, we discuss a method to trap weak and transient protein interactions for crystallization. This approach requires the structural details of at least one of the interacting partners and binding knowledge to dock the known minimum binding region (peptide) of the protein onto the other using an optimal-sized linker. Prior to crystallization, the purified linked construct should be verified for its intact folding and stability. Following structure determination, structure-guided functional studies are performed with independent, full-length unlinked proteins to validate the findings of the linked complex. We designed this approach and then validated its efficacy using a 24 amino acid minimum binding region of the intrinsically disordered, neuron-specific substrates, Neurogranin and Neuromodulin, joined via a Gly-linker to their interacting partner, Calmodulin. Moreover, the reported functional studies with independent full-length proteins confirmed the findings of the linked peptide complexes. Based on our studies, and in combination with the supporting literature, we suggest that optimized linkers can provide an environment to mimic the natural interactions between binding partners, and offer a useful strategy for structural studies to trap weak and transient interactions involved in several biological processes.

Keywords: Protein-protein interactions; co-crystallization; linked complex; transient binding.

Figure 1. Flowchart representation of the various key steps involved in the linked peptide complex method.

Figure 2. (A) Computational models of apo-CaM-Nm IQ and apo-CaM-Ng IQ complexes obtained with Deep View analysis using apo-CaM-myosin V IQ peptide complex as a template (PDB code: 2IX7). Computational models show that the IQ motif peptide interacts mainly with the C-lobe of CaM. (B) Crystal structures of apo-CaM-Nm (PDB code: 4E53) and apo-CaM-Ng (PDB code: 4E50) complexes obtained by linking the binding partners using a (Gly)₅ linker. CaM adopts an extended conformation and the IQ motif peptides interact with the C-lobe of CaM. (C) Ribbon representation of different orientations adopted by Nm (magenta) and Ng (green) IQ peptides when CaM-Nm IQ (PDB code: 4E53) and CaM-Ng IQ (PDB code: 4E50) structures were superimposed.

Figure 3. Comparison among Size Exclusion Chromatography elution profiles for CaM, CaM-(Gly)₅-Nm and CaM-(Gly)₅-Ng using 16/60 Superdex 75 prep grade column. A similar elution profile for CaM and CaM-(Gly)₅-Nm shows that the CaM and CaM linked Nm complex have similar hydrodynamic radii and indicates that the linked complex is well-folded. The CaM-(Gly)₅-Ng shows a small difference in the elution profile, indicating the possibility of a non-interacting linked complex. This was further verified using dynamic light scattering (DLS) (data not shown).

Figure 4. (A) Molecular surface and ribbon representations for the binding of Ng MBR peptide on CaM in the CaM-(Gly)₅-Ng linked peptide complex (PDB code:4E50). CaM interacts with the linked MBR peptide of the nearby molecules (symmetry-related molecule) to mimic their natural interactions. Predicted position of the linker is shown as a green dotted line. (B) Ribbon representation of phosphoprotein (P_457–507) of paramyxoviral polymerase (cyan) - nucleocapsid protein (N_486–505) (orange) (PDB code: 1T6O). C-terminus of P_457–507 is linked using an 8 amino acid-long Gly-rich linker to the N-terminus of N_486–505. But, N_486–505 linked to P_457–507 of an adjacent symmetry-related molecule is involved in the interaction. Predicted position of the linker is shown as a black dotted line. This clearly indicates that the linker does not restrict the orientation of the binding.