A Combined NMR and Computational Approach to Investigate Peptide Binding to a Designed Armadillo Repeat Protein. Issue 10 (22nd May 2015)

• Record Type: Journal Article
• Authors: Ewald, Christina; Christen, Martin T.; Watson, Randall P.; Mihajlovic, Maja; Zhou, Ting; Honegger, Annemarie; Plückthun, Andreas; Caflisch, Amedeo; Zerbe, Oliver
• Is Part Of: Journal of molecular biology. Volume 427:Issue 10(2015:May 15)
• Page Start: 1916
• Page End: 1933
• Publication Date: 2015-05-22
• Subjects: ArmRP Armadillo repeat protein -- CSP chemical shift perturbation -- HSQC heteronuclear single quantum coherence -- MD molecular dynamics -- MTSL S-(1-oxyl-2, 2, 5, 5-tetramethyl-2, 5-dihydro-1H-pyrrol-3-yl)methyl methanesulfonothioate -- NOE nuclear Overhauser enhancement -- NT neurotensin -- PRE paramagnetic relaxation enhancement -- RMSF root-mean-square fluctuation -- SEC size-exclusion chromatography -- SPR surface plasmon resonance; Armadillo repeat proteins -- protein design -- NMR -- MD simulations; Molecular biology -- Periodicals; Biology -- Periodicals; Biochemistry -- Periodicals; Bacteriology -- Periodicals; Molecular Biology -- Periodicals; Biochemistry -- Periodicals; Biologie moléculaire -- Périodiques; Biologie -- Périodiques; Biochimie -- Périodiques; Moleculaire biologie; Biochemistry; Biology; Molecular biology; Periodicals; 572.805
• DOI: 10.1016/j.jmb.2015.02.022
• Languages: English
• ISSNs: 0022-2836

Abstract

Abstract: The specific recognition of peptide sequences by proteins plays an important role both in biology and in diagnostic applications. Here we characterize the relatively weak binding of the peptide neurotensin (NT) to the previously developed Armadillo repeat protein VG_328 by a multidisciplinary approach based on solution NMR spectroscopy, mutational studies, and molecular dynamics (MD) simulations, totaling 20 μs for all MD runs. We describe assignment challenges arising from the repetitive nature of the protein sequence, and we present novel approaches to address them. Partial assignments obtained for VG_328 in combination with chemical shift perturbations allowed us to identify the repeats not involved in binding. Their subsequent elimination resulted in a reduced-size binder with very similar affinity for NT, for which near-complete backbone assignments were achieved. A binding mode suggested by automatic docking and further validated by explicit solvent MD simulations is consistent with paramagnetic relaxation enhancement data collected using spin-labeled NT. Favorable intermolecular interactions are observed in the MD simulations for the residues that were previously shown to contribute to binding in an Ala scan of NT. We further characterized the role of residues within the N-cap for protein stability and peptide binding. Our multidisciplinary approach demonstrates that an initial low-resolution picture for a low-micromolar-peptide binder can be refined through the combination of NMR, protein design, docking, and MD simulations to establish its binding mode, even in the absence of crystallographic data, thereby providing valuable information for further design. Graphical abstract: Highlights: Determining the binding mode of weak ligands is often difficult. Interplay of biochemical and biophysical methods enabled locating binding in repeat protein. Preliminary chemical shift assignments indicated which repeats were involved in binding. Subsequent elimination of repeats resulted in a smaller protein that bound the ligand equally well. Chemical shift perturbations, paramagnetic relaxation enhancements, and MD data helped to locate the binding site.