Optimization of designed armadillo repeat proteins by molecular dynamics simulations and NMR spectroscopy

Abstract

A multidisciplinary approach based on molecular dynamics (MD) simulations using homology models, NMR spectroscopy, and a variety of biophysical techniques was used to efficiently improve the thermodynamic stability of armadillo repeat proteins (ArmRPs). ArmRPs can form the basis of modular peptide recognition and the ArmRP version on which synthetic libraries are based must be as stable as possible. The 42-residue internal Arm repeats had been designed previously using a sequence-consensus method. Heteronuclear NMR revealed unfavorable interactions present at neutral but absent at high pH. Two lysines per repeat were involved in repulsive interactions, and stability was increased by mutating both to glutamine. Five point mutations in the capping repeats were suggested by the analysis of positional fluctuations and configurational entropy along multiple MD simulations. The most stabilizing single C-cap mutation Q240L was inferred from explicit solvent MD simulations, in which water penetrated the ArmRP. All mutants were characterized by temperature-and denaturant-unfolding studies and the improved mutants were established as monomeric species with cooperative folding and increased stability against heat and denaturant. Importantly, the mutations tested resulted in a cumulative decrease of flexibility of the folded state in silico and a cumulative increase of thermodynamic stability in vitro. The final construct has a melting temperature of about 85°C, 14.5°higher than the starting sequence. This work indicates that in silico studies in combination with heteronuclear NMR and other biophysical tools may provide a basis for successfully selecting mutations that rapidly improve biophysical properties of the target proteins.

Computationally Designed Armadillo Repeat Proteins for Modular Peptide Recognition

“…Figure S8). Therefore, the Rosetta model of CAR0 was used as starting structure for multiple explicit solvent MD runs (of 0.5 to 2 µs each) to identify regions with high flexibility and stabilize them, as described before [9]. The MD trajectories showed that the overall fold of model CAR0 was preserved, with an average root mean square fluctuation (RMSF) of 0.4 Å for the C α carbon atoms (the RMSD is given in Supplementary Table ST2).…”

“…for DARPins [22] and consensus-based ArmRPs [9,10]. For dArmRPs, the impact of the Y III -capping repeat was additionally tested on the original, computationally designed protein These results do not allow us to decide whether the capping repeat has an influence on the overall curvature or not.…”

“…A consensus-based design approach, which had been applied for other repeat proteins [8] and also for ArmRPs [5,9,10] (named consensus ArmRPs or cArmRPs), is expected to yield a uniform curvature, although it may not necessarily match the exact geometry desired for binding the dipeptide units. Therefore, an in silico design approach was developed in this work, based on a geometrically optimal curvature template.…”

Designing repeat proteins: a modular approach to protein design

“…Multiple copies of the unit are then linked to create a homogeneous repeat protein sequence; capping repeats are derived from naturally existing proteins. Consensus design has been the first and most widely used approach across multiple repeat protein families: ankyrin (ANK) [9][10][11][12][13], tetratricopeptide repeat (TPR) [14], armadillo (ARM) [15][16][17], leucine rich repeat (LRR) [18], pentatricopeptide repeat (PPR) [19,20], pentapeptide repeat [21], 42 residue TPR variant (42PR) [22] and HEAT-EZ [23], all have designed structures confirmed by crystallography (Table 1). Effective consensus design relies on the assumption that evolutionary conservation translates into self-compatibility of repeats and foldability of the structures.…”

“…Further restricting the MSA to narrow and homogeneous groups within the families produced successful designs in some cases [18,23]. Using structure information and molecular dynamic simulations produced stable ARM designs; ARM designs from consensus were molten globule-like [15,16]. Instead of extracting a consensus sequence, Lee and Blaber [25,26] progressively introduced mutations experimentally to a beta trefoil background to arrive at sequences that contain three identical tandem repeats.…”