Electrostatic origin of a stabilizing synergistic interaction among b-, c-, and f-residues in a trimeric coiled coil.

Coiled coils are one of most common protein quaternary structures and represent the best understood relationship between amino acid sequence and protein conformation. Whereas the roles of residues at the canonical heptad positions the a, d, e, and g are understood in precise detail, conventional approaches often assume that the solvent-exposed b-, c-, and f-positions can be varied broadly for application-specific purposes with minimal consequences. However, a growing body of evidence suggests that interactions among these b, c, and f residues can contribute substantially to coiled-coil conformational stability. In the trimeric coiled coil described here, we find that b-position Glu10 engages in a stabilizing long-range synergistic interaction with c-position Lys18 (ΔΔΔGf = -0.65 ± 0.02 kcal/mol). This favorable interaction depends strongly on the presence of two nearby f-position residues: Lys 7 and Tyr14. Extensive mutational analysis of these residues in the presence of added salt vs. denaturant suggests that this long-range synergistic interaction is primarily electrostatic in origin, but also depends on the precise location and acidity of a side-chain hydrogen-bond donor within f-position Tyr14.

A probabilistic view of protein stability, conformational specificity, and design.

Various approaches have used neural networks as probabilistic models for the design of protein sequences. These "inverse folding" models employ different objective functions, which come with trade-offs that have not been assessed in detail before. This study introduces probabilistic definitions of protein stability and conformational specificity and demonstrates the relationship between these chemical properties and the [Formula: see text] Boltzmann probability objective. This links the Boltzmann probability objective function to experimentally verifiable outcomes. We propose a novel sequence decoding algorithm, referred to as "BayesDesign", that leverages Bayes' Rule to maximize the [Formula: see text] objective instead of the [Formula: see text] objective common in inverse folding models. The efficacy of BayesDesign is evaluated in the context of two protein model systems, the NanoLuc enzyme and the WW structural motif. Both BayesDesign and the baseline ProteinMPNN algorithm increase the thermostability of NanoLuc and increase the conformational specificity of WW. The possible sources of error in the model are analyzed.

Structural guidelines for stabilization of α-helical coiled coils via PEG stapling.

Macrocyclization or stapling is one of the most well-known and generally applicable strategies for enhancing peptide/protein conformational stability and target binding affinity. However, there are limited structure- or sequence-based guidelines for the incorporation of optimal interhelical staples within coiled coils: the location and length of an interhelical staple is either arbitrarily chosen or requires significant optimization. Here we explore the impact of interhelical PEG stapling on the conformational stability and proteolytic resistance of a model disulfide-bound heterodimeric coiled coil. We demonstrate that (1) interhelical PEG staples are more stabilizing when placed farther from an existing disulfide crosslink; (2) e/g' staples are more stabilizing than f/b' or b/c' staples; (3) PEG staples between different positions have different optimal staple lengths; (4) PEG stapling tolerates variation in the structure of the PEG linker and in the mode of conjugation; and (5) the guidelines developed here enable the rational design of a stabilized PEG-stapled HER-2 affibody with enhanced conformational stability and proteolytic resistance.

Prerequisites for Stabilizing Long-Range Synergistic Interactions among b-, c-, and f-Residues in Coiled Coils.

Coiled coils are among the most abundant tertiary and quaternary structures found in proteins. A growing body of evidence suggests that long-range synergistic interactions among solvent-exposed residues can contribute substantially to coiled-coil conformational stability, but our understanding of the key sequence and structural prerequisites of this effect is still developing. Here, we show that the strength of synergistic interaction involving a b-position Glu (i), an f-position Tyr (i + 4), and a c-position Lys (i + 8) depends on the identity of the f-position residue, the length and stability of the coiled coil, and its oligomerization stoichiometry/surface accessibility. Combined with previous observations, these results map out predictable sequence- and structure-based criteria for enhancing coiled-coil stability by up to -0.58 kcal/mol per monomer (or -2.32 kcal/mol per coiled-coil tetramer). Our observations expand the available tools for enhancing coiled coil stability by sequence variation at solvent-exposed b-, c-, and f-positions and suggest the need to exercise care in the choice of substitutions at these positions for application-specific purposes.

PEGylation Increases the Strength of a Nearby NH-π Hydrogen Bond in the WW Domain.

Here we show that an NH-π interaction between a highly conserved Asn and a nearby Trp stabilizes the WW domain of the human protein Pin1. The strength of this NH-π interaction depends on the structure of the arene, with NH-π interactions involving Trp or naphthylalanine being substantially more stabilizing than those involving Tyr or Phe. Calculations suggest arene size and polarizability are key structural determinants of NH-π interaction strength. Methylation or PEGylation of the Asn side-chain amide nitrogen each strengthens the associated NH-π interaction, though likely for different reasons. We hypothesize that methylation introduces steric clashes that destabilize conformations in which the NH-π interaction is not possible, whereas PEGylation strengthens the NH-π interaction via localized desolvation of the protein surface.