Joshua L. Price

Flavonol activation defines an unanticipated ligand-binding site in the kinase-RNase domain of IRE1.

Signaling in the most conserved branch of the endoplasmic reticulum (ER) unfolded protein response (UPR) is initiated by sequence-specific cleavage of the HAC1/XBP1 mRNA by the ER stress-induced kinase-endonuclease IRE1. We have discovered that the flavonol quercetin activates yeast IRE1s RNase and potentiates activation by ADP, a natural activating ligand that engages the IRE1 nucleotide-binding cleft. Enzyme kinetics and the structure of a cocrystal of IRE1 complexed with ADP and quercetin reveal engagement by quercetin of an unanticipated ligand-binding pocket at the dimer interface of IRE1s kinase extension nuclease (KEN) domain. Analytical ultracentrifugation and crosslinking studies support the preeminence of enhanced dimer formation in quercetins mechanism of action. These findings hint at the existence of endogenous cytoplasmic ligands that may function alongside stress signals from the ER lumen to modulate IRE1 activity and at the potential for the development of drugs that modify UPR signaling from this unanticipated site.

Protein Native-State Stabilization by Placing Aromatic Side Chains in N-Glycosylated Reverse Turns

Protein reverse turns that interact with a phenlyalanine group allow stable introduction of glycan groups at asparagine residues. N-glycosylation of eukaryotic proteins helps them fold and traverse the cellular secretory pathway and can increase their stability, although the molecular basis for stabilization is poorly understood. Glycosylation of proteins at naïve sites (ones that normally are not glycosylated) could be useful for therapeutic and research applications but currently results in unpredictable changes to protein stability. We show that placing a phenylalanine residue two or three positions before a glycosylated asparagine in distinct reverse turns facilitates stabilizing interactions between the aromatic side chain and the first N-acetylglucosamine of the glycan. Glycosylating this portable structural module, an enhanced aromatic sequon, in three different proteins stabilizes their native states by –0.7 to –2.0 kilocalories per mole and increases cellular glycosylation efficiency.

Interplay among side chain sequence, backbone composition, and residue rigidification in polypeptide folding and assembly.

The extent to which polypeptide conformation depends on side-chain composition and sequence has been widely studied, but less is known about the importance of maintaining an α-amino acid backbone. Here, we examine a series of peptides with backbones that feature different repeating patterns of α- and β-amino acid residues but an invariant side-chain sequence. In the pure α-backbone, this sequence corresponds to the previously studied peptide GCN4-pLI, which forms a very stable four-helix bundle quaternary structure. Physical characterization in solution and crystallographic structure determination show that a variety of α/β-peptide backbones can adopt sequence-encoded quaternary structures similar to that of the α prototype. There is a loss in helix bundle stability upon β-residue incorporation; however, stability of the quaternary structure is not a simple function of β-residue content. We find that cyclically constrained β-amino acid residues can stabilize the folds of α/β-peptide GCN4-pLI analogues and restore quaternary structure formation to backbones that are predominantly unfolded in the absence of cyclic residues. Our results show a surprising degree of plasticity in terms of the backbone compositions that can manifest the structural information encoded in a sequence of amino acid side chains. These findings offer a framework for the design of nonnatural oligomers that mimic the structural and functional properties of proteins.

Mechanisms of transthyretin cardiomyocyte toxicity inhibition by resveratrol analogs

The transthyretin amyloidoses are a subset of protein misfolding diseases characterized by the extracellular deposition of aggregates derived from the plasma homotetrameric protein transthyretin (TTR) in peripheral nerves and the heart. We have established a robust disease-relevant human cardiac tissue culture system to explore the cytotoxic effects of amyloidogenic TTR variants. We have employed this cardiac amyloidosis tissue culture model to screen 23 resveratrol analogs as inhibitors of amyloidogenic TTR-induced cytotoxicity and to investigate their mechanisms of protection. Resveratrol and its analogs kinetically stabilize the native tetramer preventing the formation of cytotoxic species. In addition, we demonstrate that resveratrol can accelerate the formation of soluble non-toxic aggregates and that the resveratrol analogs tested can bring together monomeric TTR subunits to form non-toxic native tetrameric TTR.

Context-Dependent Effects of Asparagine Glycosylation on Pin WW Folding Kinetics and Thermodynamics

Asparagine glycosylation is one of the most common and important post-translational modifications of proteins in eukaryotic cells. N-glycosylation occurs when a triantennary glycan precursor is transferred en bloc to a nascent polypeptide (harboring the N-X-T/S sequon) as the peptide is cotranslationally translocated into the endoplasmic reticulum (ER). In addition to facilitating binding interactions with components of the ER proteostasis network, N-glycans can also have intrinsic effects on protein folding by directly altering the folding energy landscape. Previous work from our laboratories (Hanson et al. Proc. Natl. Acad. Sci. U.S.A. 2009, 109, 3131-3136; Shental-Bechor, D.; Levy, Y. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 8256-8261) suggested that the three sugar residues closest to the protein are sufficient for accelerating protein folding and stabilizing the resulting structure in vitro; even a monosaccharide can have a dramatic effect. The highly conserved nature of these three proximal sugars in N-glycans led us to speculate that introducing an N-glycosylation site into a protein that is not normally glycosylated would stabilize the protein and increase its folding rate in a manner that does not depend on the presence of specific stabilizing protein-saccharide interactions. Here, we test this hypothesis experimentally and computationally by incorporating an N-linked GlcNAc residue at various positions within the Pin WW domain, a small β-sheet-rich protein. The results show that an increased folding rate and enhanced thermodynamic stability are not general, context-independent consequences of N-glycosylation. Comparison between computational predictions and experimental observations suggests that generic glycan-based excluded volume effects are responsible for the destabilizing effect of glycosylation at highly structured positions. However, this reasoning does not adequately explain the observed destabilizing effect of glycosylation within flexible loops. Our data are consistent with the hypothesis that specific, evolved protein-glycan contacts must also play an important role in mediating the beneficial energetic effects on protein folding that glycosylation can confer.

Glycosylation of the enhanced aromatic sequon is similarly stabilizing in three distinct reverse turn contexts

Cotranslational N-glycosylation can accelerate protein folding, slow protein unfolding, and increase protein stability, but the molecular basis for these energetic effects is incompletely understood. N-glycosylation of proteins at naïve sites could be a useful strategy for stabilizing proteins in therapeutic and research applications, but without engineering guidelines, often results in unpredictable changes to protein energetics. We recently introduced the enhanced aromatic sequon as a family of portable structural motifs that are stabilized upon glycosylation in specific reverse turn contexts: a five-residue type I β-turn harboring a G1 β-bulge (using a Phe–Yyy–Asn–Xxx–Thr sequon) and a type II β-turn within a six-residue loop (using a Phe–Yyy–Zzz–Asn–Xxx–Thr sequon) [Culyba EK, et al. (2011) Science 331:571–575]. Here we show that glycosylating a new enhanced aromatic sequon, Phe–Asn–Xxx–Thr, in a type I′ β-turn stabilizes the Pin 1 WW domain. Comparing the energetic effects of glycosylating these three enhanced aromatic sequons in the same host WW domain revealed that the glycosylation-mediated stabilization is greatest for the enhanced aromatic sequon complementary to the type I β-turn with a G1 β-bulge. However, the portion of the stabilization from the tripartite interaction between Phe, Asn(GlcNAc), and Thr is similar for each enhanced aromatic sequon in its respective reverse turn context. Adding the Phe–Asn–Xxx–Thr motif (in a type I′ β-turn) to the enhanced aromatic sequon family doubles the number of proteins that can be stabilized by glycosylation without having to alter the native reverse turn type.

Structural and energetic basis of carbohydrate-aromatic packing interactions in proteins.

Carbohydrate-aromatic interactions mediate many biological processes. However, the structure-energy relationships underpinning direct carbohydrate-aromatic packing interactions in aqueous solution have been difficult to assess experimentally and remain elusive. Here, we determine the structures and folding energetics of chemically synthesized glycoproteins to quantify the contributions of the hydrophobic effect and CH-π interactions to carbohydrate-aromatic packing interactions in proteins. We find that the hydrophobic effect contributes significantly to protein-carbohydrate interactions. Interactions between carbohydrates and aromatic amino acid side chains, however, are supplemented by CH-π interactions. The strengths of experimentally determined carbohydrate CH-π interactions do not correlate with the electrostatic properties of the involved aromatic residues, suggesting that the electrostatic component of CH-π interactions in aqueous solution is small. Thus, tight binding of carbohydrates and aromatic residues is driven by the hydrophobic effect and CH-π interactions featuring a dominating dispersive component.

N-Glycosylation of Enhanced Aromatic Sequons to Increase Glycoprotein Stability

N‐glycosylation can increase the rate of protein folding, enhance thermodynamic stability, and slow protein unfolding; however, the molecular basis for these effects is incompletely understood. Without clear engineering guidelines, attempts to use N‐glycosylation as an approach for stabilizing proteins have resulted in unpredictable energetic consequences. Here, we review the recent development of three “enhanced aromatic sequons,” which appear to facilitate stabilizing native‐state interactions between Phe, Asn‐GlcNAc and Thr when placed in an appropriate reverse turn context. It has proven to be straightforward to engineer a stabilizing enhanced aromatic sequon into glycosylation‐naïve proteins that have not evolved to optimize specific protein–carbohydrate interactions. Incorporating these enhanced aromatic sequons into appropriate reverse turn types within proteins should enhance the well‐known pharmacokinetic benefits of N‐glycosylation‐based stabilization by lowering the population of protease‐susceptible unfolded and aggregation‐prone misfolded states, thereby making such proteins more useful in research and pharmaceutical applications.

Thermodynamics of proton dissociations from aqueous l-proline: apparent molar volumes and apparent molar heat capacities of the protonated cationic, zwitterionic, and deprotonated anionic forms at temperatures from 278.15 K to 393.15 K and at the pressure 0.35 MPa

Abstract Apparent molar volumes Vφ and apparent molar heat capacities Cp,φ were determined for aqueous solutions of l -proline, l -proline with equimolal HCl, and l -proline with equimolal NaOH at the pressure p=0.35 MPa. Density measurements obtained with a vibrating-tube densimeter at temperatures (278.15⩽T/K⩽368.15) were used to calculate Vφ values, and heat capacity measurements obtained with a twin fixed-cell, differential-output, power-compensation, temperature-scanning calorimeter at temperatures (278.15⩽T/K⩽393.15) were used to calculate Cp,φ values. Speciation arising from equilibrium was accounted for using Young’s Rule, and semi-empirical equations describing (Vφ, m, T) and (Cp,φ, m, T) for each aqueous equilibrium species were fitted by regression to the experimental results. From these equations, the volume change ΔrVm and heat capacity change ΔrCp,m for the protonation and deprotonation reactions were calculated. Additionally, the ΔrCp,m expression was integrated symbolically to yield values of the reaction enthalpy change ΔrHm, reaction entropy change ΔrSm, and equilibrium molality reaction quotient Q for both reactions. The results provide a much-improved thermodynamic characterization of aqueous l -proline and of its protonation and deprotonation equilibria.

Criteria for Selecting PEGylation Sites on Proteins for Higher Thermodynamic and Proteolytic Stability

PEGylation of protein side chains has been used for more than 30 years to enhance the pharmacokinetic properties of protein drugs. However, there are no structure- or sequence-based guidelines for selecting sites that provide optimal PEG-based pharmacokinetic enhancement with minimal losses to biological activity. We hypothesize that globally optimal PEGylation sites are characterized by the ability of the PEG oligomer to increase protein conformational stability; however, the current understanding of how PEG influences the conformational stability of proteins is incomplete. Here we use the WW domain of the human protein Pin 1 (WW) as a model system to probe the impact of PEG on protein conformational stability. Using a combination of experimental and theoretical approaches, we develop a structure-based method for predicting which sites within WW are most likely to experience PEG-based stabilization, and we show that this method correctly predicts the location of a stabilizing PEGylation site within the chicken Src SH3 domain. PEG-based stabilization in WW is associated with enhanced resistance to proteolysis, is entropic in origin, and likely involves disruption by PEG of the network of hydrogen-bound solvent molecules that surround the protein. Our results highlight the possibility of using modern site-specific PEGylation techniques to install PEG oligomers at predetermined locations where PEG will provide optimal increases in conformational and proteolytic stability.