Design for Solubility May Reveal Induction of Amide Hydrogen/Deuterium Exchange by Protein Self-Association.

Structural heterogeneity often constrains the characterization of aggregating proteins to indirect or low-resolution methods, obscuring mechanistic details of association. Here, we report progress in understanding the aggregation of Adnectins, engineered binding proteins with an immunoglobulin-like fold. We rationally design Adnectin solubility and measure amide hydrogen/deuterium exchange (HDX) under conditions that permit transient protein self-association. Protein-protein binding commonly slows rates of HDX; in contrast, we find that Adnectin association may induce faster HDX for certain amides, particularly in the C-terminal ß-strand. In aggregation-prone proteins, we identify a pattern of very different rates of amide HDX for residues linked by reciprocal hydrogen bonds in the native structure. These results may be explained by local loss of native structure and formation of an inter-protein interface. Amide HDX induced by self-association, detected here by deliberate modulation of propensity for such interactions, may be a general phenomenon with the potential to expose mechanisms of aggregation by diverse proteins.

Using yeast surface display to engineer a soluble and crystallizable construct of hematopoietic progenitor kinase 1 (HPK1).

Hematopoietic progenitor kinase 1 (HPK1) is an intracellular kinase that plays an important role in modulating tumor immune response and thus is an attractive target for drug discovery. Crystallization of the wild-type HPK1 kinase domain has been hampered by poor expression in recombinant systems and poor solubility. In this study, yeast surface display was applied to a library of HPK1 kinase-domain variants in order to select variants with an improved expression level and solubility. The HPK1 variant with the most improved properties contained two mutations, crystallized readily in complex with several small-molecule inhibitors and provided valuable insight to guide structure-based drug design. This work exemplifies the benefit of yeast surface display towards engineering crystallizable proteins and thus enabling structure-based drug discovery.

Ensemble Modeling and Intracellular Aggregation of an Engineered Immunoglobulin-Like Domain.

The production of recombinant proteins in Escherichia coli frequently results in the formation of insoluble protein aggregates called inclusion bodies (IBs). The determinants of IB formation remain poorly understood and are of much interest for biotechnological and research applications, as well as offering insight into disease-related in vivo protein aggregation. Here we investigate a set of engineered target-binding proteins based upon the fibronectin type III domain, and we find that variations in sequence at just three positions in a solvent-exposed loop greatly alter the extent of IB formation. The loop is analogous to the third complementarity-determining region of immunoglobulin variable domains and has been shown to be conformationally mobile. In contrast to studies of other proteins, the extent of IB formation is not explained by differences in thermal stability measured by differential scanning calorimetry. Instead, IB formation is correlated with the average local stability of the FG loop, as modeled by an ensemble of structures generated using Rosetta's kinematic closure loop reconstruction method. This correlation suggests that loop instability may promote local unfolding, exposing aggregation-prone surfaces. Consistent with this mechanism, sequence-based predictions of aggregation propensity produced by Zyggregator are also correlated with IB formation, though not with modeled loop stability. The combination of average model energy scores with sequence-based aggregation predictions accounts for the variation in IB formation remarkably well (R(2)=0.8). The comparison with experimental data validates the ensemble modeling approach, which may be applicable to dynamic protein loops involved in a wide range of phenomena.