Faculty Bibliography

Chronic myelogenous leukemia (CML) is caused by the constitutively active tyrosine kinase Bcr-Abl and treated with the tyrosine kinase inhibitor (TKI) imatinib. However, emerging TKI resistance prevents complete cure. Therefore, alternative strategies targeting regulatory modules of Bcr-Abl in addition to the kinase active site are strongly desirable. Here, we show that an intramolecular interaction between the SH2 and kinase domains in Bcr-Abl is both necessary and sufficient for high catalytic activity of the enzyme. Disruption of this interface led to inhibition of downstream events critical for CML signaling and, importantly, completely abolished leukemia formation in mice. Furthermore, disruption of the SH2-kinase interface increased sensitivity of imatinib-resistant Bcr-Abl mutants to TKI inhibition. An engineered Abl SH2-binding fibronectin type III monobody inhibited Bcr-Abl kinase activity both in vitro and in primary CML cells, where it induced apoptosis. This work validates the SH2-kinase interface as an allosteric target for therapeutic intervention.

Discriminating closely related molecules remains a major challenge in the engineering of binding proteins and inhibitors. Here we report the development of highly selective inhibitors of small ubiquitin-related modifier (SUMO) family proteins. SUMOylation is involved in the regulation of diverse cellular processes. Functional differences between two major SUMO isoforms in humans, SUMO1 and SUMO2/3, are thought to arise from distinct interactions mediated by each isoform with other proteins containing SUMO-interacting motifs (SIMs). However, the roles of such isoform-specific interactions are largely uncharacterized due in part to the difficulty in generating high-affinity, isoform-specific inhibitors of SUMO/SIM interactions. We first determined the crystal structure of a "monobody," a designed binding protein based on the fibronectin type III scaffold, bound to the yeast homolog of SUMO. This structure illustrated a mechanism by which monobodies bind to the highly conserved SIM-binding site while discriminating individual SUMO isoforms. Based on this structure, we designed a SUMO-targeted library from which we obtained monobodies that bound to the SIM-binding site of human SUMO1 with K(d) values of approximately 100 nM but bound to SUMO2 400 times more weakly. The monobodies inhibited SUMO1/SIM interactions and, unexpectedly, also inhibited SUMO1 conjugation. These high-affinity and isoform-specific inhibitors will enhance mechanistic and cellular investigations of SUMO biology.

Interactions between Src homology 2 (SH2) domains and phosphotyrosine sites regulate tyrosine kinase signaling networks. Selective perturbation of these interactions is challenging due to the high homology among the 120 human SH2 domains. Using an improved phage-display selection system, we generated a small antibody mimic (or 'monobody'), termed HA4, that bound to the Abelson (Abl) kinase SH2 domain with low nanomolar affinity. SH2 protein microarray analysis and MS of intracellular HA4 interactors showed HA4's specificity, and a crystal structure revealed how this specificity is achieved. HA4 disrupted intramolecular interactions of Abl involving the SH2 domain and potently activated the kinase in vitro. Within cells, HA4 inhibited processive phosphorylation activity of Abl and also inhibited STAT5 activation. This work provides a design guideline for highly specific and potent inhibitors of a protein interaction domain and shows their utility in mechanistic and cellular investigations.

Venomous animals immobilize prey using protein toxins that act on ion channels and other targets of biological importance. Broad use of toxins for biomedical research, diagnosis, and therapy has been limited by inadequate target discrimination, for example, among ion channel subtypes. Here, a synthetic toxin is produced by a new strategy to be specific for human Kv1.3 channels, critical regulators of immune T cells. A phage display library of 11,200 de novo proteins is designed using the alpha-KTx scaffold of 31 scorpion toxin sequences known or predicted to bind to potassium channels. Mokatoxin-1 (moka1) is isolated by affinity selection on purified target. Moka1 blocks Kv1.3 at nanomolar levels that do not inhibit Kv1.1, Kv1.2, or KCa1.1. As a result, moka1 suppresses CD3/28-induced cytokine secretion by T cells without cross-reactive gastrointestinal hyperactivity. The 3D structure of moka1 rationalizes its specificity and validates the engineering approach, revealing a unique interaction surface supported on an alpha-KTx scaffold. This scaffold-based/target-biased strategy overcomes many obstacles to production of selective toxins.

A peptide fusion tag and accompanying recombinant capture reagents have been developed on the basis of the peptide-PDZ domain interaction and affinity clamps, a new class of affinity reagent. This system allows for single-step purification under mild conditions and stable capture of a tagged protein. The subnanomolar affinity, high force resistance (>30 pN), small size ( approximately 25 kDa, approximately one-sixth of the size of IgG), and monomeric nature of the affinity clamp are all superior features for many applications, in particular single-molecule measurements.

Immobilization of a target molecule to a solid support is an indispensable step in phage display library sorting. Here we describe an immobilization method that addresses shortcomings of existing strategies. Our method is based on the use of a polyhistidine-tagged (His-tagged) target molecule and (BT)tris-NTA, a high-affinity capture reagent for His-tags that also contains a biotin moiety. (BT)tris-NTA provides a stable and reversible linkage between a His-tag and a streptavidin-coated solid support. Because His-tags are the de facto standard for recombinant protein purification, this method dramatically simplifies target preparation for phage display library sorting. Here, we demonstrate the utility of this method by selecting high-affinity binding proteins based on the fibronectin type III (FN3) scaffold to two His-tagged protein targets, yeast small ubiquitin-like modifier and maltose-binding protein. Notably, a significant number of FN3 clones binding either targets selected using the new immobilization method exhibited only very weak binding when the same target was immobilized by coating on a polystyrene surface. This suggests that the His-tag-mediated immobilization exposes epitopes that are masked by commonly used passive adsorption methods. Together, these results establish a method with the potential to streamline and enhance many binding-protein engineering experiments.

We have established a new protein-engineering strategy termed "directed domain-interface evolution" that generates a binding site by linking two protein domains and then optimizing the interface between them. Using this strategy, we have generated synthetic two-domain "affinity clamps" using PDZ and fibronectin type III (FN3) domains as the building blocks. While these affinity clamps all had significantly higher affinity toward a target peptide than the underlying PDZ domain, two distinct types of affinity clamps were found in terms of target specificity. One type conserved the specificity of the parent PDZ domain, and the other increased the specificity dramatically. Here, we characterized their specificity profiles using peptide phage-display libraries and scanning mutagenesis, which suggested a significantly enlarged recognition site of the high-specificity affinity clamps. The crystal structure of a high-specificity affinity clamp showed extensive contacts with a portion of the peptide ligand that is not recognized by the parent PDZ domain, thus rationalizing the improvement of the specificity of the affinity clamp. A comparison with another affinity clamp structure showed that, although both had extensive contacts between PDZ and FN3 domains, they exhibited a large offset in the relative position of the two domains. Our results indicate that linked domains could rapidly fuse and evolve as a single functional module, and that the inherent plasticity of domain interfaces allows for the generation of diverse active-site topography. These attributes of directed domain-interface evolution provide facile means to generate synthetic proteins with a broad range of functions.

A number of small organic molecules have been developed that bind to amyloid fibrils, a subset of which also inhibit fibrillization. Among these, the benzothiol dye Thioflavin-T (ThT) has been used for decades in the diagnosis of protein-misfolding diseases and in kinetic studies of self-assembly (fibrillization). Despite its importance, efforts to characterize the ThT-binding mechanism at the atomic level have been hampered by the inherent insolubility and heterogeneity of peptide self-assemblies. To overcome these challenges, we have developed a minimalist approach to designing a ThT-binding site in a "peptide self-assembly mimic" (PSAM) scaffold. PSAMs are engineered water-soluble proteins that mimic a segment of beta-rich peptide self-assembly, and they are amenable to standard biophysical techniques and systematic mutagenesis. The PSAM beta-sheet contains rows of repetitive amino acid patterns running perpendicular to the strands (cross-strand ladders) that represent a ubiquitous structural feature of fibril-like surfaces. We successfully designed a ThT-binding site that recapitulates the hallmarks of ThT-fibril interactions by constructing a cross-strand ladder consisting of contiguous tyrosines. The X-ray crystal structures suggest that ThT interacts with the beta-sheet by docking onto surfaces formed by a single tyrosine ladder, rather than in the space between adjacent ladders. Systematic mutagenesis further demonstrated that tyrosine surfaces across four or more beta-strands formed the minimal binding site for ThT. Our work thus provides structural insights into how this widely used dye recognizes a prominent subset of peptide self-assemblies, and proposes a strategy to elucidate the mechanisms of fibril-ligand interactions.

Though beta-rich self-assemblies comprise a major structural class of polypeptides, a detailed understanding of the determinants of their structure and stability is lacking. In particular, the roles of repetitive stretches of side chains running the long axis of these beta-sheets, termed "cross-strand ladders," remain poorly characterized due to the inherently insoluble and heterogeneous nature of self-assemblies. To overcome these experimental challenges, we have established a complementary experimental system termed "peptide self-assembly mimics" (PSAMs). The PSAMs capture a defined number of self-assembly-like peptide repeats within a soluble beta-rich protein, making structural and energetic studies possible. In this work, we investigated the role of cross-strand ladders containing aromatic residues, which are prominent in self-assembling peptides. A combination of solution data and high-resolution crystal structures revealed that a single cross-strand ladder consisting solely of Tyr significantly stabilized, rigidified, and flattened the PSAM beta-sheet. These characteristics would stabilize each beta-sheet layer of a self-assembly and direct sheet conformations compatible with lamination. Our results therefore provide a rationale for the abundance of aromatic amino acids in fibril-forming peptides and establish important roles of cross-strand Tyr ladders in the structure and stability of beta-rich peptide self-assemblies.

Stability evaluation of many mutants can lead to a better understanding of the sequence determinants of a structural motif and of factors governing protein stability and protein evolution. The traditional biophysical analysis of protein stability is low throughput, limiting our ability to widely explore sequence space in a quantitative manner. In this study, we have developed a high-throughput library screening method for quantifying stability changes, which is based on protein fragment reconstitution and yeast surface display. Our method exploits the thermodynamic linkage between protein stability and fragment reconstitution and the ability of the yeast surface display technique to quantitatively evaluate protein-protein interactions. The method was applied to a fibronectin type III (FN3) domain. Characterization of fragment reconstitution was facilitated by the co-expression of two FN3 fragments, thus establishing a yeast surface two-hybrid method. Importantly, our method does not rely on competition between clones and thus eliminates a common limitation of high-throughput selection methods in which the most stable variants are recovered predominantly. Thus, it allows for the isolation of sequences that exhibit a desired level of stability. We identified more than 100 unique sequences for a beta-bulge motif, which was significantly more informative than natural sequences of the FN3 family in revealing the sequence determinants for the beta-bulge. Our method provides a powerful means for the rapid assessment of the stability of many variants, for the systematic assessment of the contribution of different factors to protein stability, and for enhancement of the protein stability.