Here we have constructed a single-chain T-cell receptor (scTCR) scaffold with high stability and soluble expression efficiency by directed evolution and yeast surface display. We evolved scTCRs in parallel for either enhanced resistance to thermal denaturation at 46 degrees C, or improved intracellular processing at 37 degrees C, with essentially equivalent results. This indicates that the efficiency of the consecutive kinetic processes of membrane translocation, protein folding, quality control, and vesicular transport can be well predicted by the single thermodynamic parameter of thermal stability. Selected mutations were recombined to create an scTCR scaffold that was stable for over an hour at 65 degrees C, had solubility of over 4 mg ml(-1), and shake-flask expression levels of 7.5 mg l(-1), while retaining specific ligand binding to peptide-major histocompatibility complexes (pMHCs) and bacterial superantigen. These properties are comparable to those for stable single-chain antibodies, but are markedly improved over existing scTCR constructs. Availability of this scaffold allows engineering of high-affinity soluble scTCRs as antigen-specific antagonists of cell-mediated immunity. Moreover, yeast displaying the scTCR formed specific conjugates with antigen-presenting cells (APCs), which could allow development of novel cell-to-cell selection strategies for evolving scTCRs with improved binding to various pMHC ligands in situ.

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The heterodimeric ␣␤ T cell receptor (TCR) for antigen is the key determinant of T cell specificity. The structure of the TCR is very similar to that of antibodies, but the engineering of TCRs by directed evolution with combinatorial display libraries has not been accomplished to date. Here, we report that yeast surface display of a TCR was achieved only after the mutation of specific variable region residues. These residues are located in two regions of the TCR, at the interface of the ␣-and ␤-chains and in the ␤-chain framework region that is thought to be in proximity to the CD3 signal-transduction complex. The mutations are encoded naturally in many antibody variable regions, indicating specific functional differences that have not been appreciated between TCRs and antibodies. The identification of these residues provides an explanation for the inherent difficulties in the display of wild-type TCRs compared with antibodies. Yeastdisplayed mutant TCRs bind specifically to the peptide͞MHC antigen, enabling engineering of soluble T cell receptors as specific T cell antagonists. This strategy of random mutagenesis followed by selection for surface expression may be of general use in the directed evolution of other eukaryotic proteins that are refractory to display.T cell receptors (TCRs) and antibodies have evolved to recognize different classes of ligands. Antibodies function as membrane-bound and soluble proteins that bind to soluble antigens, whereas TCRs function only as membrane-bound molecules that bind to cell-associated peptide͞MHC antigens. All of the energy of the antibody:antigen interaction focuses on the foreign antigen, whereas a substantial fraction of the energy of the TCR:peptide͞MHC interaction seems to be directed at the self-MHC molecule (1). In addition, antibodies can have ligand-binding affinities that are orders of magnitude higher than those of TCRs, largely because of the processes of somatic mutation and affinity maturation. In their normal cellular context, TCRs do not undergo somatic mutation and the processes of thymic selection seem to operate by maintaining a narrow window of affinities (2). The association of TCRs at the cell surface with the accessory molecules CD4 or CD8 also may influence the functional affinity of TCRs (3). Despite these differences, the three-dimensional structures of the two proteins are remarkably similar, with the hypervariable regions forming loops on a single face of the molecule that contacts the antigen (4-7).Based on their structural similarities, it is somewhat surprising that there have been significant differences in the success of producing soluble and surface-displayed forms of the extracellular domains of TCRs and antibodies in heterologous expression systems. Many antibodies have now been expressed at high yield and solubility as either intact or Fab-fragment forms or as single-chain (sc) fragment-variable (Fv) proteins. In addition, there are numerous antigen-binding Fv fragments that have been isolated de novo and͞or improved through...

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When two proteins associate they form a molecular interface that is a structural and energetic mosaic. Within such interfaces, individual amino acid residues contribute distinct binding energies to the complex. In combination, these energies are not necessarily additive, and significant positive or negative cooperative effects often exist. The basis of reliable algorithms to predict the specificities and energies of protein-protein interactions depends critically on a quantitative understanding of this cooperativity. We have used a model protein-protein system defined by an affinity maturation pathway, comprising variants of a T cell receptor V␤ domain that exhibit an overall affinity range of ϳ1500-fold for binding to the superantigen staphylococcal enterotoxin C3, in order to dissect the cooperative and additive energetic contributions of residues within an interface. This molecular interaction has been well characterized previously both structurally, by x-ray crystallographic analysis, and energetically, by scanning alanine mutagenesis. Through analysis of group and individual maturation and reversion mutations using surface plasmon resonance spectroscopy, we have identified energetically important interfacial residues, determined their cooperative and additive energetic properties, and elucidated the kinetic and thermodynamic bases for molecular evolution in this system. The summation of the binding free energy changes associated with the individual mutations that define this affinity maturation pathway is greater than that of the fully matured variant, even though the affinity gap between the end point variants is relatively large. Two mutations in particular, both located in the complementarity determining region 2 loop of the V␤ domain, exhibit negative cooperativity.Specific associations between heterologous proteins are required for many essential cellular processes. The biophysical factors that govern these protein-protein interactions, including van der Waals interactions, hydrogen bonding, hydrophobic packing, shape and charge complementarity, allostery, plasticity, and cooperativity (1-9), have been the subject of intensive study for decades. However, the formulation of reliable algorithms to predict the specificities and energies of these molecular interactions from protein structures alone has not been realized. Structural and energetic analyses of model proteinprotein interactions have yielded some quantitative correlations on which to base such algorithms (1, 5, 10 -15), but they remain far from comprehensive as some biophysical factors have resisted quantification or generalization.Many studies have sought to establish such correlations based on the analysis of single-site residue mutations, which arose either naturally or by site-directed mutagenesis. Although this strategy can provide correlations for some factors that govern interactions, it is not possible to probe many of the more complex biophysical factors such as plasticity and cooperativity in this manner. Nonetheless, the interfaces o...

Yeast surface display and sorting by flow cytometry have been used to isolate mutants of an scFv that is specific for the Vbeta8 region of the T cell receptor. Selection was based on equilibrium binding by two fluorescently labeled probes, a soluble Vbeta8 domain and an antibody to the c-myc epitope tag present at the carboxy-terminus of the scFv. The mutants that were selected in this screen included a scFv with threefold increased affinity for the Vbeta8 and scFv clones that were bound with reduced affinities by the anti-c-myc antibody. The latter finding indicates that the yeast display system may be used to map conformational epitopes, which cannot be revealed by standard peptide screens. Equilibrium antigen binding constants were estimated within the surface display format, allowing screening of isolated mutants without necessitating subcloning and soluble expression. Only a relatively small library of yeast cells (3 x 10[5]) displaying randomly mutagenized scFv was screened to identify these mutants, indicating that this system will provide a powerful tool for engineering the binding properties of eucaryotic secreted and cell surface proteins.

Although protein-protein interactions are involved in nearly all cellular processes, general rules for describing affinity and selectivity in protein-protein complexes are lacking, primarily because correlations between changes in protein structure and binding energetics have not been well determined. Here, we establish the structural basis of affinity maturation for a protein-protein interaction system that we had previously characterized energetically. This model system exhibits a 1500-fold affinity increase. Also, its affinity maturation is restricted by negative intramolecular cooperativity. With three complex and six unliganded variant X-ray crystal structures, we provide molecular snapshots of protein interface remodeling events that span the breadth of the affinity maturation process and present a comprehensive structural view of affinity maturation. Correlating crystallographically observed structural changes with measured energetic changes reveals molecular bases for affinity maturation, intramolecular cooperativity, and context-dependent binding.

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