Protein Engineering Potential Inhibitor of Detrimental Immune Responses

On the surface of immune cells, class II major histocompatibility complex proteins (MHCII) present antigenic peptides for CD4+ T cell recognition, which initiate a variety of antigen-specific immune responses such as antibody response or cytotoxic T cell activation. In people with with auto-immune diseases including but not limited to type 1 diabetes, multiple sclerosis, and rheumatoid arthritis, detrimental immune responses occur after the presentation of antigenic peptides. A single-chain, minimal MHCII (scm-MHCII) has been designed to retain its function as an antigen-presenting protein with a simplified structure that can be easily produced and manipulated in a laboratory by recombinant microbial expression. By applying directed evolution and selection for protein stability quantified using yeast surface display (YSD), we have engineered a mutant library which may contain highly stable mutants capable of functioning as a highly specific inhibitor of T cell-mediated immune responses with the potential to be applied to treating a variety auto-immune diseases

Autocatalytic Activation of Influenza Hemagglutinin

Enveloped viruses contain surface proteins that mediate fusion between the viral and target cell membranes following an activating stimulus. Acidic pH induces the influenza virus fusion protein hemagglutinin (HA) via irreversible refolding of a trimeric conformational state leading to exposure of hydrophobic fusion peptides on each trimer subunit. Herein, we show that cells expressing fowl plague virus HA demonstrate discrete switching behavior with respect to the HA conformational change. Partially activated states do not exist at the scale of the cell, activation of HA leads to aggregation of cell surface trimers, and newly synthesized HA refold spontaneously in the presence of previously activated HA. These observations imply a feedback mechanism involving self-catalyzed refolding of HA and thus suggest a mechanism similar to the autocatalytic refolding and aggregation of prions

Post-translational Regulation of Expression and Conformation of an Immunoglobulin Domain in Yeast Surface Display

Display of heterologous proteins on the surface of Saccharomyces cerevisiae is increasingly being exploited for directed evolution because of straightforward cell screens. However, yeast post-translationally modifies proteins in ways that must be factored into library engineering and refinement. Here, we express the extracellular immunoglobulin domain of an ubiquitous mammalian membrane protein, CD47, which is implicated in cancer, immunocompatibility, and motility. CD47 has multiple sites of glycosylation and a core disulfide bond. We assess the effects of both of these post-translational modifications on expression and antibody binding. CD47’s extracellular domain is fused to the yeast mating protein Aga2p on the cell wall, and the resulting fusion protein binds several key antibodies, including a conformation-sensitive antibody. Site-by-site mutagenesis of CD47’s five N-linked glycosylation sites progressively decreases expression levels on yeast, but folding appears stable. Cysteine mutations disrupt the expected core disulfide, and also decrease protein expression levels, though not to the extent seen with complete deglycosylation. However, with the core disulfide mutants, antibody binding proves to be lower than expression levels might indicate and glycosylation is clearly reduced compared to wild-type. The results indicate that glycosylation regulates heterologous display on yeast more than core disulfides do and thus suggest bounds on directed evolution by post-translational processing

Understanding Conformational Regulation of the Integrin I-domain for Design of Chimeric Protein Switches

Within all complex biological processes intricate proteins are expressed to complete every niche and necessary task. Many express multiple allosterically regulated conformational states, with protein function regulated by effector molecules and other ligands. One such protein is the LFA-1 surface integrin protein and its inserted domain, the I-domain. We Isolated the I-domain for investigation of determining binding properties and understanding conformational regulations of affinity changes to its target ligand ICAM-1, for further use in chimeric protein switch design. A large change in binding affinity was found through the deletion of a sub-sequence of amino acids in I-domain known as the α7 helix. Our investigation shows that, when the α7 helix is deleted, I-domain converts into a permanent high affinity state in which binding affinity to ICAM-1 was increased, and this state can be reversed by co-expression with soluble α7 helix peptide. These results conclude that the α7 helix stabilizes the I domain in its low affinity conformation in a ligand-like manner, allowing relaxation to the high affinity conformation upon disruption of α7 helix interaction. While deletion of the α7 helix yields higher binding affinity in I-domain it cannot be applied in design of chimeric protein switches due to its permanent conformational state. Because of this, our switch design has a focus of allosterically regulating the I-domain and α7 helix through utilizing on/off switching of conformational states. I-domain is fused with EF3 and EF4 hands of calmodulin, which then regulates binding affinity to ICAM-1 through interaction with α7 helix, when the EF hands’ natural ligand peptides are present. Currently, mutant switches are being developed to alter EF hand binding specificity which, when bound to new target ligands, will cause an increase in I-domain-ICAM-1 binding affinity in switch molecules. The results of these allosteric regulations highlight the potential of chimeric protein switches for design of environmentally responsive targeting agents and suggest that, through directed evolution, regulated binding to a range of novel targets could be achieved for therapeutic intervention

Progress in the development and application of computational methods for probabilistic protein design

Proteins exhibit a wide range of physical and chemical properties, including highly selective molecular recognition and catalysis, and are also key components in biological metabolic, catabolic, and signaling pathways. Given that proteins are well-structured and can now be rapidly synthesized, they are excellent targets for engineering of both molecular structure and biological function. Computational analysis of the protein design problem allows scientists to explore sequence space and systematically discover novel protein molecules. Nonetheless, the complexity of proteins, the subtlety of the determinants of folding, and the exponentially large number of possible sequences impede the search for peptide sequences compatible with a desired structure and function. Directed search algorithms, which identify directly a small number of sequences, have achieved some success in identifying sequences with desired structures and functions. Alternatively, one can adopt a probabilistic approach. Instead of a finite number of sequences, such calculations result in a probabilistic description of the sequence ensemble. In particular, by casting the formalism in the language of statistical mechanics, the site-specific amino acid probabilities of sequences compatible with a target structure may be readily identified. The computational probabilities are well suited for both de novo protein design of particular sequences as well as combinatorial, library-based protein engineering. The computed site-specific amino acid profile may be converted to a nucleotide base distribution to allow assembly of a partially randomized gene library. The ability to synthesize readily such degenerate oligonucleotide sequences according to the prescribed distribution is key to constructing a biased peptide library genuinely reflective of the computational design. Herein we illustrate how a standard DNA synthesizer can be used with only a slight modification to the synthesis protocol to generate a pool of degenerate DNA sequences, which encodes a predetermined amino acid distribution with high fidelity

Structural Coupling Between FKBP12 and Buried Water

Globular proteins often contain structurally well-resolved internal water molecules. Previously, we reported results from a molecular dynamics study that suggested that buried water (Wat3) may play a role in modulating the structure of the FK506 binding protein-12 (FKBP12) (Park and Saven, Proteins 2005; 60:450-463). In particular, simulations suggested that disrupting a hydrogen bond to Wat3 by mutating E60 to either A or Q would cause a structural perturbation involving the distant W59 side chain, which rotates to a new conformation in response to the mutation. This effectively remodels the ligand-binding pocket, as the side chain in the new conformation is likely to clash with bound FK506. To test whether the protein structure is in effect modulated by the binding of a buried water in the distance, we determined high-resolution (0.92-1.29 A) structures of wild-type FKBP12 and its two mutants (E60A, E60Q) by X-ray crystallography. The structures of mutant FKBP12 show that the ligand-binding pocket is indeed remodeled as predicted by the substitution at position 60, even though the water molecule does not directly interact with any of the amino acids of the binding pocket. Thus, these structures support the view that buried water molecules constitute an integral, noncovalent component of the protein structure. Additionally, this study provides an example in which predictions from molecular dynamics simulations are experimentally validated with atomic precision, thus showing that the structural features of protein-water interactions can be reliably modeled at a molecular level

Yeast Surface Display of a Noncovalent MHC Class II Heterodimer Complexed with Antigenic Peptide

Microbial protein display technologies have enabled directed molecular evolution of binding and stability properties in numerous protein systems. In particular, dramatic improvements to antibody binding affinity and kinetics have been accomplished using these tools in recent years. Examples of successful application of display technologies to other immunological proteins have been limited to date. Herein, we describe the expression of human class II major histocompatibility complex allele (MHCII) HLA-DR4 on the surface of Saccharomyces cerevisiae as a noncovalently associated heterodimer. The yeast-displayed MHCII is fully native as assessed by binding of conformationally specific monoclonal antibodies; failure of antibodies specific for empty HLA-DR4 to bind yeast-displayed protein indicates antigenic peptide is bound. This report represents the first example of a noncovalent protein dimer displayed on yeast and of successful display of wildtype MHCII. Results further point to the potential for using yeast surface display for engineering and analyzing the antigen binding properties of MHCII

Protein-Protein Fusion Catalyzed by Sortase A

Chimeric proteins boast widespread use in areas ranging from cell biology to drug delivery. Post-translational protein fusion using the bacterial transpeptidase sortase A provides an attractive alternative when traditional gene fusion fails. We describe use of this enzyme for in vitro protein ligation and report the successful fusion of 10 pairs of protein domains with preserved functionality — demonstrating the robust and facile nature of this reaction

An affinity matured minibody for PET imaging of prostate stem cell antigen (PSCA)-expressing tumors

PurposeProstate stem cell antigen (PSCA), a cell surface glycoprotein expressed in normal human prostate and bladder, is over-expressed in the majority of localized prostate cancer and most bone metastases. We have previously shown that the hu1G8 minibody, a humanized anti-PSCA antibody fragment (single-chain Fv-C(H)3 dimer, 80 kDa), can localize specifically and image PSCA-expressing xenografts at 21 h post-injection. However, the humanization and antibody fragment reformatting decreased its apparent affinity. Here, we sought to evaluate PET imaging contrast with affinity matured minibodies.MethodsYeast scFv display, involving four rounds of selection, was used to generate the three affinity matured antibody fragments (A2, A11, and C5) that were reformatted into minibodies. These three affinity matured anti-PSCA minibodies were characterized in vitro, and following radiolabeling with (124)I were evaluated in vivo for microPET imaging of PSCA-expressing tumors.ResultsThe A2, A11, and C5 minibody variants all demonstrated improved affinity compared to the parental (P) minibody and were ranked as follows: A2 > A11 > C5 > P. The (124)I-labeled A11 minibody demonstrated higher immunoreactivity than the parental minibody and also achieved the best microPET imaging contrast in two xenograft models, LAPC-9 (prostate cancer) and Capan-1 (pancreatic cancer), when evaluated in vivo.ConclusionOf the affinity variant minibodies tested, the A11 minibody that ranked second in affinity was selected as the best immunoPET tracer to image PSCA-expressing xenografts. This candidate is currently under development for evaluation in a pilot clinical imaging study

Identification and Characterization of Ixodes scapularis Antigens That Elicit Tick Immunity Using Yeast Surface Display

Repeated exposure of rabbits and other animals to ticks results in acquired resistance or immunity to subsequent tick bites and is partially elicited by antibodies directed against tick antigens. In this study we demonstrate the utility of a yeast surface display approach to identify tick salivary antigens that react with tick-immune serum. We constructed an Ixodes scapularis nymphal salivary gland yeast surface display library and screened the library with nymph-immune rabbit sera and identified five salivary antigens. Four of these proteins, designated P8, P19, P23 and P32, had a predicted signal sequence. We generated recombinant (r) P8, P19 and P23 in a Drosophila expression system for functional and immunization studies. rP8 showed anti-complement activity and rP23 demonstrated anti-coagulant activity. Ixodes scapularis feeding was significantly impaired when nymphs were fed on rabbits immunized with a cocktail of rP8, rP19 and rP23, a hall mark of tick-immunity. These studies also suggest that these antigens may serve as potential vaccine candidates to thwart tick feeding