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The epidermal growth factor receptor (EGFR) plays a central role in the progression of many solid tumors. We used this validated target to analyze the de novo design of EGFR-binding peptides and their application for the delivery of complex payloads via rational design of a viral vector. Peptides were computationally designed to interact with the EGFR dimerization interface. Two new peptides and a reference (EDA peptide) were chemically synthesized, and their binding ability characterized. Presentation of these peptides in each of the 60 capsid proteins of recombinant adeno-associated viruses (rAAV) via a genetic based loop insertion enabled targeting of EGFR overexpressing tumor cell lines. Furthermore, tissue distribution and tumor xenograft specificity were analyzed with systemic injection in chicken egg chorioallantoic membrane (CAM) assays. Complex correlations between the targeting of the synthetic peptides and the viral vectors to cells and in ovo were observed. Overall, these data demonstrate the potential of computational design in combination with rational capsid modification for viral vector targeting opening new avenues for viral vector delivery and specifically suicide gene therapy.

The design and selection of peptides targeting cellular proteins is challenging and often yields candidates with undesired properties. Therefore we deployed a new selection system based on the twin-arginine translocase (TAT) pathway of Escherichia coli, named hitchhiker translocation (HiT) selection. A pool of α-helix encoding sequences was designed and selected for interference with the coiled coil domain (CC) of a melanoma-associated basic-helix-loop-helix-leucine-zipper (bHLHLZ) protein, the microphthalmia associated transcription factor (MITF). One predominant sequence (iM10) was enriched during selection and showed remarkable protease resistance, high solubility and thermal stability while maintaining its specificity. Furthermore, it exhibited nanomolar range affinity towards the target peptide. A mutation screen indicated that target-binding helices of increased homodimer stability and improved expression rates were preferred in the selection process. The crystal structure of the iM10/MITF-CC heterodimer (2.1Å) provided important structural insights and validated our design predictions. Importantly, iM10 did not only bind to the MITF coiled coil, but also to the markedly more stable HLHLZ domain of MITF. Characterizing the selected variants of the semi-rational library demonstrated the potential of the innovative bacterial selection approach., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The twin-arginine translocation (TAT) pathway of the bacterial cytoplasmic membrane mediates translocation only of proteins that accomplished a native-like conformation. We deploy this feature in modular selection systems for directed evolution, in which folding helpers as well as dimeric or oligomeric protein-protein interactions enable TAT-dependent translocation of the resistance marker TEM β-lactamase (βL). Specifically, we demonstrate and analyze selection of (i) enhancers for folding by direct TAT translocation selection of a target protein interposed between the TorA signal sequence and βL, (ii) dimeric or oligomeric protein-protein interactions by hitchhiker translocation (HiT) selection of proteins fused to the TorA signal sequence and to the βL, respectively and (iii) heterotrimeric protein-protein interactions by combining HiT with protein fragment complementation selection of proteins fused to two split βL fragments and TorA, respectively. The lactamase fragments were additionally engineered for improved activity and stability. Applicability was benchmarked with interaction partners of known affinity and multimerization whereby cellular fitness correlated well with biophysical protein properties. Ultimately, the HiT selection was employed to identify peptides, which specifically bind to leukemia- and melanoma-relevant target proteins (MITF and ETO) by coiled-coil or tetra-helix-bundle formation with high affinity. The various versions of TAT selection led to inhibiting peptides (iPEPs) of disease-promoting interactions and enabled so far difficult to achieve selections.

Phage display with filamentous phages is widely applied and well developed, yet proteins requiring a cytoplasmic environment for correct folding still defy attempts at functional display. To extend applicability of phage display, we employed the twin-arginine translocation (TAT) pathway to incorporate proteins fused to the C-terminal domain of the geneIII protein into phage particles. We investigated functionality and display level of fluorescent proteins depending on the translocation pathway, which was the TAT, general secretory (SEC) or signal recognition particle (SRP) pathway mediated by the TorA, PelB or DsbA signal sequences, respectively. Importantly, for green fluorescent protein, yellow fluorescent protein and cyan fluorescent protein, only TAT, but not SEC or SRP, translocation led to fluorescence of purified phage particles, although all three proteins could be displayed regardless of the translocation pathway. In contrast, the monomeric red fluorescent protein mCherry was functionally displayed regardless of the translocation pathway. Hence, correct folding and fluorophor formation of mCherry is not limited to the cytosol. Furthermore, we successfully displayed firefly luciferase as well as an 83 kDa argonaute protein, both containing free cysteines. This demonstrates broad applicability of the TAT-mediated phagemid system for the display of proteins requiring cytoplasmic factors for correct folding and should prove useful for the display of proteins requiring incorporation of co-factors or oligomerization to gain function.

Improving enzyme stability is a highly desirable design step in generating enzymes able to function under extreme conditions, such as elevated temperatures, while having the additional benefit of being less susceptible to cleavage by proteases. For these reasons, many different approaches and techniques have been devised in constructing such proteins, but the results to date have been of mixed success. Here, we present a robust method involving the terminal truncation, random mutagenesis and fragmentation, recombination, elongation, and finally, selection at physiological temperatures, to generate an enzyme with improved stability. Three cycles of directed evolution comprising of random mutagenesis, DNA shuffling, and selection at 37 degrees C were used, using the bacterial enzyme TEM-1 beta-lactamase as a model protein to yield deletion mutants with in vivo ampicillin resistance levels comparable to wild-type (wt) enzyme. Kinetic studies demonstrate the selected mutant to have a significantly improved thermostability relative to its wt counterpart. Elongation of this mutant to the full-length gene resulted in a beta-lactamase variant with dramatically increased thermostability. This technique was so fruitful that the evolved enzyme retained its maximum catalytic activity even 20 degrees C above its wt parent protein optimum. Thus, structural perturbation by terminal truncation and subsequent compensation by directed evolution at physiological temperatures is a fast, efficient, and highly effective way to improve the thermostability of proteins without the need for selecting at elevated temperatures.

Coiled coil motifs are, despite their apparent simplicity, highly specific, and play a significant role in the understanding of tertiary structure and its formation. The most commonly observed of the coiled coils, the parallel dimeric, is yet to be fully characterized for this structural class in general. Nonetheless, strict rules have emerged for the necessity of specific types of amino acids at specific positions. In this chapter, we discuss this system in light of existing coiled coil structures and in applying rules to coiled coils that are to be designed or optimized. Understanding and expanding on these rules is crucial in using these motifs, which play key roles in virtually every cellular process, to act as drug-delivery agents by sequestering other proteins that are not behaving natively or that have been upregulated (for example, by binding to coiled coil domains implicated in oncogenesis). The roles of the a and d "hydrophobic" core positions and the e and g "electrostatic" edge positions in directing oligomerization and pairing specificity are discussed. Also discussed is the role of these positions in concert with the b, c, and f positions in maintaining alpha-helical propensity, helix solubility, and dimer stability.

The non-random chromosomal translocations t(10;11)(p13;q23) and t(10;11)(p13;q14-21) result in leukemogenic fusion proteins comprising the coiled coil domain of the transcription factor AF10 and the proteins MLL or CALM, respectively, and subsequently cause certain types of acute leukemia. The AF10 coiled-coil domain, which is crucial for the leukemogenic effect, has been shown to interact with GAS41, a protein previously identified as the product of an amplified gene in glioblastoma. Using sequential synthetic peptides, we mapped the potential AF10/GAS41 interaction site, which was subsequently be used as scaffold for a library targeting the AF10 coiled-coil domain. Using phage display, we selected a peptide that binds the AF10 coiled-coil domain with higher affinity than the respective coiled-coil region of wild-type GAS41, as demonstrated by phage ELISA, CD, and PCAs. Furthermore, we were able to successfully deploy the inhibitory peptide in a mammalian cell line to lower the expression of Hoxa genes that have been described to be overexpressed in these leukemias. This work dissects molecular determinants mediating AF10-directed interactions in leukemic fusions comprising the N-terminal parts of the proteins MLL or CALM and the C-terminal coiled-coil domain of AF10. Furthermore, it outlines the first steps in recognizing and blocking the leukemia-associated AF10 interaction in histiocytic lymphoma cells and therefore, may have significant implications in future diagnostics and therapeutics. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Phage display with filamentous phages is widely applied and well developed, yet proteins requiring a cytoplasmic environment for correct folding still defy attempts at functional display. To extend applicability of phage display, we employed the twin-arginine translocation (TAT) pathway to incorporate proteins fused to the C-terminal domain of the geneIII protein into phage particles. We investigated functionality and display level of fluorescent proteins depending on the translocation pathway, which was the TAT, general secretory (SEC) or signal recognition particle (SRP) pathway mediated by the TorA, PelB or DsbA signal sequences, respectively. Importantly, for green fluorescent protein, yellow fluorescent protein and cyan fluorescent protein, only TAT, but not SEC or SRP, translocation led to fluorescence of purified phage particles, although all three proteins could be displayed regardless of the translocation pathway. In contrast, the monomeric red fluorescent protein mCherry was functionally displayed regardless of the translocation pathway. Hence, correct folding and fluorophor formation of mCherry is not limited to the cytosol. Furthermore, we successfully displayed firefly luciferase as well as an 83 kDa argonaute protein, both containing free cysteines. This demonstrates broad applicability of the TAT-mediated phagemid system for the display of proteins requiring cytoplasmic factors for correct folding and should prove useful for the display of proteins requiring incorporation of co-factors or oligomerization to gain function. [ABSTRACT FROM AUTHOR]

Activator protein-1 (AP-1) is a crucial transcription factor implicated in numerous cancers. For this reason, nine homologues of the AP-1 leucine zipper region have been characterized: Fos (c-Fos, FosB, Fra1, and Fra2), Jun (c-Jun, JunB, and JunD), and semirational library-designed winning peptides FosW and JunW. The latter two were designed to specifically target c-Fos or c-Jun. They have been identified by using protein-fragment complementation assays combined with growth competition. This assay removes nonspecific, unstable, and protease susceptible library members from the pool, leaving winners with excellent drug potential. Thermal melts of all 45 possible dimeric interactions have been surveyed, with the FosW-c-Jun complex displaying a melting temperature (Tm) of 63°C, compared to only 16°C for wild-type c-Fos-c-Jun interaction. This impressive 70,000-fold KD decrease is largely due to optimized core packing, α-helical propensity, and electrostatics. Contrastingly, due to a poor c-Fos core, c-Fos-JunW dimerizes with lower affinity. However the Tm far exceeds wild-type c-Fos-c-Jun and averaged JunW and c-Fos, indicating a preference over either homodimer. Finally, and with wider implications, we have compiled a method for predicting interaction of parallel, dimeric coiled coils, using our Tm data as a training set, and applying it to 59 bZIP proteins previously reported. Our algorithm, unlike others to date, accounts for helix propensity, which is found to be integral in coiled coil stability. Indeed, in applying the algorithm to these 592 bZIP interactions, we were able to correctly identify 92% of all strong interactions and 92% of all noninteracting pairs. [ABSTRACT FROM AUTHOR]

To investigate how electrostatic interactions restrict the associations of coiled coils, we improved a heterodimeric coiled coil (WinZip-A1B1) by in vivo selection and, alternatively, by rational design. Selection from libraries encoding variable edge (g and e) residues enriched g/e′ ion pairs, but the optimum selected heterodimers unexpectedly retained two predicted repulsive g/e′ pairs. The best genetically selected heterodimer displayed similar thermodynamic stability and specificity as a rationally designed dimer with predicted ion pairs at all edge positions. This rationally designed pair, however, was less effective than the best genetically selected pair in mediating dimerization in vivo. Thus, the effects of predicted charge pairs depend on sequence context, and complementary charges at the edge positions rationalize only a fraction of the sequences that form stable, specific coiled coils. [Copyright &y& Elsevier]

The stability of proteins is paramount for their therapeutic and industrial use and, thus, is a major task for protein engineering. Several types of chemical and physical stabilities are desired, and discussion revolves around whether each stability trait needs to be addressed separately and how specific and compatible stabilizing mutations act. We demonstrate a stepwise perturbation-compensation strategy, which identifies mutations rescuing the activity of a truncated TEM β-lactamase. Analyses relating structural stress with the external stresses of heat, denaturants, and proteases reveal our second-site suppressors as general stability centers that also improve the full-length enzyme. A library of lactamase variants truncated by 15 N-terminal and three C-terminal residues (Bla-NΔ15CΔ3) was subjected to activity selection and DNA shuffling. The resulting clone with the best in vivo performance harbored eight mutations, surpassed the full-length wild-type protein by 5.3 °C in Tm, displayed significantly higher catalytic activity at elevated temperatures, and showed delayed guanidine- induced denaturation. The crystal structure of this mutant was determined and provided insights into its stability determinants. Stepwise reconstitution of the N- and C-termini increased its thermal, denaturant, and proteolytic resistance successively, leading to a full-length enzyme with a Tm, increased by 15.3 °C and a half-denaturation concentration shifted from 0.53 to 1.75 M guanidinium relative to that of the wild type. These improvements demonstrate that iterative truncation-optimization cycles can exploit stability-trait linkages in proteins and are exceptionally suited for the creation of progressively stabilized variants and/or downsized proteins without the need for detailed structural or mechanistic information. [ABSTRACT FROM AUTHOR]