Katja M. Arndt

Coiled Coil Domains: Stability, Specificity, and Biological Implications

The coiled coil is a common structural motif, formed by approximately 3 ± 5% of all amino acids in proteins. Typically, it consists of two to five -helices wrapped around each other into a left-handed helix to form a supercoil. Whereas regular -helices go through 3.6 residues for each complete turn of the helix, the distortion imposed upon each helix within a left-handed coiled coil lowers this value to around 3.5. Thus a heptad repeat occurs every two turns of the helix. 3] The coiled coil was first described by Crick in 1953. He noted that -helices pack together 20 away from parallel whilst wrapping around each other, with their side chains packing TMin a knobs-into-holes manner∫. The same year, Pauling and Corey put forward a model for -keratin. It was some 20 years later that the sequence of rabbit skeletal tropomyosin was published, and another twenty until the first structure of the leucine zipper motif was solved by Alber and co-workers. These last discoveries pushed the coiled-coil field into the spotlight, as it became apparent that they are found in important structures that are involved in crucial interactions such as transcriptional control. The most commonly observed type of coiled coil is left-handed; here each helix has a periodicity of seven (a heptad repeat), with anywhere from two (in designed coiled coils) to 200 of these repeats in a protein. This repeat is usually denoted (a-b-c-d-e-fg)n in one helix, and (a -b -c -d -e -f -g )n in the other (Figure 1). In this model, a and d are typically nonpolar core residues found at the interface of the two helices, whereas e and g are solventexposed, polar residues that give specificity between the two helices through electrostatic interactions. Similarly in righthanded coiled coils, an eleven-residue repeat is observed (undecatad repeat). 11] The apparent simplicity of the structure with its heptad periodicity has led to extensive studies. Here we aim to outline the importance of individual amino acids in maintaining -helical structure (intramolecular interactions) within individual helices, whilst promoting specific coiled-coil interactions (intermolecular interactions) of correct oligomeric state and orientation. The PV Hypothesis

An in vivo library-versus-library selection of optimized protein-protein interactions.

We describe a rapid and efficient in vivo library-versus-library screening strategy for identifying optimally interacting pairs of heterodimerizing polypeptides. Two leucine zipper libraries, semi-randomized at the positions adjacent to the hydrophobic core, were genetically fused to either one of two designed fragments of the enzyme murine dihydrofolate reductase (mDHFR), and cotransformed into Escherichia coli. Interaction between the library polypeptides reconstituted enzymatic activity of mDHFR, allowing bacterial growth. Analysis of the resulting colonies revealed important biases in the zipper sequences relative to the original libraries, which are consistent with selection for stable, heterodimerizing pairs. Using more weakly associating mDHFR fragments, we increased the stringency of selection. We enriched the best-performing leucine zipper pairs by multiple passaging of the pooled, selected colonies in liquid culture, as the best pairs allowed for better bacterial propagation. This competitive growth allowed small differences among the pairs to be amplified, and different sequence positions were enriched at different rates. We applied these selection processes to a library-versus-library sample of 2.0 × 106 combinations and selected a novel leucine zipper pair that may be appropriate for use in further in vivo heterodimerization strategies.

THE FIRST CONSTANT DOMAIN (CH1 AND CL) OF AN ANTIBODY USED AS HETERODIMERIZATION DOMAIN FOR BISPECIFIC MINIANTIBODIES

Bispecific miniantibodies were constructed by genetically fusing the CH1 domain of an IgG1 to the C‐terminus of a single‐chain Fv fragment (scFv‐425), specific for the EGF receptor, and fusing the CL domain of a kappa light chain to the C‐terminus of a scFv specific for CD2 (scFv‐M1). An efficient dicistronic gene arrangement for functional expression in Escherichia coli was constructed. Immunoblots demonstrated correct domain assembly and the formation of the natural CH1‐CL disulfide bridge. Gel filtration confirmed the correct size, sandwich ELISAs demonstrated bispecific functionality, and SPR biosensor measurements determined binding to EGF‐R in comparison to bivalent constructs. Bispecific anti‐EGF‐R/anti‐CD2 miniantibodies are candidates for the immunotherapy of cancer.

A dimeric bispecific miniantibody combines two specificities with avidity

Bispecific antibodies extend the capabilities of nature and might be applied in immunotherapy and biotechnology. By fusing the gene of a single‐chain Fv (scFv) fragment to a helical dimerization domain, followed by a second scFv fragment of different specificity, we were able to express a functional protein in E. coli, which is bispecific and has two valencies for each specificity. The dimeric bispecific (DiBi) miniantibody preserves the natural avidity of antibodies in a very small‐sized molecule of only 120 kDa. The generality of the principle was shown with a scFv fragment binding the EGF‐receptor (named scFv 425) in three combinations with scFv fragments either directed against CD2 (ACID2.M1), phosphorylcholine (McPC603) or fluorescein (FITC‐E2). Binding was analyzed by sandwich surface plasmon resonance biosensor (BIAcore) measurements.

Photocontrol of Coiled-Coil Proteins in Living Cells**

Light switching of the activity of a coiled-coil protein, the AP-1 transcription factor, in living cells was made possible by the introduction of a designed azobenzene-cross-linked dominant negative peptide, XAFosW (red and yellow in the picture). In the dark, XAFosW showed decreased helical content and decreased affinity for target Jun proteins (green); irradiation at 365 nm enhanced helicity and target affinity.

Nucleotide exchange and excision technology (NExT) DNA shuffling: a robust method for DNA fragmentation and directed evolution

DNA shuffling is widely used for optimizing complex properties contained within DNA and proteins. Demonstrated here is the amplification of a gene library by PCR using uridine triphosphate (dUTP) as a fragmentation defining exchange nucleotide with thymidine, together with the three other nucleotides. The incorporated uracil bases were excised using uracil-DNA-glycosylase and the DNA backbone subsequently cleaved with piperidine. These end-point reactions required no adjustments. Polyacrylamide urea gels demonstrated adjustable fragmentation size over a wide range. The oligonucleotide pool was reassembled by internal primer extension to full length with a proofreading polymerase to improve yield over Taq. We present a computer program that accurately predicts the fragmentation pattern and yields all possible fragment sequences with their respective likelihood of occurrence, taking the guesswork out of the fragmentation. The technique has been demonstrated by shuffling chloramphenicol acetyltransferase gene libraries. A 33% dUTP PCR resulted in shuffled clones with an average parental fragment size of 86 bases even without employment of a fragment size separation, and revealed a low mutation rate (0.1%). NExT DNA fragmentation is rational, easily executed and reproducible, making it superior to other techniques. Additionally, NExT could feasibly be applied to several other nucleotide analogs.

Selectively infective phage (SIP) technology: scope and limitations

We review here the selectively infective phage (SIP) technology, a powerful tool for the rapid selection of protein-ligand and peptide-ligand pairs with very high affinities. SIP is highly suitable for discriminating between molecules with subtle stability and folding differences. We discuss the preferred types of applications for this technology and some pitfalls inherent in the in vivo SIP method that have become apparent in its application with highly randomized libraries, as well as some precautions that should be taken in successfully applying this technology.

Improved stability of the Jun-Fos Activator Protein-1 coiled coil motif: A stopped-flow circular dichroism kinetic analysis.

Two c-Jun leucine zipper variants that bind with high affinity to c-Fos have been selected using semirational design combined with protein-fragment complementation assays (JunW) or phage display selection (JunWPh1). Enriched winners differ from each other in only two of ten semi-randomized positions, with ΔTm values of 28 °C and 37 °C over wild-type. cFos-JunW, cFos-JunWPh1, and two intermediate mutants (cFos-JunWQ21R and cFos-JunWE23K) display biphasic kinetics in the folding direction, indicating the existence of a folding intermediate. The first reaction phase is fast and concentration-dependent, showing that the intermediate is readily populated and dimeric. The second phase is independent of concentration and is exponential. In contrast, in the unfolding direction, all molecules display two-state kinetics. Collectively this implies a transition state between unfolded helices and dimeric intermediate that is readily traversed in both directions. We demonstrate that the added stability of cFos-JunWPh1 relative to cFos-JunW is achieved via a combination of kinetic rate changes; cFos-JunWE23K has an increased initial dimerization rate, prior to the major transition state barrier while cFos-JunWQ21R displays a decreased unfolding rate. The former implies that improved hydrophobic burial and helix-stabilizing mutations exert their effect on the initial, rapid, monomer-collision event. In contrast, electrostatic interactions exert their effect late in the folding pathway. Although our focus is the leucine zipper region of the oncogenic transcription factor Activator Protein-1, coiled coils are ubiquitous and highly specific in their recognition of partners. Consequently, generating kinetic-based rules to predict and engineer their stability is of major significance in peptide-based drug design and nano-biotechnology.

[17] Protein fusions to coiled-coil domains

Publisher Summary Oligomerization plays key roles in protein function and regulation. As a consequence, powerful experimental tools have been created with defined, chimeric multimers made by genetic fusions to heterologous oligomerization domains. Coiled coils provide versatile fusion partners. They are particularly small domains with predictable quaternary structure and adjustable stability. Numerous coiled-coil fusions have been constructed to achieve diverse experimental aims. The use of coiled coils to probe these functions will continue to grow with increasing knowledge of the principles of helical associations. In vivo applications that target natural coiled-coil domains offer particularly fruitful prospects. Expressing coiled-coil motifs alone or in combination with inactivating domains may be used increasingly to generate dominant negative proteins. Fusions to fluorescent molecules such as green fluorescent protein (GFP) can yield new methods to label coiled-coil structures. This chapter reviews the principles of coiled-coil structure and describes fusion domain sequences and their several applications.

Efficient phage display of intracellularly folded proteins mediated by the TAT pathway

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.