Human FoxP Transcription Factors as Tractable Models of the Evolution and Functional Outcomes of Three-Dimensional Domain Swapping

The association of two or more proteins to adopt a quaternary complex is one of the most widespread mechanisms by which protein function is modulated. In this scenario, three-dimensional domain swapping (3D-DS) constitutes one plausible pathway for the evolution of protein oligomerization that exploits readily available intramolecular contacts to be established in an intermolecular fashion. However, analysis of the oligomerization kinetics and thermodynamics of most extant 3D-DS proteins shows its dependence on protein unfolding, obscuring the elucidation of the emergence of 3D-DS during evolution, its occurrence under physiological conditions, and its biological relevance. Here, we describe the human FoxP subfamily of transcription factors as a feasible model to study the evolution of 3D-DS, due to their significantly faster dissociation and dimerization kinetics and lower dissociation constants in comparison to most 3D-DS models. Through the biophysical and functional characterization of FoxP proteins, relevant structural aspects highlighting the evolutionary adaptations of these proteins to enable efficient 3D-DS have been ascertained. Most biophysical studies on FoxP suggest that the dynamics of the polypeptide chain are crucial to decrease the energy barrier of 3D-DS, enabling its fast oligomerization under physiological conditions. Moreover, comparison of biophysical parameters between human FoxP proteins in the context of their minute sequence differences suggests differential evolutionary strategies to favor homoassociation and presages the possibility of heteroassociations, with direct impacts in their gene regulation function.

Plasticity engineering of plant monoterpene synthases and application for microbial production of monoterpenoids

Plant monoterpenoids with structural diversities have extensive applications in food, cosmetics, pharmaceuticals, and biofuels. Due to the strong dependence on the geographical locations and seasonal annual growth of plants, agricultural production for monoterpenoids is less effective. Chemical synthesis is also uneconomic because of its high cost and pollution. Recently, emerging synthetic biology enables engineered microbes to possess great potential for the production of plant monoterpenoids. Both acyclic and cyclic monoterpenoids have been synthesized from fermentative sugars through heterologously reconstructing monoterpenoid biosynthetic pathways in microbes. Acting as catalytic templates, plant monoterpene synthases (MTPSs) take elaborate control of the monoterpenoids production. Most plant MTPSs have broad substrate or product properties, and show functional plasticity. Thus, the substrate selectivity, product outcomes, or enzymatic activities can be achieved by the active site mutations and domain swapping of plant MTPSs. This makes plasticity engineering a promising way to engineer MTPSs for efficient production of natural and non-natural monoterpenoids in microbial cell factories. Here, this review summarizes the key advances in plasticity engineering of plant MTPSs, including the fundamental aspects of functional plasticity, the utilization of natural and non-natural substrates, and the outcomes from product isomers to complexity-divergent monoterpenoids. Furthermore, the applications of plasticity engineering for improving monoterpenoids production in microbes are addressed.

Regulation of Dishevelled DEP domain swapping by conserved phosphorylation sites

Wnt signals bind to Frizzled receptors to trigger canonical and noncanonical signaling responses that control cell fates during animal development and tissue homeostasis. All Wnt signals are relayed by the hub protein Dishevelled. During canonical (β-catenin–dependent) signaling, Dishevelled assembles signalosomes via dynamic head-to-tail polymerization of its Dishevelled and Axin (DIX) domain, which are cross-linked by its Dishevelled, Egl-10, and Pleckstrin (DEP) domain through a conformational switch from monomer to domain-swapped dimer. The domain-swapped conformation of DEP masks the site through which Dishevelled binds to Frizzled, implying that DEP domain swapping results in the detachment of Dishevelled from Frizzled. This would be incompatible with noncanonical Wnt signaling, which relies on long-term association between Dishevelled and Frizzled. It is therefore likely that DEP domain swapping is differentially regulated during canonical and noncanonical Wnt signaling. Here, we use NMR spectroscopy and cell-based assays to uncover intermolecular contacts in the DEP dimer that are essential for its stability and for Dishevelled function in relaying canonical Wnt signals. These contacts are mediated by an intrinsically structured sequence spanning a conserved phosphorylation site upstream of the DEP domain that serves to clamp down the swapped N-terminal α-helix onto the structural core of a reciprocal DEP molecule in the domain-swapped configuration. Mutations of this phosphorylation site and its cognate surface on the reciprocal DEP core attenuate DEP-dependent dimerization of Dishevelled and its canonical signaling activity in cells without impeding its binding to Frizzled. We propose that phosphorylation of this crucial residue could be employed to switch off canonical Wnt signaling.

CirPred, the first structure modeling and linker design system for circularly permuted proteins

Background This work aims to help develop new protein engineering techniques based on a structural rearrangement phenomenon called circular permutation (CP), equivalent to connecting the native termini of a protein followed by creating new termini at another site. Although CP has been applied in many fields, its implementation is still costly because of inevitable trials and errors. Results Here we present CirPred, a structure modeling and termini linker design method for circularly permuted proteins. Compared with state-of-the-art protein structure modeling methods, CirPred is the only one fully capable of both circularly-permuted modeling and traditional co-linear modeling. CirPred performs well when the permutant shares low sequence identity with the native protein and even when the permutant adopts a different conformation from the native protein because of three-dimensional (3D) domain swapping. Linker redesign experiments demonstrated that the linker design algorithm of CirPred achieved subangstrom accuracy. Conclusions The CirPred system is capable of (1) predicting the structure of circular permutants, (2) designing termini linkers, (3) performing traditional co-linear protein structure modeling, and (4) identifying the CP-induced occurrence of 3D domain swapping. This method is supposed helpful for broadening the application of CP, and its web server is available at http://10.life.nctu.edu.tw/CirPred/ and http://lo.life.nctu.edu.tw/CirPred/.

PAM-interacting domain swapping is extensively utilized in nature to evolve CRISPR-Cas9 nucleases with altered PAM specificities

It has been anticipated that protospacer adjacent motif (PAM) specificity of the CRISPR-Cas9 nucleases protospacer appears to be modular. Here we present the finding that naturally occurring domain swapping has been extensively involved in altering its PAM specificities. Sequence analysis of streptococcal Cas9 sequences revealed conservation of three distinct PAM-interacting motifs, with phylogenetic analysis of full-length Cas9 and the PID demonstrating that PAM interacting domain (PID) domain swapping was extensively utilized to diversify its PAM specificity. An extended analysis of 582 representative Cas9 sequences revealed that this PIDswapping was broadly present in most of the investigated genera. Mimicking the natural PID domain swapping, a functional chimeric enzyme, based on the scaffold of compact Staphylococcus aureus Cas9, with novel NNAAAA PAM specificity was developed. In summary, our findings shed new light on a rich source of exchangeable PID domains in Cas9 genes, which can be mined for domain swapping aiming to an effective PAM refinement.

Exploitation of Novel Bt ICPs for the Management of Helicoverpa armigera (Hübner) in Cotton (Gossypium hirsutum L.): A Transgenic Approach

Cotton is a commercial crop of global importance. The major threat challenging the productivity in cotton has been the lepidopteron insect pest Helicoverpa armigera or cotton bollworm which voraciously feeds on various plant parts. Biotechnological interventions to manage this herbivore have been a universally inevitable option. The advent of plant genetic engineering and exploitation of Bacillus thuringiensis (Bt) insecticidal crystal proteins (ICPs) marked the beginning of plant protection in cotton through transgenic technology. Despite phenomenal success and widespread acceptance, the fear of resistance development in insects has been a perennial concern. To address this issue, alternate strategies like introgression of a combination of cry protein genes and protein-engineered chimeric toxin genes came into practice. The utility of chimeric toxins produced by domain swapping, rearrangement of domains, and other strategies aid in toxins emerging with broad spectrum efficacy that facilitate the avoidance of resistance in insects toward cry toxins. The present study demonstrates the utility of two Bt ICPs, cry1AcF (produced by domain swapping) and cry2Aa (produced by codon modification) in transgenic cotton for the mitigation of H. armigera. Transgenics were developed in cotton cv. Pusa 8–6 by the exploitation of an apical meristem-targeted in planta transformation protocol. Stringent trait efficacy-based selective screening of T1 and T2 generation transgenic plants enabled the identification of plants resistant to H. armigera upon deliberate challenging. Evaluation of shortlisted events in T3 generation identified a total of nine superior transgenic events with both the genes (six with cry1AcF and three with cry2Aa). The transgenic plants depicted 80–100% larval mortality of H. armigera and 10–30% leaf damage. Molecular characterization of the shortlisted transgenics demonstrated stable integration, inheritance and expression of transgenes. The study is the first of its kind to utilise a non-tissue culture-based transformation strategy for the development of stable transgenics in cotton harbouring two novel genes, cry1AcF and cry2Aa for insect resistance. The identified transgenic events can be potential options toward the exploitation of unique cry genes for the management of the polyphagous insect pest H. armigera.