Engineered biosynthetic assembly lines could revolutionize the sustainable production of bioactive natural product analogues. While yeast display is a proven, powerful tool for altering the substrate specificity of gatekeeper adenylation domains in nonribosomal peptide synthetases (NRPSs), comparable strategies for other components of these megaenzymes have not been described. Here we report a high-throughput approach for engineering condensation (C) domains responsible for peptide elongation. We show that a 120-kDa NRPS module, displayed in functional form on yeast, can productively interact with an upstream module, provided in solution, to produce amide products tethered to the yeast surface. Using this system to screen a large C domain library, we reprogrammed a surfactin synthetase module to accept a fatty acid donor, increasing catalytic efficiency for this noncanonical substrate >40-fold. Since C domains can function as selectivity filters in NRPSs, this methodology should facilitate precision engineering of these molecular assembly lines.

No abstract

No abstract

Introduction: Chimeric Antigen Receptors (CARs) are designed to be inserted in the effector immune cells (T cells) membrane, conferring specificity to determined tumor cells. These receptors consist of three portions: an ectodomain, a transmembrane domain and an endodomain. The latter two are related to signal transduction and cytotoxic response. The ectodomain is generally formed by a single chain fragment variable (scFv) that recognizes a receptor on tumor cells, being responsible for the affinity and specificity to its antigen. Molecular Dynamics (MD) was used to study the scFv-antigen interface to understand how this interaction affects the CAR action. The antigen CD19 is a B cell receptor with no significant homology to other known correlated proteins, so it is a perfect biomarker for lymphoma diagnosis and then for CAR-mediated immunotherapy. Objective: To build the scFv of an anti-CD19 antibody, to analyze through MD simulation the complex scFv-CD19 structural stability in water and to determine energetic components involved in the formation of the scFv-CD19 interface. Methodology: The anti-CD19 scFv was built from crystallographic data (PDB 6AL5) containing its VL and VH domains structures and from a built Whitlow linker (GSTSGSGKPGSGEGSTKG) that connects these two antibody portions. The scFv was submitted to MD for 300 ns at CHARMM36 force field with 25205 TIP3P water model, 310 K and physiological concentration of 0.15 M (74 Na+ and 73 Cl-). Then, the scFv-CD19 complex (CD19 also obtained from PDB 6AL5) was simulated for 300 ns at same MD parameters. The structural stability was determined by Root Mean Square Deviation (RMSD). The Intermolecular Interaction Potential (IIP), which is nonbonded Coulomb and Lennard-Jones interactions, between scFv and CD19 residues was measured along the simulation. The Gromacs package 5.1.3 was used during equilibration, molecular trajectory acquisition and analysis. Results: The simulation time was enough to stabilize the scFv and CD19 structures in water. The scFv achieves stability in water after 200 ns with a RMSD of 0.2 ± 0,02 nm, the VL and VH domains are stable since the beginning of the simulation with the same RMSD of 0.09 ± 0,01 nm. As expected, the Whitlow linker RMSD is higher than VL and VH domains, due to its majority composition of glycine and serine flexible residues. The CD19 structure achieves stability after 100 ns with a RMSD of 0.44 ± 0,04 nm. Conclusion: A stable scFv-CD19 complex was obtained, allowing us to identify the CD19 binding site and how structural and energetic components are involved in scFv-CD19 interaction. Therefore, it is expected to obtain an improved anti-CD19 CART cell, that will be tested in vitro and in vivo further on.

The CYFIP2 protein (cytoplasmic FMR1-interacting protein 2) is part of the WAVE regulatory complex (WRC). CYFIP2 was recently correlated to neurological disorders by the association of the R87C variant with early infantile epileptic encephalopathy (EIEE) patients. In this set of syndromes, the epileptic spasms and seizures since early childhood lead to impaired neurological development in children. Inside the WRC, the variant residue is at the CYFIP2 and WAVE1 protein interface. Thus, the hypothesis is that the R87C modification weakens this interaction, allowing the WRC complex’s constant activation. This work aimed to investigate the impacts of the mutation on the structure of the WRC complex through molecular dynamics simulation. For that, we constructed WRC models containing WAVE1-NCKAP1 proteins complexed with WT or R87C CYFIP2. Our simulations showed a flexibilization of the loop comprising residues 80–110 due to the loss of contacts between internal residues in the R87C CYFIP2 as well as the key role of residues R/C87, E624, and E689 in structural modification. These data could explain the mechanism by which the mutation impairs the stability and proper regulation of the WRC.