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Newly Born proteins, devoid of detectable homology to any other proteins, known as orphan proteins, occur in a single species or within a taxonomically restricted gene family. They are generated by expression of novel Open Reading Frames, and appear throughout evolution. We used the recently developed programs for predicting protein structures, RoseTTAFold and AlphaFold2, to compare such Newly Born proteins to random polypeptides generated by shuffling sequences of native proteins, which have been called ‘ Never Born’ proteins. The two programs were used to compare the structures of two sets of four Never Born proteins, one set that had been expressed and shown to be intrinsically disordered, and a second set that had been shown experimentally to possess substantial secondary structure. Since the programs rely to a large extent on multisequence alignment, the models generated were scored as being of low quality. However, a significant pattern emerged when the models generated by RoseTTAFold were examined. Specifically, all four members of Group 1 were shown to be very extended, as would be expected for intrinsically disordered proteins. In contrast, all four members of Group 2 appeared to be compact, and possessed substantial secondary structure. As a further control, both programs predicted unfolded structures for three well characterized intrinsically disordered proteins. The two programs were used to predict the structures of two orphan proteins whose crystal structures have been solved, both of which display novel folds. RoseTTAFold predicted both structures very well, whereas AlphaFold2 predicted only one well. The two programs were used to predict the structures of five orphan proteins with well-identified biological functions, one of which is predicted to be intrinsically disordered, and four to be folded. Both programs displayed the intrinsically disordered protein as an unfolded structure. RoseTTAFold displayed all four of those predicted to be folded as compact folded structures, with apparent novel folds, as determined by Dali and Foldseek. It is plausible that new biological functions may be implemented by orphan proteins due to their novel folds.

Pleiotropic intervention may be a requirement for effective limitation of the progression of multifactorial diseases such as Alzheimer's Disease. One approach to such intervention is to design a single chemical entity capable of acting on two or more targets of interest, which are accordingly known as Multi-Target Directed Ligands (MTDLs). We recently described donecopride, the first MTDL able to simultaneously inhibit acetylcholinesterase and act as an agonist of the 5-HT

The active site of acetylcholinesterase (AChE) from Torpedo californica is located 20 A from the enzyme surface at the bottom of a narrow gorge. To understand the role of this gorge in the function of AChE, we have studied simulations of its molecular dynamics. When simulations were conducted with pure water filling the gorge, residues in the vicinity of the active site deviated quickly and markedly from the crystal structure. Further study of the original crystallographic data suggests that a bis-quaternary decamethonium (DECA) ion, acquired during enzyme purification, residues in the gorge. There is additional electron density within the gorge that may represent small bound cations. When DECA and 2 cations are placed within the gorge, the simulation and the crystal structure are dramatically reconciled. The small cations, more so than DECA, appear to stabilize part of the gorge wall through electrostatic interactions. This part of the gorge wall is relatively thin and may regulate substrate, product, and water movement through the active site.

International audience; A soluble, monomeric form of acetylcholinesterase from mouse (mAChE), truncated at its carboxyl-terminal end, was generated from a cDNA encoding the glycophospholipid-linked form of the mouse enzyme by insertion of an early stop codon at position 549. Insertion of the cDNA behind a cytomegalovirus promoter and selection by aminoglycoside resistance in transfected HEK cells yielded clones secreting large quantities of mAChE into the medium. The enzyme sediments as a soluble monomer at 4.8 S. High levels of expression coupled with a one-step purification by affinity chromatography have allowed us to undertake a crystallographic study of the fasciculin-mAChE complex. Complexes of two distinct fasciculins, Fasl-mAChE and Fas2-mAChE, were formed prior to the crystallization and were characterized thoroughly. Single hexagonal crystals, up to 0.6 mm × 0.5 mm × 0.5 mm, grew spontaneously from ammonium sulfate solutions buffered in the pH 7.0 range. They were found by electrophoretic migration to consist entirely of the complex and diffracted to 2.8 A resolution. Analysis of initial X-ray data collected on Fas2-mAChE crystals identified the space group as P6122 or P6522 with unit cell dimensions a = b = 75.5 Å, c = 556 Å, giving a Vm value of 3.1 Å3/Da (or 60% of solvent), consistent with a single molecule of Fas2-AChE complex (72 kDa) per asymmetric unit. The complex Fasl-mAChE crystallizes in the same space group with identical cell dimensions.