Background: PDZ domain is a well-conserved, structural protein domain found in hundreds of signaling proteins that are otherwise unrelated. PDZ domains can bind to the C-terminal peptides of different proteins and act as glue, clustering different protein complexes together, targeting specific proteins and routing these proteins in signaling pathways. These domains are classified into classes I, II and III, depending on their binding partners and the nature of bonds formed. Binding specificities of PDZ domains are very crucial in order to understand the complexity of signaling pathways. It is still an open question how these domains recognize and bind their partners. Results: The focus of the current study is two folds: 1) predicting to which peptides a PDZ domain will bind and 2) classification of PDZ domains, as Class I, II or I-II, given the primary sequences of the PDZ domains. Trigram and bigram amino acid frequencies are used as features in machine learning methods. Using 85 PDZ domains and 181 peptides, our model reaches high prediction accuracy (91.4%) for binary interaction prediction which outperforms previously investigated similar methods. Also, we can predict classes of PDZ domains with an accuracy of 90.7%. We propose three critical amino acid sequence motifs that could have important roles on specificity pattern of PDZ domains. Conclusions: Our model on PDZ interaction dataset shows that our approach produces encouraging results. The method can be further used as a virtual screening technique to reduce the search space for putative candidate target proteins and drug-like molecules of PDZ domains.

Single chain antibody fragments (scFvs) are favored in diagnostic and therapeutic fields thanks to their small size and the availability of various engineering approaches. Linker between variable heavy (V_H) and light (V_L) chains of scFv covalently links these domains and it can affect scFv’s bio-physical/chemical properties and in vivo activity. Thus, scFv linker design is important for a successful scFv construction, and flexible linkers are preferred for a proper pairing of V_H–V_L. The flexibility of the linker is determined by length and sequence content and glycine-serine (GS) linkers are commonly preferred for scFvs based on their highly flexible profiles. Despite the advantage of this provided flexibility, GS linkers carry repeated sequences which can cause problems for PCR-based engineering approaches and immunogenicity. Here, two different linkers, a repetitive GS linker and an alternative non-repetitive linker with similar flexibility but lower immunogenicity are employed to generate anti-Vascular Endothelial Growth Factor scFvs derived from bevacizumab. Our findings highlight a better in vitro profile of the non-repetitive linker such as a higher monomer ratio, higher thermal stability while there was no significant difference in in vivo efficacy in a zebrafish embryonic angiogenesis model. This is the first study to compare in vivo efficacy of scFvs with different linkers in a zebrafish model.

Mechanistic details of intramembrane aspartyl protease (IAP) chemistry, which is central to many biological and pathogenic processes, remain largely obscure. Here, we investigated the in vitro kinetics of a microbial intramembrane aspartyl protease (mIAP) fortuitously acting on the renin substrate angiotensinogen and the C-terminal transmembrane segment of amyloid precursor protein (C100), which is cleaved by the presenilin subunit of γ-secretase, an Alzheimer disease (AD)-associated IAP. mIAP variants with substitutions in active-site and putative substrate-gating residues generally exhibit impaired, but not abolished, activity toward angiotensinogen and retain the predominant cleavage site (His–Thr). The aromatic ring, but not the hydroxyl substituent, within Tyr of the catalytic Tyr–Asp (YD) motif plays a catalytic role, and the hydrolysis reaction incorporates bulk water as in soluble aspartyl proteases. mIAP hydrolyzes the transmembrane region of C100 at two major presenilin cleavage sites, one corresponding to the AD-associated Aβ42 peptide (Ala–Thr) and the other to the non-pathogenic Aβ48 (Thr–Leu). For the former site, we observed more favorable kinetics in lipid bilayer–mimicking bicelles than in detergent solution, indicating that substrate–lipid and substrate–enzyme interactions both contribute to catalytic rates. High-resolution MS analyses across four substrates support a preference for threonine at the scissile bond. However, results from threonine-scanning mutagenesis of angiotensinogen demonstrate a competing positional preference for cleavage. Our results indicate that IAP cleavage is controlled by both positional and chemical factors, opening up new avenues for selective IAP inhibition for therapeutic interventions.