CAIRA: Catalytic associated irregular residue analyser

For the semi-automated identification of potential cleavage specificity-modulating SNVs we developed a program, called CAIRA (Catalytic Associated Irregular Residue Analyser). Entering the UniProt ID of a protease, CAIRA takes the predicted AlphaFold structure, generates a user-definable radius (e.g. 20 Å) around the active site and filters the COSMIC database for cancer-associated SNVs within this radius, which might have cleavage specificity-modulating effects. CAIRA can be used with all types of proteases (metallo, serine, aspartyl and cysteine proteases). To further assess their potential impact, SNV-affected amino acids are labelled in the protein structure of a downloadable pdb-file.

Methods
CAIRA was programmed using python. Protein structures were obtained from AlphaFold. Cleavage specificity logos were generated as described above using cleavage site specificity data from MEROPS the Peptidase Database. Cancer-associated SNVs data was taken from the COSMIC database (cancer.sanger.ac.uk;). Catalytically important amino acid residue(s) within the active site were extracted from UniProt annotation  and are used as the centre of the sphere. For proteases with more than one catalytically important amino acid residue within the active site (aspartyl, serine and cysteine proteases) CAIRA uses the middle between the relevant residues as the centre of the sphere. In the beta version, the ‘use binding cavity (beta)’ button can also be additionally used to switch to a version in which the binding cavity is imitated and SNVs can be output within it. For this purpose, cylinders of adjustable size are placed around the chemical bonds of the backbone of the protease's propeptide and amino acids of the protease that lie with at least one atom within these cylinders are filtered out and checked for annotated SNVs. The logo of CAIRA was designed with the help of Adobe Firefly.