The Involvement of the Cryptococcal Yeast Kexin Protease in the Proteolytic Activation of the Uncleaved Influenza Virus Hemagglutinin Glycoprotein

Abstract

ABSTRACT Infectious diseases typically involve a single pathogen harming its host. However, in clinical settings, co‐infections also occur. This study investigated the potential interaction between Cryptococcus (C.) neoformans and influenza virus by assessing whether the cryptococcal yeast kexin protease (Kex2p) could activate the uncleaved hemagglutinin glycoprotein. Protein‐protein docking was performed using HADDOCK and independently validated using AlphaFold3. 6x His‐tagged truncated KEX2 gene was expressed in E. coli BL 21 (DE3), and the recombinant protein was purified using nickel ion affinity chromatography. Successful protein production was confirmed by sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis, anti‐His immunoblotting, and mass spectrometry. Recombinant Kex2p was evaluated in a biochemical peptide cleavage assay using a fluorogenic 12‐mer peptide mimicking the hemagglutinin HA1/HA2 site as substrate, with furin serving as the reference protease. HADDOCK predicted favorable binding of Kex2p to hemagglutinin (HADDOCK score = −119.2 ± 8.7; RMSD = 1.1 ± 0.2 Å), comparable to the furin‐hemagglutinin complex (HADDOCK score = −115.9 ± 5.7; RMSD = 2.1 ± 1.3 Å). AlphaFold 3 independently reproduced the HADDOCK‐predicted binding orientations, yielding moderate‐confidence complexes for hemagglutinin‐furin (ipTM = 0.50, pTM = 0.56; RMSD = 3.634 Å) and hemagglutinin‐Kex2p (ipTM = 0.41, pTM = 0.58; RMSD = 3.243 Å). In enzymatic assays, recombinant Kex2p exhibited a significantly higher Vmax than furin (p = 0.0411), indicating greater catalytic activity toward the hemagglutinin cleavage peptide. These findings provide the first biochemical and complementary computational evidence that C. neoformans Kex2p may recognize influenza hemagglutinin, supporting its potential role in hemagglutinin activation during cryptococcal‐influenza co‐infection.