Cuticle-degrading proteases are involved in the breakdown of cuticle/eggshells of nematodes or insects, a hard physical barrier against fungal infections. Understanding the 3-dimensional structures of these proteins can provide crucial information for improving the effectiveness of these fungi in biocontrol applications, e. g., by targeted protein engineering. However, the structures of these proteases remain unknown. Here, we report the structures of two cuticle-degrading proteases from two species of nematophagous fungi. The two structures were solved with X-ray crystallography to resolutions of 1.65 angstrom (Ver112) and 2.1 angstrom (PL646), respectively. Crystal structures of PL646 and Ver112 were found to be very similar to each other, and similar to that of proteinase K from another fungus Tritirachium album. Differences between the structures were found among residues of the substrate binding sites (S1 and S4). Experimental studies showed that the enzymes differed in hydrolytic activity to synthetic peptide substrates. Our analyses of the hydrophobic/hydrophilic and electrostatic features of these two proteins suggest that their surfaces likely play important roles during fungal infection against nematodes. The two crystal structures provide a solid basis for investigating the relationship between structure and function of cuticle-degrading proteases.-Liang, L., Lou, Z., Ye, F., Yang, J., Liu, S., Sun, Y., Guo, Y., Mi, Q., Huang, X., Zou, C., Meng, Z., Rao, Z., Zhang, K.-Q. The crystal structures of two cuticle-degrading proteases from nematophagous fungi and their contribution to infection against nematodes. FASEB J. 24, 1391-1400 (2010). www.fasebj.org