Extended Intermolecular Interactions in a Serine Protease–Canonical Inhibitor Complex Account for Strong and Highly Specific Inhibition

Autoproteolytic stability of a trypsin from the marine crab Cancer pagurus

“…The open reading frame (ORF) consisted of 705 bp, coding for a mature and active protein of 235 residues with a calculated molecular mass of 25.7 kDa and a theoretical isoelectric point (pI) of 4.38. These parameters are close to those of mature trypsins from other crustaceans [8,10]. Almost 30 % of the deduced C.p.TryIII amino acid sequence was additionally confirmed by internal mass spectrometry sequencing (Fig.…”

“…In this respect we found the conserved residues His57, Asp102, and Ser195 which form the catalytic triad, as well as Gly216 and Gly226, which determine the trypsin specificity. The expected evolutionary distance of C.p.TryIII to trypsins from mammals and other vertebrates (Table 1) was confirmed by specific features that are only characteristic for crustacean trypsins: (i) three sequence insertions; (ii) a highly acidic pI due to an increased amount of Asp/Glu residues, while the numbers of Lys/Arg residues were slightly reduced; (iii) a decreased and, for some species including C. pagurus, impaired number of Cys residues [5,[7][8][9][10]. C.p.TryIII has eight cysteines in conserved positions, which indicate a comparable pattern of the associated disulphide bonds.…”

“…Crustacean trypsins were already isolated and characterised from crayfishes [4][5][6], shrimps [7,8], and king crab [9]. Moreover, the X-ray structures of two crustacean serine proteases [10,11] have been solved. Interestingly, crustacean trypsins turned out to be more stable than their mammalian counterparts.…”

Crystallization and preliminary X-ray diffraction studies of trypsin-like proteases from the gastric fluid of the marine crabCancer pagurus

“…Moreover, attempts to determine the molecular structures of C.p.TryII and C.p.TryIII by the molecular-replacement (MR) method are planned using the structure of a trypsin from the crayfish P. leptodactylus as a starting model (PDB code 2f91; Fodor et al, 2005). Based on similarity searches including the available structures of trypsins from different species, a typical trypsin-like fold containing two -barrel domains is expected for the structures of both C. pagurus trypsins.…”

“…Comparative structural analysis of vertebrate and crustacean trypsins would be useful in order to elucidate the molecular mechanisms of enhanced activity and stability. Only one crystal structure of a trypsin from a crustacean species, that from the freshwater crayfish Pontastacus leptodactylus, has been published to date (Fodor et al, 2005). Here, we describe for the first time the purification, crystallization and preliminary crystallographic analysis of two trypsin-like proteases from the marine crustacean species C. pagurus.…”