An ancient mechanism of arginine-specific substrate cleavage: What's 'up' with NSP4?


Department of Early Discovery Biochemistry, Genentech Inc, 1 DNA Way, South San Francisco, CA 94080, USA. Electronic address: [Email]


The recently discovered neutrophil serine protease 4 (NSP4) is the fourth member of the NSP family, which includes the well-studied neutrophil elastase, proteinase 3 and cathepsin G. Like the other three NSP members, NSP4 is synthesized by myeloid precursors in the bone marrow and, after cleavage of the two-amino acid activation peptide, is stored as an active protease in azurophil granules of neutrophils. Based on its primary amino acid sequence, NSP4 is predicted to have a shallow S1 specificity pocket with elastase-like substrate specificity. However, NSP4 was found to preferentially cleave after an arginine residue. Structural studies resolved this paradox by revealing an unprecedented mechanism of P1-arginine recognition. In contrast to the canonical mechanism in which the P1-arginine residue points down into a deep S1 pocket, the arginine side chain adopts a surface-exposed 'up' conformation in the NSP4 active site. This conformation is stabilized by the Phe190 residue, which serves as a hydrophobic platform for the aliphatic portion of the arginine side chain, and a network of hydrogen bonds between the arginine guanidium group and the NSP4 residues Ser192 and Ser216. This unique configuration allows NSP4 to cleave even after naturally modified arginine residues, such as citrulline and methylarginine. This non-canonical mechanism, characterized by the hallmark 'triad' Phe190-Ser192-Ser216, is largely preserved throughout evolution starting with bony fish, which appeared about 400 million years ago. Although the substrates and physiological role of NSP4 remain to be determined, its remarkable evolutionary conservation, restricted tissue expression and homology to other neutrophil serine proteases anticipate a function in immune-related processes.


Elastase,Neutrophil,Serine protease,Substrate specificity,Trypsin,

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