Non-combinatorial library screening reveals subsite cooperativity and identifies new high-efficiency substrates for kallikrein-related peptidase 14
de Veer, Simon J., Swedberg, Joakim E., Parker, Edward A., & Harris, Jonathan M. (2012) Non-combinatorial library screening reveals subsite cooperativity and identifies new high-efficiency substrates for kallikrein-related peptidase 14. Biological Chemistry, 393(5), pp. 331-341.
An array of substrates link the tryptic serine protease, kallikrein-related peptidase 14 (KLK14), to physiological functions including desquamation and activation of signaling molecules associated with inflammation and cancer. Recognition of protease cleavage sequences is driven by complementarity between exposed substrate motifs and the physicochemical signature of an enzyme's active site cleft. However, conventional substrate screening methods have generated conflicting subsite profiles for KLK14. This study utilizes a recently developed screening technique, the sparse matrix library, to identify five novel high-efficiency sequences for KLK14. The optimal sequence, YASR, was cleaved with higher efficiency (k(cat)/K(m)=3.81 ± 0.4 × 10(6) M(-1) s(-1)) than favored substrates from positional scanning and phage display by 2- and 10-fold, respectively. Binding site cooperativity was prominent among preferred sequences, which enabled optimal interaction at all subsites as indicated by predictive modeling of KLK14/substrate complexes. These simulations constitute the first molecular dynamics analysis of KLK14 and offer a structural rationale for the divergent subsite preferences evident between KLK14 and closely related KLKs, KLK4 and KLK5. Collectively, these findings highlight the importance of binding site cooperativity in protease substrate recognition, which has implications for discovery of optimal substrates and engineering highly effective protease inhibitors.
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|Item Type:||Journal Article|
|Keywords:||phage display; positional scanning synthetic combinatorial library; serine protease; sparse matrix library; substrate specificity|
|Subjects:||Australian and New Zealand Standard Research Classification > MEDICAL AND HEALTH SCIENCES (110000)|
|Divisions:||Current > Schools > School of Biomedical Sciences|
Current > Schools > School of Chemistry, Physics & Mechanical Engineering
Current > QUT Faculties and Divisions > Faculty of Health
Current > Institutes > Institute of Health and Biomedical Innovation
|Deposited On:||06 Jul 2012 08:52|
|Last Modified:||13 Jun 2013 00:54|
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