Crystallization and preliminary X-ray analysis of a Kunitz-type inhibitor, textilinin-1 fromPseudonaja textilis textilis

Abstract

Textilinin-1 (Txln-1), a Kunitz-type serine protease inhibitor, is a 59-amino-acid polypeptide isolated from the venom of the Australian Common Brown snake Pseudonaja textilis textilis. This molecule has been suggested as an alternative to aprotinin, also a Kunitz-type serine protease inhibitor, for use as an antibleeding agent in surgical procedures. Txln-1 shares only 47% amino-acid identity to aprotinin; however, six cysteine residues in the two peptides are in conserved locations. It is therefore expected that the overall fold of these molecules is similar but that they have contrasting surface features. Here, the crystallization of recombinant textilinin-1 (rTxln-1) as the free molecule and in complex with bovine trypsin (229 amino acids) is reported. Two organic solvents, phenol and 1,4-butanediol, were used as additives to facilitate the crystallization of free rTxln-1. Crystals of the rTxln-1-bovine trypsin complex diffracted to 2.0 Å resolution, while crystals of free rTxln-1 diffracted to 1.63 Å resolution.

Crystal structure of textilinin‐1, a Kunitz‐type serine protease inhibitor from the venom of the Australian common brown snake (Pseudonaja textilis)

“…This high number of solvent‐exposed hydrophobic residues is somewhat surprising, given that the molecule is soluble up to 40 mg·mL −1 for crystallization studies, suggesting that textilinin‐1 may form oligomers at higher concentrations. Eleven different reagent conditions yielded crystals of textilinin‐1 [15]. All of the crystals tested by X‐ray diffraction yielded a conserved set of unit cell parameters and space group, indicating consistent packing.…”

“…It was expressed in inclusion bodies as a fusion protein where a proprietary N‐terminal expression enhancer sequencer (TV) was joined to an internal tryptophan residue (W) followed by the 59 amino acid mature protein. The TVW moiety was then cleaved, and the protein was purified and crystallized as described previously [15].…”

“…The X‐ray data for free textilinin‐1 were collected and processed as described previously [15]. The program phaser [37] was used to determine the structure of textilinin‐1.…”

Textilinin‐1, an alternative anti‐bleeding agent to aprotinin: Importance of plasmin inhibition in controlling blood loss

“…The serine protease inhibitor, textilinin‐1, from the venom of the Australian eastern brown snake, Pseudonaja textilis is related in sequence to aprotinin and exhibits a three‐dimensional structure typical of Kunitz/BPTI‐type inhibitors (Masci et al , 2000; Filippovich et al , 2002; Millers et al , 2006). In preliminary studies, textilinin‐1 was found to strongly inhibit plasmin and trypsin (Flight et al , 2005) and to reduce blood loss in an animal model (Masci et al , 2000).…”

Selective Vulnerability of Neuronal Subtypes in ALS: A Fertile Ground for the Identification of Therapeutic Targets

“…Although the role of MMP-9 in the context of ALS is still poorly understood, it might be involved in the disruption of the neuronal extracellular matrix interaction (reviewed by [51]). MMPs have also been shown to promote the inflammatory process [52]. Kaplan et al [38] found that MMP-9 is highly expressed in the FF MNs, in contrast to the S MNs, which have undetectable MMP-9 levels ( Figure 2D).…”