Reactive Centre Loop Dynamics and Serpin Specificity

Abstract

Serine proteinase inhibitors (serpins), typically fold to a metastable native state and undergo a major conformational change in order to inhibit target proteases. However, conformational labiality of the native serpin fold renders them susceptible to misfolding and aggregation, and underlies misfolding diseases such as α1-antitrypsin deficiency. Serpin specificity towards its protease target is dictated by its flexible and solvent exposed reactive centre loop (RCL), which forms the initial interaction with the target protease during inhibition. Previous studies have attempted to alter the specificity by mutating the RCL to that of a target serpin, but the rules governing specificity are not understood well enough yet to enable specificity to be engineered at will. In this paper, we use conserpin, a synthetic, thermostable serpin, as a model protein with which to investigate the determinants of serpin specificity by engineering its RCL.Replacing the RCL sequence with that from α1-antitrypsin fails to restore specificity against trypsin or human neutrophil elastase. Structural determination of the RCL-engineered conserpin and molecular dynamics simulations indicate that, although the RCL sequence may partially dictate specificity, local electrostatics and RCL dynamics may dictate the rate of insertion during protease inhibition, and thus whether it behaves as an inhibitor or a substrate.Engineering serpin specificity is therefore substantially more complex than solely manipulating the RCL sequence, and will require a more thorough understanding of how conformational dynamics achieves the delicate balance between stability, folding and function required by the exquisite serpin mechanism of action.

Overview of serpin B9 and its roles in cancer (Review)

“…All serpins share a conserved tertiary structure made of three β-sheets, eight or nine α-helices and a reactive centre loop (RCL), which contains 17 amino acids and is located between the A and C β-sheets (11). The RCL is the main action site located in the protease recognition domain close to the C-terminus of serpin (12) and determines the specificity of serpin inhibitory functions (13,14). Previous research has demonstrated that there are two types of serpin inhibitory mechanisms, termed the inhibitory pathway and the substrate pathway (Fig.…”

Engineering the serpin α1‐antitrypsin: A diversity of goals and techniques

“…89,90 Replacing conserpin RCL residues P7-P1 0 with P7-P2 0 from α 1 -AT, so that the RCL length and identity were closer to a natural serpin, improved SI values although complexes continued to dissociate faster than wild type α 1 -AT. 90,91 Interestingly, the protein remained unable to efficiently inhibit the primary cognate proteinase neutrophil elastase, which may be due to changes in RCL structural dynamics and the modified electrostatic potential of the serpin body. 91 These studies further stress the important balance between the inhibitory properties of α 1 -AT and its stability, which remain challenging engineering independent of one another.…”

“…90,91 Interestingly, the protein remained unable to efficiently inhibit the primary cognate proteinase neutrophil elastase, which may be due to changes in RCL structural dynamics and the modified electrostatic potential of the serpin body. 91 These studies further stress the important balance between the inhibitory properties of α 1 -AT and its stability, which remain challenging engineering independent of one another.…”

“…Functional expression of wild type recombinant α 1 -AT in CHO and HEK293 cells has been reported, although without pharmacokinetic information. 90,91 Amann et al engineered CHO cells, combining CRISPR/Cas9-mediated disruption of 10 rodent glycosylation-related genes and overexpression of human St6 β galactoside α-2,6-sialyltransferase 1 (ST6GAL1), to produce recombinant α 1 -AT with N-linked glycan structures more closely resembling those in plasma-derived α 1 -AT than in standard CHO-derived α 1 -AT. 108 Several groups have attempted to prolong α 1 -AT t 1/2 via glycoengineering, which is the addition or modification of glycans on the protein.…”

“…Even in the case of the synthetic thermostable conserpin, it remains difficult to alter specificity productively due to the many demands of the serpin mechanism. 91 In the medium term, it is likely that combining different approaches, such as phage display or serpin-proteinase docking data to select α 1 -AT variants and then reconfiguring them into long-lasting injectable protein drugs, will be undertaken. In the shorter term, it is likely that protein engineering modifications improving the pharmacokinetics of wild type α 1 -AT will make feasible clinical trials for new indications.…”