Variable and Conserved Regions of Secondary Structure in the β-Trefoil Fold: Structure Versus Function

Blaber, Michael

doi:10.3389/fmolb.2022.889943

Cited by 2 publications

(2 citation statements)

References 66 publications

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“…In the inhibitors cospin and cnispin the trypsin‐binding sites are located in the loops β2–β3 and β11–β12, which demonstrates the plasticity of the fold that can use several loops with different conformations to inhibit proteases (Avanzo Caglič et al, 2014). This functional role and conformational plasticity of the loops seem to be patterns in all β‐trefoil proteins (Blaber, 2022).…”

Section: Discussionmentioning

confidence: 99%

Canonical or noncanonical? Structural plasticity of serine protease‐binding loops in Kunitz‐STI protease inhibitors

et al. 2023

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The Kunitz‐Soybean Trypsin Inhibitor (Kunitz‐STI) family is a large family of proteins with most of its members being protease inhibitors. The versatility of the inhibitory profile and the structural plasticity of these proteins, make this family a promising scaffold for designing new multifunctional proteins. Historically, Kunitz‐STI inhibitors have been classified as canonical serine protease inhibitors, but new inhibitors with novel inhibition mechanisms have been described in recent years. Different inhibition mechanisms could be the result of different evolutionary pathways. In the present work, we performed a structural analysis of all the crystallographic structures available for Kunitz‐STI inhibitors to characterize serine protease‐binding loop structural features and locations. Our study suggests a relationship between the conformation of serine protease‐binding loops and the inhibition mechanism, their location in the β‐trefoil fold, and the plant source of the inhibitors. The classical canonical inhibitors of this family are restricted to plants from the Fabales order and bind their targets via the β4–β5 loop, whereas serine protease‐binding loops in inhibitors from other plants lie mainly in the β5–β6 and β9–β10 loops. In addition, we found that the β5–β6 loop is used to inhibit two different families of serine proteases through a steric blockade inhibition mechanism. This work will help to change the general perception that all Kunitz‐STI inhibitors are canonical inhibitors and proteins with protease‐binding loops adopting noncanonical conformations are exceptions. Additionally, our results will help in the identification of protease‐binding loops in uncharacterized or newly discovered inhibitors, and in the design of multifunctional proteins.

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Section: Discussionmentioning

confidence: 99%

Canonical or noncanonical? Structural plasticity of serine protease‐binding loops in Kunitz‐STI protease inhibitors

et al. 2023

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“…Beyond the demonstrated success of engineering core residues, this approach may allow protein stabilization while generally maintaining function. For example, core residue engineering may minimally effect function in β-trefoil proteins, as β-trefoils achieve ligand-binding through surface loop residues ( Figure 1 ) ( Brych et al, 2004 ; Olsen et al, 2004 ; Gosavi et al, 2008 ; Broom et al, 2012 ; Terada et al, 2017 ; Blaber, 2022 ) and display considerable plasticity in their core packing arrangements ( Murzin et al, 1992 ; Ponting and Russell, 2000 ; Longo and Blaber, 2013 ; Blaber, 2022 ). So, in the absence of allosteric affects, β-trefoil proteins with desirable functionality but only moderate stability may gain kinetic and thermodynamic stability from the replacement of core residues with those of another β-trefoil or from de novo core repacking.…”

Section: Discussionmentioning

confidence: 99%

Engineering the kinetic stability of a β-trefoil protein by tuning its topological complexity

Anderson

Jayanthi²,

Gosavi³

et al. 2023

Front. Mol. Biosci.

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Kinetic stability, defined as the rate of protein unfolding, is central to determining the functional lifetime of proteins, both in nature and in wide-ranging medical and biotechnological applications. Further, high kinetic stability is generally correlated with high resistance against chemical and thermal denaturation, as well as proteolytic degradation. Despite its significance, specific mechanisms governing kinetic stability remain largely unknown, and few studies address the rational design of kinetic stability. Here, we describe a method for designing protein kinetic stability that uses protein long-range order, absolute contact order, and simulated free energy barriers of unfolding to quantitatively analyze and predict unfolding kinetics. We analyze two β-trefoil proteins: hisactophilin, a quasi-three-fold symmetric natural protein with moderate stability, and ThreeFoil, a designed three-fold symmetric protein with extremely high kinetic stability. The quantitative analysis identifies marked differences in long-range interactions across the protein hydrophobic cores that partially account for the differences in kinetic stability. Swapping the core interactions of ThreeFoil into hisactophilin increases kinetic stability with close agreement between predicted and experimentally measured unfolding rates. These results demonstrate the predictive power of readily applied measures of protein topology for altering kinetic stability and recommend core engineering as a tractable target for rationally designing kinetic stability that may be widely applicable.

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Variable and Conserved Regions of Secondary Structure in the β-Trefoil Fold: Structure Versus Function

Cited by 2 publications

References 66 publications

Canonical or noncanonical? Structural plasticity of serine protease‐binding loops in Kunitz‐STI protease inhibitors

Canonical or noncanonical? Structural plasticity of serine protease‐binding loops in Kunitz‐STI protease inhibitors

Engineering the kinetic stability of a β-trefoil protein by tuning its topological complexity

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