Engineered Domain Swapping as an On/Off Switch for Protein Function

Ha, Jeung Hoi; Karchin, Joshua M.; Walker-Kopp, Nancy; Castañeda, Carlos; Loh, Stewart N.

doi:10.1016/j.chembiol.2015.09.007

Cited by 28 publications

(43 citation statements)

References 38 publications

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“…1C). In the present case of the SF and RF constructs, the NFM and CFM mutations are themselves slightly destabilizing1728, so we did not introduce any secondary mutations for the purpose of favoring the NFM-CFM complex.…”

Section: Resultsmentioning

confidence: 99%

“…In doing so the lever protein becomes the hinge region of the dimer and is thus placed outside of the structure of the target protein. We further showed that this design can be used to turn on and off activity of the target domain17. Two separate lever-target fusion proteins were made wherein the function of the target domain (ribose binding protein, or RBP) was knocked out by point mutations N-terminal and C-terminal to the lever (ubiquitin) insertion site.…”

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confidence: 99%

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Small Molecule-Induced Domain Swapping as a Mechanism for Controlling Protein Function and Assembly

Karchin

Namitz

et al. 2017

Sci Rep

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Domain swapping is the process by which identical proteins exchange reciprocal segments to generate dimers. Here we introduce induced domain swapping (INDOS) as a mechanism for regulating protein function. INDOS employs a modular design consisting of the fusion of two proteins: a recognition protein that binds a triggering molecule, and a target protein that undergoes a domain swap in response to binding of the triggering ligand. The recognition protein (FK506 binding protein) is inserted into functionally-inactivated point mutants of two target proteins (staphylococcal nuclease and ribose binding protein). Binding of FK506 to the FKBP domain causes the target domain to first unfold, then refold via domain swap. The inactivating mutations become ‘swapped out’ in the dimer, increasing nuclease and ribose binding activities by 100-fold and 15-fold, respectively, restoring them to near wild-type values. INDOS is intended to convert an arbitrary protein into a functional switch, and is the first example of rational design in which a small molecule is used to trigger protein domain swapping and subsequent activation of biological function.

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Small Molecule-Induced Domain Swapping as a Mechanism for Controlling Protein Function and Assembly

Karchin

Namitz

et al. 2017

Sci Rep

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“…Thus, the protein design approach for domain swapping has focused on engineering the putative hinge loop . The engineering strategy varies from a domain insertion to a hydrophobic shortsequence insertion . Protein domain insertion enables the incorporation of a rigid element that is suitable for inducing domain swapping, although the size is relatively large.…”

Section: Figurementioning

confidence: 99%

Domain‐Swapping Design by Polyproline Rod Insertion

et al. 2019

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During domain swapping, proteins mutually interconvert structural elements to form a di‐/oligomer. Engineering this process by design is important for creating a higher order protein assembly with minimal modification. Herein, a simple design strategy is shown for domain‐swapping formation by loop deletion and insertion of a polyproline rod. Crystal structures revealed the formation of the domain‐swapped dimers and polyproline portion formed a polyproline II (PPII) structure. Small‐angle X‐ray scattering demonstrated that an extended orientation of domain‐swapped dimer was retained in solution. It is found that a multiple of three of inserting proline residue is favored for domain swapping because of the helical nature of PPII. The rigid nature of the polyproline rod enables precise control of the interdomain distance and orientation.

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“…The free energy is computed for the binding of an unbound Aβ peptide (P 1 ) to the fibril template (P 2 − P 4 ). small peptide chains 63 or proteins, 64 and by stabilizing the domain swapped dimers by the formation of disulfide bonds. 65 Simulations 66 also revealed that functionally active partially folded protein conformations can form domain swapped dimers leading to the hypothesis that domain swapping can be a consequence of protein function imposing constraints on the structrure.…”

Section: Introductionmentioning

confidence: 99%

Cosolvent Effects on the Growth of Protein Aggregates Formed by a Single Domain Globular Protein and an Intrinsically Disordered Protein

Mondal

Reddy

2018

Preprint

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Cosolvents modulate the stability of protein conformations and exhibit contrasting effects on the kinetics of aggregation by globular proteins and intrinsically disordered proteins (IDPs). The growth of ordered protein aggregates, after the initial nucleation step is believed to proceed through a dock-lock mechanism. We have studied the effect of two denaturants (guanidinium chloride (GdmCl) and urea) and four protective osmolytes (trimethylamine N-oxide (TMAO), sucrose, sarcosine, and sorbitol) on the free energy surface (FES) of the dock-lock growth step of protein aggregation using a coarse-grained protein model and metadynamics simulations. We have used the proteins cSrc-SH3 and Aβ 9−40 as model systems representing globular proteins and IDPs, respectively. The effect of cosolvents on protein conformations is taken into account using the molecular transfer model (MTM). The computed FES shows that protective osmolytes stabilize the compact aggregates, while denaturants destabilize them for both cSrc-SH3 and Aβ 9−40 . However, protective osmolytes increase the effective energy barrier for the multi-step domain swapped dimerization of cSrc-SH3, which is critical to the growth of protein aggregates by globular proteins, thus slowing down overall aggregation rate. Contrastingly, denaturants decrease the effective barrier height for cSrc-SH3 dimerization, and hence enhances the aggregation rate in globular proteins.The simulations further show that cSrc-SH3 monomers unfold before dimerization and the barrier to monomer unfolding regulates the effective rate of agrgegation. In the case of IDP, Aβ 9−40 , protective osmolytes decrease and denaturants increase the effective barriers in the dock-lock mechanism of fibril growth, leading to faster and slower growth kinetics, respectively.

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Engineered Domain Swapping as an On/Off Switch for Protein Function

Cited by 28 publications

References 38 publications

Small Molecule-Induced Domain Swapping as a Mechanism for Controlling Protein Function and Assembly

Small Molecule-Induced Domain Swapping as a Mechanism for Controlling Protein Function and Assembly

Domain‐Swapping Design by Polyproline Rod Insertion

Cosolvent Effects on the Growth of Protein Aggregates Formed by a Single Domain Globular Protein and an Intrinsically Disordered Protein

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