Ligand-induced conformational switch in an artificial bidomain protein scaffold

Abstract

Artificial proteins binding any predefined “target” protein can now be efficiently generated using combinatorial libraries based on robust protein scaffolds. αRep is such a family of artificial proteins, based on an α-solenoid protein repeat scaffold. The low aggregation propensity of the specific “binders” generated from this library opens new protein engineering opportunities such as the creation of biosensors within multidomain constructions. Here, we have explored the properties of two new types of artificial bidomain proteins based on αRep structures. Their structural and functional properties are characterized in detail using biophysical methods. The results clearly show that both bidomain proteins adopt a closed bivalve shell-like conformation, in the ligand free form. However, the presence of ligands induces a conformational transition, and the proteins adopt an open form in which each domain can bind its cognate protein partner. The open/closed equilibria alter the affinities of each domain and induce new cooperative effects. The binding-induced relative domain motion was monitored by FRET. Crystal structures of the chimeric proteins indicate that the conformation of each constituting domain is conserved but that their mutual interactions explain the emergent properties of these artificial bidomain proteins. The ligand-induced structural transition observed in these bidomain proteins should be transferable to other αRep proteins with different specificity and could provide the basis of a new generic biosensor design.

An Artificial Hemoprotein with Inducible Peroxidase‐ and Monooxygenase‐Like Activities

“…Molecular modeling identified that this cavity was wide enough for the anchoring of even larger metal complexes such as meso‐tetraphenylporphyrin. Moreover, in a posterior study, we also observed that large conformational changes occur when in presence of a specific protein effector bA3‐2 and that the interdomain region can largely open and close in an inducible manner [35] . Thus, from an enzyme designer point of view, the artificial αRep proteins gather the required properties for the development of ArMs dedicated to the catalysis of oxidation reactions.…”

“…This lack in peroxidase activity suggested that the Mn III ‐NHMal‐TPP cofactor was buried too deeply in the cavity of the bidomain protein, thus preventing the OPD to approach the porphyrin periphery and deliver electrons to the H 2 O 2 derived oxidative species. To facilitate the access to the metalloporphyrin cofactor, we then thought about widening the opening of the cavity using another protein from the αRep library, named bA3‐2, that was previously selected by phage display [32] and was recently found to be able to bind tightly to the bidomain A3A3′ proteins and to open their bivalve shell‐like conformation [35] . The reaction was then performed under the same conditions as described above but in the presence of 40 μ m bA3‐2, and effectively, a peroxidase activity was observed in the presence of 4 m m imidazole with an initial rate of 278 μ m DAP μmol −1 BH‐MnTPP min −1 , and 14.5 μ m DAP was produced after 4 h. (Figure 3 A, trace d).…”

“…Moreover,i naposterior study,w ea lso observed that large conformational changes occur when in presence of as pecific protein effector bA3-2 andt hat the interdomainr egion can largely open and close in an inducible manner. [35] Thus, from an enzymed esigner point of view,t he artificial aRep proteins gather the required properties for the development of ArMs dedicatedt ot he catalysis of oxidation reactions. They appear convenient to fully accommodate aM n-tetraphenylporphyrin, are compatible to covalenta nchoring and also display conformationalc hanges at the large scale that could be controlled by an adequate molecular inducer.…”

“…It is the first example of an artificial hemoprotein in which the synthetic metallotetraaryl-porphyrin cofactor is covalentlya nchored to ac ysteine residue in its protein binding pocket and which activity is controlled by the addition of the specific aRep protein binder, bA3-2. [35] This species triggersn ot only ap eroxidase activity, which allows the efficient oxidation of severalt ypical peroxidase cosubstrates, but also of am onooxygenase activity that allows the selective oxidation of thioanisole into sulfoxide. The best activity was obtained with His 6 -bA3-2, the protein bearing aH is 6 -Tag that also acted as ac o-catalyst in thesereactions.…”

“…The work reported in this manuscript is a major proof‐of‐concept of artificial P450 induced systems. It is the first example of an artificial hemoprotein in which the synthetic metallo‐tetraaryl‐porphyrin cofactor is covalently anchored to a cysteine residue in its protein binding pocket and which activity is controlled by the addition of the specific αRep protein binder, bA3‐2 [35] . This species triggers not only a peroxidase activity, which allows the efficient oxidation of several typical peroxidase cosubstrates, but also of a monooxygenase activity that allows the selective oxidation of thioanisole into sulfoxide.…”