Alpha repeat proteins (αRep) as expression and crystallization helpers

Chevrel, Anne; Mesneau, Agnès; Sanchez, Dyana; Celma, Louisa; Quevillon‐Chéruel, Sophie; Cavagnino, Andrea; Nessler, Sylvie; Sierra-Gallay, I. Li de la; Tilbeurgh, Herman van; Minard, Philippe; Valerio-Lepiniec, Marie; Urvoas, Agathe

doi:10.1016/j.jsb.2017.08.002

Cited by 16 publications

(15 citation statements)

References 38 publications

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“…It displays a variable number of α-helical repeats carrying five highly randomized amino acid positions, which form a hypervariable surface ensuring specific recognition of the target protein (Guellouz et al., 2013). The selected YabT(∆TM)-specific αREP binder called bE8 is composed of two internal repeats framed by additional N- and C-Cap α-helical fragments (Chevrel et al., 2017).…”

Section: Resultsmentioning

confidence: 99%

“…The bE8 binder, which displays the classical αREP fold (Urvoas et al., 2010), interacts as expected with YabT(∆TM) through its concave face. Fifteen out of the eighteen variable residues of bE8 are involved in YabT(∆TM) binding (Chevrel et al., 2017). From the kinase point of view, the interaction mainly occurs through residues Arg162 from loop β6–β7, Gln131 from helix αE, and Ser78 from loop αC-β4 (Figure 4B).…”

Section: Resultsmentioning

confidence: 99%

“…A large library of 10 9 artificial protein scaffolds called αREPs has been screened using phage display to select αREP variants displaying high affinity for YabT(∆TM) (Chevrel et al., 2017). The YabT-specific αREP binder called bE8 has been produced with a N-terminal 6xHis-tag and purified using IMAC.…”

Section: Methodsmentioning

confidence: 99%

“…Short and long forms of bE8-YabT(∆TM) fusion protein were constructed by inserting a short linker of 10 residues (SGGGGSGGGG) or a long linker of 32 residues (GSAGSAAGSGGASGGGGSGGGGSAGSAAGSGG) connecting 6xHis-bE8 to the N-terminus YabT(∆TM) (residues 1–315) (Chevrel et al., 2017).…”

Section: Methodsmentioning

confidence: 99%

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Structural Analysis of the Hanks-Type Protein Kinase YabT From Bacillus subtilis Provides New Insights in its DNA-Dependent Activation

et al. 2019

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YabT is a serine/threonine kinase of the Hanks family from Bacillus subtilis, which lacks the canonical extracellular signal receptor domain but is anchored to the membrane through a C-terminal transmembrane helix. A previous study demonstrated that a basic juxtamembrane region corresponds to a DNA-binding motif essential for the activation of YabT trans-autophosphorylation. YabT is expressed during spore development and localizes to the asymmetric septum where it specifically phosphorylates essential proteins involved in genome maintenance, such as RecA, SsbA, and YabA. YabT has also been shown to phosphorylate proteins involved in protein synthesis, such as AbrB and Ef-Tu, suggesting a possible regulatory role in the progressive metabolic quiescence of the forespore. Finally, cross phosphorylations with other protein kinases implicate YabT in the regulation of numerous other cellular processes. Using an artificial protein scaffold as crystallization helper, we determined the first crystal structure of this DNA-dependent bacterial protein kinase. This allowed us to trap the active conformation of the kinase domain of YabT. Using NMR, we showed that the basic juxtamembrane region of YabT is disordered in the absence of DNA in solution, just like it is in the crystal, and that it is stabilized upon DNA binding. In comparison with its closest structural homolog, the mycobacterial kinase PknB allowed us to discuss the dimerization mode of YabT. Together with phosphorylation assays and DNA-binding experiments, this structural analysis helped us to gain new insights into the regulatory activation mechanism of YabT.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Structural Analysis of the Hanks-Type Protein Kinase YabT From Bacillus subtilis Provides New Insights in its DNA-Dependent Activation

et al. 2019

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“…Each protein member of the αRep library has the same general architecture but is endowed with a unique binding surface made by the juxtaposition of hypervariable residues 24 . Specific αRep binders for a wide range of arbitrarily predefined target proteins with unrelated sequences and structures have been selected by phage display or protein complementation assay 25 . This suggests that the αRep library is a general source of specific reagents.…”

Section: Introductionmentioning

confidence: 99%

Ligand-induced conformational switch in an artificial bidomain protein scaffold

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Méo

Aumont-Niçaise

et al. 2019

Sci Rep

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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.

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