Structural Characterization of Two Alternate Conformations in a Calbindin D_9k-Based Molecular Switch

Abstract

We have demonstrated that calbindin D9k can be converted into a calcium-sensing switch (calbindin-AFF) by duplicating the C-terminal half of the protein (residues 44–75) and appending it to the N-terminus (creating residues 44′–75′). This re-engineering results in a ligand-driven interconversion between two native folds: the wild-type structure (N) and a circularly permuted form (N′). The switch between N and N′ is predicted to involve exchange of the 44–75 and 44′–75′ segments, possibly linked to their respective folding and unfolding. Here we present direct structural evidence supporting the existence of N and N′. To isolate the N′ and N conformations, we introduced the knockdown Ca2+ binding mutation Glu → Gln at position 65 (E65Q mutant) or at the analogous position 65′ (E65′Q mutant). E65Q and E65′Q are therefore expected to adopt conformations N′ and N, respectively, in the presence of calcium. Though the amino acid sequences of E65Q and E65′Q differ at only these two positions, nuclear magnetic resonance resonance assignments, chemical shifts, and paramagnetic relaxation enhancement data reveal that they take on separate structures when bound to calcium. Both proteins are comprised of a well-folded domain and a disordered region. However, the segment that is disordered in E65Q (residues 44–75) is folded in E65′Q, and the region that is disordered in E65′Q (residues 44′–75′) is structured in E65Q. The results demonstrate that the N ⇆ N′ conformational change is mediated by a mutually exclusive folding reaction in which folding of one segment of the protein is coupled to unfolding of another segment, and vice versa.

Structures and energies of the transition between two conformations of the alternate frame folding calbindin-D9kprotein: a theoretical study

“…In detail, for the rst conformational transition step from AFF-D 9k -N 0 to Im via E and TS1 conformations, the distances between the EF2 0 and EF1 hands rst keep at 10.5Å during the simulation beginning time; oppositely, the distances between the EF2 region and EF1 hand decrease from 23.1Å in AFF-D 9k -N 0 to 17.0Å in E during simulation times of 0$3 ns due to the unmoving of stable EF2 0 hand and the up-moving of the exible EF2 loop structure at the transition beginning. 28 9 Mass center distances between the EF2 region and the EF1 hand (blue), between the EF2 0 and the EF1 hands (black), and between the EF2 0 hand and EF2 region (red) along the transition pathway from the simulation of AFF-D 9k -N 0 to AFF-D 9k -N; the roseate letters included in the grey areas denote the AFF-D 9k -N 0 , E, TS1, Im, TS2 and AFF-D 9k -N conformations.…”

“…21 Unlike other members of this superfamily, such as calmodulin and troponin C, perform their regulatory function through substantial conformational rearrangements, the WT calbindin-D 9k protein is unable to undergo a large conformational change upon to practise regulatory function. [26][27][28][29][30][31] The protein conformational switches applied by AFF strategy are in response to a signaling event such as ligand binding etc. [26][27][28][29][30][31] The protein conformational switches applied by AFF strategy are in response to a signaling event such as ligand binding etc.…”

“…The AFF calbindin-D 9k protein could adopt two distinctly different conformations in Ca 2+ -free form, i.e., N 0 and N conformations, with similar stabilities in native condition. [26][27][28] The N conformation of the AFF calbindin-D 9k protein possesses two EF-hands (assigned as EF1 hand of Lys1-Met43 and EF2 hand of Ser44-Gln75), the structure of which is similar to that of the WT calbindin-D 9k protein, an unstructured EF region of Ser44 0 -Gln75 0 (assigned as EF2 0 region) and the linker (see Fig. 26 The folding component of N conformation of the AFF calbindin-D 9k protein is comprised of the WT amino acid sequence, and adopts the WT protein structure; while the folding component of N 0 conformation of the AFF calbindin-D 9k protein with the circularly permuted structure consists of the original sequence of residues 1-43, the duplicate parent sequence and the linker to bridge the original N-and C-termini of the WT calbindin-D 9k protein.…”

“…[26][27][28] The N conformation of the AFF calbindin-D 9k protein possesses two EF-hands (assigned as EF1 hand of Lys1-Met43 and EF2 hand of Ser44-Gln75), the structure of which is similar to that of the WT calbindin-D 9k protein, an unstructured EF region of Ser44 0 -Gln75 0 (assigned as EF2 0 region) and the linker (see Fig. [26][27][28] Especially, a surface loop of Phe36-Met43, connecting the EF1 and EF2 hands, can be as the only possible site for circular permutation in the AFF strategy. Its mutually exclusive folding N 0 conformation possesses the circularly permuted folding structure of EF1 and EF2 0 hands, and an unstructured EF2 region (see Fig.…”

Influence of Secondary-Structure Folding on the Mutually Exclusive Folding Process of GL5/I27 Protein: Evidence from Molecular Dynamics Simulations

“…Such a biosensor is a protein containing duplicated segments in which only one of the two fragments at a time is incorporated in the folded protein. Stratton et al developed the alternate frame folding (AFF) strategy from a wild-type fold (N) to its circularly-permuted fold (N’) to build a switchable protein based on conformational changes [33,34,35,36,37,38]. Although MEF and AFF are incorporated into the folding-unfolding switch design, experimental studies on their switch mechanisms and the details of conformational variations at atomic level are limited so far.…”