Hobiger K et al. (2013), The linker pivot in Ci-VSP: the key to unlock c...

XB-ART-48002

PLoS One 2013 Jul 01;87:e70272. doi: 10.1371/journal.pone.0070272.

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The linker pivot in Ci-VSP: the key to unlock catalysis.

Hobiger K , Utesch T , Mroginski MA , Seebohm G , Friedrich T .

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In the voltage-sensitive phosphatase Ci-VSP, conformational changes in the transmembrane voltage sensor domain (VSD) are transduced to the intracellular catalytic domain (CD) leading to its dephosphorylation activity against membrane-embedded phosphoinositides. The linker between both domains is proposed to be crucial for the VSD-CD coupling. With a combined approach of electrophysiological measurements on Xenopus oocytes and molecular dynamics simulations of a Ci-VSP model embedded in a lipid bilayer, we analyzed how conformational changes in the linker mediate the interaction between the CD and the activated VSD. In this way, we identified specific residues in the linker that interact with well-defined amino acids in one of the three loops forming the active site of the protein, named TI loop. With our results, we shed light into the early steps of the coupling process between the VSD and the CD, which are based on fine-tuned electrostatic and hydrophobic interactions between the linker, the membrane and the CD.

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Species referenced: Xenopus
Genes referenced: gnl3 kcnq2 kcnq3 pten tbx2

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	Figure 2. Flipping mechanism of R253 between D400 and E402 in the WT model.Interaction interface between the linker (blue backbone) and the TI loop (beige backbone) in the WT model. Membrane lipids are represented as cyan-colored sticks. A typical snapshot of the most frequently occurring state is shown in (A). Here, salt bridges between R253 and D400 strongly stabilize the contact between linker and TI loop. In this state, K252 is able to interact with E402. (B) The stable configuration between R253 and D400 breaks several times during the 50 ns long MD simulation whereby R253 gets in contact with E402. In this constellation of salt bridges, the interaction between K252 and the TI loop is interrupted.
	Figure 3. Models of WT and mutants reveal differences in the interface between linker and TI loop.Representative snapshots showing the linker-TI loop interface of (A) the WT, (B) NEUT, (C) ALA, and (D) D400A model. For clarity, only K252, R253, Y255, D400X, E402X (X denotes the inserted mutation residue, Fig. 1C) and F401 are shown as indicated in ball-and-stick representation. Furthermore, the backbones of the linker and the TI loop are represented as blue and beige cartoon, respectively. For a better orientation, the membrane lipids are shown as cyan sticks. For the NEUT model, PI(4,5)P2 molecules interacting with R253 are indicated, with C atoms in black.
	Figure 4. Phosphatase activities of TI loop mutants.(A) Representative currents traces of Ci-VSP WT and denoted mutants that were co-expressed with KCNQ2/KCNQ3 potassium channels in Xenopus oocytes. Channel currents were recorded in response to a depolarization pulse from a holding potential of −80 to+80 mV for the indicated time interval. (B–C) From the resulting current traces, maximal and minimal current amplitudes (Imax and Imin) were determined during the depolarization phase at+80 mV. Inhibition ratios calculated with the values of Imin and Imax are plotted with the corresponding time durations required for each mutant to inhibit the channel currents to 50% (τ50%). Inhibition ratio of 0.5 (corresponding to 50%) is highlighted as dashed line.
	Figure 5. TI loop mutations differently affect the voltage sensor dynamics.On-sensing currents of Ci-VSP were recorded from a holding potential of −60 mV in response to variable test pulses between −40 to+160 mV at maximum (increment: 20 mV, duration: 500 ms). Off-sensing currents were monitored by stepping back to −50 mV after the test potential phase (off-pulse duration: 500 ms). (A–D) Representative full current traces are shown for the WT and the denoted mutants. From these signals, selected off-currents are zoomed out (40 mV increments). As described in Materials and Methods, Qoff,all-V-distributions were calculated by integrating the off-sensing currents. These distributions are plotted against the potential of the preceding test pulse phase. From the whole amount of translocated sensing charges (Qoff,all), the fast (Qoff,fast) and the slow (Qoff,slow) component was determined as described in Materials and Methods. All calculated Qoff-values were normalized to the Qoff,all-value corresponding to the test potential of+120 mV. Qoff,all-V-distributions were approximated with a Boltzmann-type function (see Materials and Methods). Parameters of voltage-dependence (V0.5, zq) are given in Table S3.
	Figure 6. Interaction between linker and TI loop influences the structural integrity of the active site.(A–C) Electrostatic potential surfaces of the phosphatase domain calculated with the APBS tool [26] are shown. Representations of (A) the wild type model at 0 and after 50 ns of the MD simulation, (B) the crystallographic structures by Liu et al. of the IP3 bound conformation (PDB 3V0H) and (C) the PO43–bound state (3V0G) [14]. Negatively and positively charged regions are colored in red and blue, respectively. The yellow element marks the linker residues 252–257. Encircled in yellow is the immediate environment of the catalytic cysteine C363. (D) The aligned backbone structures of the phosphatase domain of the WT (grey) and the corresponding TI loop mutant (yellow) are shown. The P loop containing C363 is highlighted with a dashed circle in red. The linker motif K252-K257 of the mutants is indicated in cyan. Additionally, the figure contains the rmsd values for the structural deviation between wild type and the respective mutant.
	Figure 1. Topology scheme of Ci-VSP. ( A ) Topology scheme of Ci-VSP with the transmembrane voltage sensor (VSD) and the intracellular catalytic domain (CD), which comprises the phosphatase (PD) and the C2-domain. VSD and CD are connected via the linker sequence M240–K257. ( B ) Three-dimensional structure of the Ci-VSP CD based on the crystal structure by Liu et al. (PDB entry 3V0H) [14]. The P- (orange), the TI- (blue), and the WPD loop (yellow) form the active site of the CD. The linker (colored in red, with R253 shown as sticks) is oriented toward the TI loop, as proposed by Liu et al. [14]. ( C ) Amino acid alignment of Ci-VSP’s linker (M240–K257), active site (H362–R369), and the TI loop (R398–S414) with regions of the homolog PTEN (Hs, Homo sapiens). Amino acid identities are highlighted with a gray background. Putative interacting partners in the linker and the TI loop are denoted with blue and red asterisks, respectively. Single and multiple altered Ci-VSP mutants are aligned as well (in red letters: mutated positions). In the single neutralization mutants, the X stands for N (in case aspartate was the native residue) or for Q (in case of glutamate).

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