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. 2007 Dec;15(12):1591-602.
doi: 10.1016/j.str.2007.10.014.

Phosphorylation-induced conformational switching of CPI-17 produces a potent myosin phosphatase inhibitor

Masumi Eto  1 , Toshio Kitazawa, Fumiko Matsuzawa, Sei-Ichi Aikawa, Jason A Kirkbride, Noriyoshi Isozumi, Yumi Nishimura, David L Brautigan, Shin-Ya Ohki
Affiliations

Phosphorylation-induced conformational switching of CPI-17 produces a potent myosin phosphatase inhibitor

Masumi Eto et al. Structure. 2007 Dec.

Abstract

Phosphorylation of endogenous inhibitor proteins for type-1 Ser/Thr phosphatase (PP1) provides a mechanism for reciprocal coordination of kinase and phosphatase activities. A myosin phosphatase inhibitor protein CPI-17 is phosphorylated at Thr38 through G-protein-mediated signals, resulting in a >1000-fold increase in inhibitory potency. We show here the solution NMR structure of phospho-T38-CPI-17 with rmsd of 0.36 +/- 0.06 A for the backbone secondary structure, which reveals how phosphorylation triggers a conformational change and exposes an inhibitory surface. This active conformation is stabilized by the formation of a hydrophobic core of intercalated side chains, which is not formed in a phospho-mimetic D38 form of CPI-17. Thus, the profound increase in potency of CPI-17 arises from phosphorylation, conformational change, and hydrophobic stabilization of a rigid structure that poses the phosphorylated residue on the protein surface and restricts its hydrolysis by myosin phosphatase. Our results provide structural insights into transduction of kinase signals by PP1 inhibitor proteins.

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Figures

Figure 1
Figure 1. Inhibitory domain of CPI-17 family
(A) Amino acid sequence of CPI-17 family of PP1 inhibitor proteins. Asterisk indicates Thr38, and arrows indicate Trp55, Ile77, Leu80, and Leu81. Boxes show regions in helices A to D. (B) 1H-15N HSQC spectrum of 13C/15N-labeled P-CPI-17 (1 mM). Red, gray and blue indicate amide resonances of P-, U-, and D38-CPI-17(22–120), respectively. Resonance peaks of T38, V39 and V41 were connected by arrows, from U-form to P- or D38-form.
Figure 2
Figure 2. Solution NMR structure of phospho-CPI-17(22–120)
(A) Stereo view of the backbone of 20 superimposed structures obtained by NMR analysis. Red indicates the region of α-helix. (B) Ribbon representation of an energy-minimized average structure. In the ribbon model, the flexible N-terminal 31 residues are not depicted. T38 and residues involved in hydrophobic clustering are drawn with ball-and-stick side chains labeled with residue numbers to facilitate identification, and the helices are labeled with upper case letters. (C) Space-filling representation with molecular surface potential, superimposed on backbone structure (green). Red and blue on surface indicate regions of negative and positive charges, respectively. A side chain of P-T38 is drawn as ball-and-stick.
Figure 3
Figure 3. Conformational transition of CPI-17 upon phosphorylation
U-CPI-17 (1J2M) (left) D38-CPI-17 (1J2N) (middle) and P-T38-CPI-17 (right) are displayed as ribbon structures. Side chains of hydrophobic residues that tether Helix A to D are shown as ball-and-stick for emphasis.
Figure 4
Figure 4. Formation of the hydrophobic core in P-CPI-17
(A) Top, middle, and bottom panels represent the structures of U-, D38-, and P-T38-CPI-17, respectively. Hydrophobic side chains responsible for the condensed conformation are displayed as Corey, Pauling and Koltun colored model. (B) Thermal stability of CPI-17 proteins monitored by circular dichroism (CD). Vertical axis shows relative change of the molar ellipticity at 222 nm at indicated temperature. CD spectrum was measured with 20 μM protein; P-T38-CPI-17 (open circle), U-CPI-17 (closed circle), D38-CPI-17 (Rectangle), and P-T38-CPI-17 (Y41A) (cross), in 50 mM potassium phosphate buffer, pH 7.0. Mean values from triplicate assays are shown.
Figure 5
Figure 5. Effects of the mutation in the hydrophobic core on the inhibitory potency of CPI-17
(A) Inhibition of myosin phosphatase purified from pig aorta. The myosin phosphatase activity without inhibitor proteins was set as 100 %. Data are averaged from two independent assays done in duplicate. (B) Force measurement of vas deferens smooth muscle strip. Panels represent the time-dependent force trace with U-CPI-17: wild type, D73A/E74A, I77A/L80A/L81A and W55A. The rabbit vas deferens smooth muscle strips were permeabilized with beta-escin. The CPI-17 proteins were doped into the tissue after the addition of PDBu. The extent of contraction in the presence of 1 μM Ca2+ is set as 100 % value.
Figure 6
Figure 6. Interaction of P-CPI-17 with PP1•MYPT1 complex
(A) The coordinates of MYPT1(1–299)•PP1 complex (1S70) in Protein Data Bank were used for computer modeling of the complex. MYPT1 (yellow) and PP1 (cyan) are depicted in a surface model, and P-T38-CPI-17 is drawn as a ribbon structure. The positive charge of MYPT1 Asp5 is shown as red, and the side chains of phospho-Thr38 and Arg43/44 in CPI-17 are shown as sticks. (B) Binding assay of P-T38-CPI-17 was performed with synthetic peptides corresponding to MYPT1 segments (1–19) and (24–41), immobilized on agarose beads. Asp5 is indicated by arrowhead in the sequence. Bound P-T38-CPI-17 was quantified by immunoblotting with anti-CPI-17 antibody (top). Mean values from 3 independent experiments are represented as a bar graph. Student’s t test was used to assess significance and * and ** indicate p > 0.15 and P < 0.03 respectively.
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