Structure of the pseudosubstrate recognition site of chicken smooth muscle myosin light chain kinase

BB

ELSEVIER

Biochimica et Biophysica Acta 1292 (1996) 106-112

Biochi~ic~a et BiophysicaA~ta

Structure of the pseudosubstrate recognition site of chicken smooth muscle myosin light chain kinase Julian A. Barden

a,*,

Poonam Sehgal a, Bruce E. Kemp b

a The Universi~ of Sydney, Department of Anatomy and Histology, Sydney, N.S.W. 2006, Australia b St. Vincent's Institute of Medical Research, Melbourne, N.S.W. 3065, Australia

Received 27 March 1995; revised 18 July 1995; accepted 25 July 1995

Abstract

The structure of the chicken smooth muscle myosin light chain kinase pseudosubstrate sequence MLCK(774-807)amide was studied using two-dimensional proton NMR spectroscopy. Resonance assignments were made with the aid of totally correlated and nuclear Overhauser effect spectroscopy. A distance geometry algorithm was used to process the body of NMR distance and angle data and the resulting family of structures was further refined using dynamic simulated annealing. The major structural features determined include two helical segments extending from Asp-777 to Lys-785 and from Arg-790/Met-791 to Trp-800 connected by a turn region from Leu-786 to Asp-789 enabling the helices to interact in solution. The C-terminal helix incorporates the bulk of the pseudosubstrate recognition site which is partially overlapped by the calmodulin binding site while the N-terminal helix forms the bulk of the connecting peptide. The demonstrated turn between the helices may assist in enabling the autoregulatory or pseudosubstrate recognition sequence to be rotated out of the active site of the catalytic core following calmodulin binding. Keywords: Myosin light chain kinase; Pseudosubstrate; NMR; Structure; (Chicken gizzard); (Muscle)

1. Introduction

Myosin light chain kinase (MLCK) has an important function in regulating smooth muscle contraction [1]. MLCK phosphorylates Ser-19 in chicken smooth muscle myosin light chain [2] but in doing so exhibits a high degree of tissue specificity [1] with exogenous substrates being phosphorylated to only a very small extent [3]. The enzyme is a calmodulin binding protein with the chicken gizzard sequence 796-815 known to bind calmodulin stoichiometrically with an estimated dissociation constant of about 1 nM [4]. This calmodulin binding site appears to occupy and partially overlap the C-terminal segment of the sequence 780-808 constituting the autoregulatory domain [5]. Peptides from the N-terminal portion of the autoregulatory domain such as 774-787 are unable to inhibit the kinase, whereas peptides from within the pseudosub-

* Corresponding author. Fax: +61 2 5522026; e-mail: [email protected]. 0167-4838/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 01 6 7 - 4 8 3 8 ( 9 5 ) 0 0 1 7 1-9

strate sequence such as 787-807 remain potent inhibitors [5]. These data strongly suggest that the autoregulatory region of MLCK is identical to the pseudosubstrate domain. The pseudosubstrate sequence contains the same number and spacing of basic residues as that found in the myosin light chain surrounding the site of phosphorylation at Ser-19. It was thus proposed that the pseudosubstrate sequence binds to the active site in the catalytic core of MLCK thus maintaining the enzyme in an inactive conformation [6]. When Ca 2+. calmodulin binds immediately adjacent to the pseudosubstrate sequence the active site becomes exposed to the myosin light chain substrate. The suggestion has been made that calmodulin binding may induce an extension in a-helical structure [7]. This conformational change may itself induce a rotation of the pseudosubstrate away from the active site. Alternatively, calmodulin binding may induce the rotation of the pseudosubstrate away from the active site by means of a hinge between the pseudosubstrate and the peptide connecting the pseudosubstrate to the catalytic core.

J.A. Barden et al. / Biochimica et BiophysicaActa 1292 (1996) 106-112

The synthetic peptide '783-804 acts as a substrate antagonist [6] inhibiting the calmodulin-dependent activation of the chicken gizzard MLCK with an IC50 of 46 nM. Inhibition of the 61 kDa constitutively active MLCK fragment 282-779 by synthetic peptides and studies with proteinases have revealed the pseudosubstrate encompasses Ser787-Lys 8°2 [8,9] indicating that autoinhibition of MLCK is due entirely to the pseudosubstrate sequence. The recent determination of the twitch in kinase structure provides strong support for the intrasteric model of protein kinase regulation [10]. Chicken gizzard MLCK(787-807) is a potent inhibitor of the constitutively active MLCK core with a K i of 11.7 nM, whereas the longer p eptide containing both the pseudosubstrate and connecting sequences (774-807) exhibited a K i of 0.33 nM [11]. This indicates that the connecting peptide is a significant contributor to the binding of the pseudosubstrate. The extended binding of the pseudosubstrate and connecting peptides to the catalytic core appear to explain why some mutation experiments appeared inconsistent with the pseudosubstrate model [ 12-14]. This paper seeks to establish the stabilized solution structure of the synthetic peptide Leu774-Val 8°7 incorporating all the pseudosubstrate domain as well as the connecting peptide Leu774-Leu786. The solution conditions chosen for this study included 10% F3EtOH, since the structure in water alone showed only nascent helix. The structure of PTHrP(1-34) had been successfully stabilized under these conditions [15,16], comparing closely with the structure of PTH(1-34) in water [17] and without inducing additional helical content. These conditions also have been found to mimic those in the presence of lipid vesicles [ 18,19]. The pseudosubstrate structure is compared with that of the calmodulin binding peptidke 796-815 determined in complex with calmodulin [20]. Knowledge of the structure of the pseudosubstrate is directed towards elucidating the likely mechanism of binding to and release of the pseudosubstrate from the active site. 2. Materials and methods

Chicken gizzard MLCK(774-807)amide was synthesized with an Applied Biosystems 430A peptide synthesizer using t-butyloxycarbonyl amino-acid derivatives [21]. Glu, Ser and Thr side chains were protected with dinitrophenol, Lys side chains with chlorobenzyl and Arg side chains with the nitro group. Dinitrophenol deprotection (20% 2-mercaptoethanol in dimethylformamide) of the peptide was undertaken before cleavage from the benzhydrylamine resin with anhydrous HF containing 10% ( v / v ) anisole [22]. The peptide was extracted with acetonitrile (60% v/v)/trifluoroacetic acid solvent (0.1% v / v ) in water, then rotary-evaporated, lyophilized from water and purified using reverse-phase HPLC. Residual trifluoroacetic acid was removed using G-10 gel filtration and the peptide lyophilized.

107

2.1. Proton NMR spectroscopy

Peptide samples were made up at 2.2 mM at pH 4.1 in 55 mM KCI to which was added 10% F3EtOH-d 2 making the final peptide concentration 2.0 mM. Samples were placed in 5 mm precision tubes. Spectra were recorded at 293 K and 400 MHz in the Fourier transform mode with quadrature detection using a Bruker AMX-400 spectrometer without sample spinning. Spin systems were assigned using two-dimensional TOCSY spectra obtained with a MLEV-17 pulse sequence [23] with 2.5 ms trim pulses and a mixing time of 100 ms. The spin-lock field strength was close to 9 kHz. The water resonance was suppressed using continuous coherent irradiation throughout the relaxation (1.8 s) and mixing periods [24]. Two-dimensional NOESY experiments with a mixing time of 250 ms recorded using the time-proportional-phase-increments method [25] were used to aid in making sequence-specific resonance assignments as well as for estimations of proton-proton distance constraints. This mixing time was found to be sufficiently long to allow NOE intensity build-up while not suffering from spin diffusion complications. An F2 time-domain of 4 K was acquired with 512 separate free induction decays, each of 160 scans. The data were zero-filled to 4 X 1 K and apodized using Gaussian multiplication in F2 and shifted sine-bell in F1 prior to Fourier transformation. Resolution in F2 was increased to 1.07 Hz/point in DQFCOSY spectra. Third-order polynomial functions were employed to correct baselines. Chemical shifts were referenced to trimethylsilylpropanesulfonic acid. 2.2. Distance geometry calculations

NOE cross-peaks were separated into the conservative distance categories 0.3, 0.35, 0.4 and 0.45 nm depending on their intensity from volume integrals and calibrated using the Thr-803 a C H / / 3 C H cross-peak. Corrections for pseudoatoms were applied where stereospecific identification directly from the spectra was not obtained. The torsion angles (th) were constrained within the range - 9 0 ° to - 30° for all values of the coupling constant 3J~cHNH of 5 Hz or lower. These were measured variously using highresolution one-dimensional NMR spectra or high-resolution two-dimensional DQF-COSY spectra. A total of 2000 distance geometry structures were calculated from random starting structures using the program DIANA [26] on a Silicon Graphics RS4000 work station while molecular graphics were processed using the program INSIGHT II operating on a Silicon Graphics Indigo 2 workstation. 2.3. Dynamic simulated annealing

The best 20 distance geometry structures possessing the lowest penalty values were refined in X-PLOR 3.0 using a dynamic simulated annealing protocol [27]. Non-bonded interactions were not considered in the DIANA structures

108

J.A. Barden et al. / Biochimica et Biophysica Acta 1292 (1996) 106-112

0.5 ~ . V34

A33 O

K~ ~ .

5

Q2

"

"?° T30 @1~1 0 ~, 00~l~ I( | 124 A;I ,I'

ca~

I

I 8.6

8.4

1.5

I

/+

•

8.8

1

8.2

8.0

5 ppm 7.8 p p m

NH Chemical Shift Fig. 1. The aliphatic fingerprint region of a TOCSY spectrum at 400 MHz (mixing time 100 ms) showing all the relay scalar connectivities between the backbone NH protons and the respective side chain protons in chicken gizzard MLCK(774-807)amide in 10% F3EtOH-d 2 and 50 mM KC1 at a concentration of 2.0 mM, pH 4.1 and 293 K. Several spin system connectivities have been illustrated.

3. Results Part of a TOCSY NMR spectrum of Leu774-Gln-LysAsp-Thr-Lys-Asn-Met-Glu-Ala-Lys-Lys-Leu-Ser-LysAsp-Arg-Met-Lys-Lys-Tyr-Met-Ala-Arg-Arg-Lys-TrpGln-Lys-Thr-Gly-His-Ala-ValS°7-amide in 50 mM KCI and 10% F3EtOH-d 2 is shown in Fig. 1 recorded with a mixing time of 100 ms. Of the 34 spin systems in the peptide, the unique residues Leu-774 (due to lack of NH), Asn-780 and Glu-782 (after pH titration) and Gly-804 could be assigned sequence specifically from the spectrum. All the remaining spin systems could be assigned to either residue or at least spin system type aided by detection of coherence transfers as far apart as those between the NH backbone and the C protons of Lys separated by seven bonds. Two-dimensional NOESY correlations between the CH o~i proton and the NHi+ ~ protons from sequential residues and between sequential NH protons (Fig. 2) were used to specifically assign the remainder of the spin systems. A selection of sequential NH connectivities between the C-terminal residues is illustrated in Fig. 2. These are often indicators of the presence of a-helix. In this way, sequence-specific assignments could be made of all the spin systems. Several entry points to this backbone 'walk' were available [29]. The complete list of chemical shifts is shown in Table 1. Sequential and short-range inter-residue backbone NOEs are summarized in Fig. 3. The amino-acid sequence is presented in single letter code. Dihedral angles in residues possessing low 3 j coupling constants ( < 5 Hz) were confined to values of - 60 ° + 30 ° from reference to the Karplus equation. These included Lys-779, Met781-Ala 783, Lys 785-

- - 7.9

- - 8.0

and consequently they possessed poor potential energies. In the initial stage of the simulation 500 cycles of energy minimization were applied. Standard parameters were used to constrain covalent geometry [28]. Non-bonded interactions were modelled with a repel function which ignored electrostatic interactions. Stage 2 of the simulation involved heating the molecule to 1000 K by assigning high kinetic energies to the atoms from a Maxwellian distribution. The dynamic trajectory of the molecule was then followed for 50 ps in steps of 2 fs following which the NOE restraint term was linearly increased over 25 ps in 2 fs steps. This placed a gradually increased weight on the covalent geometry. Stage 4 involved cooling the molecule from 1000 K to 300 K over 10 ps. Equilibration of the molecule then commenced at 300 K for 1 ps. Each structure was then refined with 2000 cycles of energy minimization.

--8.1

r~

- - 8.2 "~

I~

22/23

30/31

#oo I

I

8.5

8.4

I

I

--8.3 ~ - - 8.4

-- 8.5 p p m

I

8.3 8.2 8.1 N H C h e m i c a l Shift

I 8.0

I 7.9ppm

Fig. 2. A section of a two-dimensional NOESY spectrum of MLCK(774807)amide (z m = 250 ms) showing examples of sequential NHi-NHi+ 1 connectivities between residues in the C-terminal segment, indicative of the presence of helix. Solution conditions are as listed in the legend to Fig. 1.

J.A. Barden et al. / Biochimica et Biophysica Acta 1292 (1996) 106-112

109

Table 1 JH Assignments and chemical shift data for human MLCK(1-34)amide in 10% F3EtOH-d2/90% H20, 50 mM KCI, pH 4.1 at 293 K Residue

NH

aH

fl H

Other

Leu-774 Gln-775 Lys-776 Asp-777 Thr-778 Lys-779 Asn-780 Met-781 Glu-782 Ala-783 Lys-784 Lys-785 Leu-786 Ser-787 Lys-788 Asp-789 Arg-790 Met-791 Lys-792 Lys-793 Tyr-794 Met-795 Ala-796 Arg-797 Arg-798 Lys-799 Trp-800 Gln-801 Lys-802 Thr-803 Gly-804 His-805 Ala-806 Val-807

8.78 8.51 8.45 8.11 8.36 8.32 8.25 8.32 8.12 8.03 8.09 8.13 8.08 8.08 8.31 8.07 8.14 8.56 7.95 8.10 8.26 7.97 7.89 7.99 8.06 7.98 8.03 8.20 8.01 8.34 8.24 8.38 8.10

4.02 4.42 4.29 4.74 4.33 4.23 4.63 4.35 4.19 4.20 4.18 4.21 4.30 4.33 4.17 4.50 4.14 4.41 4.29 4.12 4. 4.24 4.21 4.17 4.15 4.17 4.62 4.20 4.25 4.33 3.89, 3.91 4.67 4.33 4.09

1.72 2.02, 2.09 1.82, 1.77 2.73, 2.82 4.29 1.81, 1.85 2.79, 2.86 2.12, 2.12 2.03, 2.10 1.44 1.68, 1.68 1.87, 1.90 1.70, 1.74 3.94, 4.02 1.80, 1.84 2.72, 2.76 1.89, 1.93 2.17, 2.09 1.77, 1.84 1.78, 1.82 2.90, 3.14 2.07, 2.07 1.45 1.78, 1.86 1.67, 1.71 1.80, 1.84 3.25, 3.34 1.90, 2.00 1.77, 1.85 4.22

y C H 1.65; ~CH 3 0.95, 0.95 y C H 2 2.37, 2.37 y C H 2 1.45; 3CH 2 1.66, 1.70; ¢CH 2 2.99, 2.99 y C H 3 1.23 y C H 2 1.42, 1.47; 6CH 2 1.67, 1.70; s C H 2 3.00, 3.00 3NH 6.92, 7.70 y C H 2 2.56, 2.65; ~CH 3 2.07 y C H 2 2.40, 2.43 y C H 2 1.40, 1.43; 6CH 2 1.49, 1.53; e C H 2 2.99, 2.99 y C H 2 1.54, 1.58; 6CH 2 1.70, 1.72; ¢CH 2 2.98, 2.98 "yCH 2 1.58; ~CH 3 0.86, 0.90 TCH 2 1.27, 1.33; 6CH 2 1.72, 1.73; ¢CH 2 2.91, 2.91 y C H 2 1.63, 1.75; 3CH 2 3.21, 3.23; NH 7.24 y C H 2 2.61, 2.67; 8CH 3 2.09 y C H 2 1.41, 1.47; 8CH 2 1.69, 1.71; e C H 2 3.01, 3.01 y C H 2 1.31, 1.33; y C H 2 1.62, 1.64; eCH 2 2.94, 2.94 C2, 6H 7.10, 7.15; C3, 5H 6.79, 6.81 y C H 2 2.62, 2.71; ~CH 3 2.09 y C H 2 1.59, 1.64; 8CH 2 3.12, 3.15; NH 7.18 y C H 2 1.51, 1.55; 3CH 2 3.05, 3.08; NH 7.11 3,CH 2 1.40, 1.51; 8CH 2 1.68, 1.72; e C H 2 3.00, 3.00 C2H 7.22; C4H 7.58; C5H 7.12; C6H 7.19; C7H 7.46; NH 10.11 y C H 2 2.21, 2.26 y C H 2 1.42, 1.47; 8CH 2 1.66, 1.70; ¢CH 2 2.98, 2.98 TCH 3 1.20

3.08, 3.22 1.36 2.05

2H 8.54; 4H 7.23 y C H 3 0.93, 0.97

Ser 787, Asp-789, Met-791 and Lys793-Arg798. The relative intensity of the inter-residue NOEs is represented by the thickness of the connecting bars with the shortest inter-pro774 LQKD ~N

780 TKNMEAKKLS

m m m m _ _ _ _

* _m ISN

m - - m

790 KDRMKKYMA

m m m m m m _ _ _ _ m

m

* mmm_*

mn--

m . m m m m m m _ _

iNi+3 0~i~ i+3

ton distances reflected in the thickest bars and the largest upper distance limits reflected in the thinnest bars. An asterisk indicates the likely presence of an NOE which

mm

_

mmmm

m m

m m m

m

800 RRKWQKTGHAV m

m

m

mmm

m m

m

m

807

mm--mmm

* * m__mmmmmmmmmmmmmmm

m

-

-

_

m

m

n

m

_

_

n m

mmm

m '

m

i j Fig. 3. A summary of the interresidue NOE connectivities observed for MLCK(774-807)amide. The solid bars show the presence of an NOE with the thickness being proportional tc~ the intensity of the NOE. The presence of helix in the region Asp-777 to Lys-785 and Arg-790/Met-791 to Trp-800 or beyond is strongly suggested by the pattern of NOEs, particularly the medium range NOEs otiNi+ 3 and ai fli+3' Overlapped NH resonances which probably have a corresponding NOE are shown by an asterisk. The NOEs labelled i, j include: Gln-2/Gln-28 and Gln-2/Lys-29.

110

J.A. Barden et al. / Biochimica et Biophysica Acta 1292 (1996) 106-112

cannot be detected due to overlap degeneracy. Weak or absent sequential H a i / N H i ÷ ~ cross-peaks combined with strong sequential N H i / N H i + ~ cross-peaks are indicators of the presence of c~-helix [30], a conclusion supported by the presence of several medium-range NOEs such as H oti/NHi+ 3. Some longer range NOEs were measured, including Gln-775 H f l J G l n - 8 0 1 HN and Gln-775 H fl2/Lys-802 HN. Other long range NOEs between distant side-chain protons were difficult to positively identify. A total of 136 distance and 15 angle constraints were used in the distance geometry algorithm DIANA [26] to 200

100-

Sir

T!

ii ! '

,

-I00-

-200 770

780

790 Residue Number

800

810

8~0

810

2OO

i

!.

i 770

ASP777

5

Fig. 5. Stereo view of the backbone of 20 MLCK(774-807)amide structures superimposed over the N-terminal domain Asp777-Lys785. The consistency of the structures is reflected in the r.m.s, deviation of 5 4 + 7 pm in this region. The complete backbone of only one structure is shown for simplicity, since superposition was not possible for more than half the molecule at once due to flexibility in the region 786-789 joining the Nand C-terminal segments.

(a)

o-

LEU774

•

! 7~0

7~0 Residue Number

Fig. 4. (a) The torsion angles (4,) for each residue in each of the 20 best structures. The segments 779-788 and 792-800 appear particularly well-conserved. (b) The torsion angles (1/') for each of the structures showing similar conservation in the central segments of the molecule, Much greater conformational averaging is seen at the ends of the peptide as expected.

generate 2000 structures from random starting conformations. These calculations yielded several structures which satisfied all the NMR distance constraints within 5 pm and all angle constraints. Of these, the 20 structures with the lowest penalty values were refined further with dynamic simulated annealing (X-PLOR 3) tO include non-bonded interactions. These all displayed good covalent geometry. The mean r.m.s, deviation from ideal bond lengths was 0.10 + 0.01 pm, while the r.m.s, deviation from ideal bond angles was 0.141 + 0.021 °. The mean Lennard-Jones potential of - 2 9 4 + 84 kJ mol-1 indicated that the nonbonded contacts were good. This family of refined and energy minimized structures were superimposed and the root mean square (r.m.s.) deviation of backbone atoms measured throughout all segments in the molecule. Two distinct regions were found to possess low r.m.s, deviations, Asp-777 to Lys-785 and Lys-788 to Trp-800. Plots of the dihedral angles ~b and ~ from the 20 refined structures are presented in Fig. 4a and b, respectively. Some conformational averaging is present at both the Nand C-terminal ends but two regions within the peptide, Lys779-Lys788 and Lys792-Trp 8°° maintain a consistent conformation. The pairing of ~ b / ~ angles in these regions show results clearly typical of helical structures. The most prominent features observed in the structures is a helical segment extending from Asp-777 to Lys-785, a turn region from Leu-786 to Asp-789 and a second helical segment from Arg-790/Met791-Trp 8°°. A two-step superposition process was required to overlay the models due to the flexibility in the segment Leu786-Ser 787. A stereo view of the backbone of 20 structures superimposed over the backbone atoms in the N-terminal helix 777-785 is shown in Fig. 5. Only the complete backbone of one structure is shown since the regions outside this segment did not superimpose. A similar presentation of the C-terminal region 788-800 is shown in Fig. 6. The mean r.m.s. deviation between these structures and the mean structure

J.A. Barden et al. / Biochimica et Biophysica Acta 1292 (1996) 106-112 VAL807

LEU774

LYS788

Fig. 6. A stereo view of the ba,:kbone atoms of MLCK(774-807)amide superimposed over the C-terminal domain LysTSS-Trp8°°. The structures exhibit an r.m.s, deviation of 69 4-11 pm in this longer segment. A helix is again apparent from Arg79°-Trp 8°° with a more nascent helical structure indicated in the C-terminal residues.

in the N-terminal region is 54 ___7 pm while in the Cterminal domain the r.m.s, deviation is 69 4- 11 pm indicating the conformational space defined by the molecule is quite well-defined. Two-step superpositions were found necessary in other peptides of similar length in a range of solvents such as PTH [17,31] and PTHrP [15,16] possessing hinges connecting more rigid domains. No H-bonds were assumed in the structure calculations.

4. Discussion The most prominent structural features in the autoregulatory peptide are the two helices separated by a mobile turn segment. The structure of the N-terminal helix 777-785 forming the major part of the connecting peptide joining the pseudosubstrate sequence to the bulk of the catalytic core is well-defined. A slightly longer C-terminal helix 790/791-800 incorporates the segment known to form the autoregulatory sequence of chicken gizzard MLCK [8,9]. The carboxy-terminal limit of the pseudosubstrate inhibitory region was found to be Lys-802 [8]. Reference to Fig. 6 indicates that the C-terminal helix may well extend to Lys-802 in a longer peptide. The structure of the C-terminal segment is affected in all likelihood by the interaction between residues at the C-terminal end of the pseudosubstrate segment and the N-terminal end of the connecting peptide. It is unlikely that such an interaction would occur in the same sequence within the intact enzyme, since the two helical segments would be kept separated by N- and C-terminal connections to the remaining structure. This is consistent with the findings that the residues Ala796-Ser 815 form a helix when bound to calmodulin [20,32]. The crystal structure of the equivalent cAPK inhibitor peptide PKI(5-24) has be,en determined in complex with

111

the cAPK catalytic subunit [33,34]. The N-terminal portion of the inhibitor PKI(5-17) forms an amphipathic a-helix which binds to a hydrophobic groove on the major lobe of catalytic cAPK while the so-called consensus site (18-24) forms an extended structure which binds in a cleft between the two lobes [34]. The PKI(5-24) peptide has a similar function to the chicken gizzard MLCK pseudosubstrate and should thus exhibit a similar tertiary structure. Sequence comparison between the two peptides reveals that MLCK(789-808) in the pseudosubstrate peptide is equivalent to PKI(5-24) [ 11]. A comparison of the structures of the PKI(5-24) crystal structure with segment 789-807 in the pseudosubstrate peptide (Fig. 6) reveals a high degree of similarity. The PKI helix (5-17) is duplicated in the pseudosubstrate peptide and the C-terminal segment PKI(18-24) is similarly extended in the pseudosubstrate (801-807). This close structural equivalence implies that the MLCK pseudosubstrate sequence is likely to interact with the active site in the catalytic core in a similar way to the demonstrated interaction of PKI with cAPK [34]. The structure of the pseudosubstrate peptide revealed in this work is in complete agreement with earlier results using peptides overlapping the pseudosubstrate peptide sequence at the C-terminal end forming the calmodulin binding site. This peptide has been shown to form a helix in complex with calmodulin after the central helix is disrupted [35-37]. The connecting peptide in the pseudosubstrate sequence MLCK(774-786) appears largely helical and may therefore bind to the catalytic core in a similar fashion to that expected for the C-terminal helical segment, i.e., interacting with a groove on the catalytic core. This added tight binding acts to increase the K i of MLCK (774-807) about 30-fold compared with the pseudosubstrate sequence MLCK(787-807) alone [11]. An average structure of the pseudosubstrate and connecting peptides is shown in Fig. 7 with the backbone represented by the ribbon and the side Y794

Fig. 7. A view of an average structure of MLCK(774-807)amide with the backbone conformation shown with the ribbon. Several side chains have been included using reduced diameter ball-and-stick representation which shows the residues affected by the folding back of the N-terminal helix. Trp-800 is involved in calmodulin binding.

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J.A. Barden et al. / Biochimica et Biophysica Acta 1292 (1996) 106-112

chains shown with a ball-and-stick representation for clarity. The connecting peptide folds back over the side chains of Ser-787, Arg-790, Tyr-794, Arg-797, Trp-800 and Lys802. Trp-800 is one of the residues important for the binding of calmodulin [14]. The binding of calmodulin is reported to increase ahelix content in the calmodulin binding site on MLCK [7] while the segment 799-813 in the peptide 796-815 is c~-helical when complexed with calmodulin [20,32]. The results in this paper indicate that the segment N-terminal to the calmodulin binding peptide is also helical so that the segment containing both the pseudosubstrate and calmodulin binding sites may form a single helix from Arg790/Met791-Leu 813 in the presence of calmodulin. An extension of the helix following calmodulin binding may well act to rotate the pseudosubstrate away from the enzyme by utilizing the flexible bend between the helices in the pseudosubstrate and connecting peptides, thereby exposing the site to the myosin light chain substrate. This may be analogous to the way the central helix within calmodulin acts as a flexible connector between the two globular domains [20].

Acknowledgements This research was supported by grants from the National Health and Medical Research Council of Australia.

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