Modular structure of smooth muscle Myosin light chain kinase: hydrodynamic modeling and functional implications

Abstract

Smooth muscle myosin light chain kinase (smMLCK) is a calcium-calmodulin complex-dependent enzyme that activates contraction of smooth muscle. The polypeptide chain of rabbit uterine smMLCK (Swiss-Prot entry P29294) contains the catalytic/regulatory domain, three immunoglobulin-related motifs (Ig), one fibronectin-related motif (Fn3), a repetitive, proline-rich segment (PEVK), and, at the N-terminus, a unique F-actin-binding domain. We have evaluated the spatial arrangement of these domains in a recombinant 125 kDa full-length smMLCK and its two catalytically active C-terminal fragments (77 kDa, residues 461-1147, and 61 kDa, residues 461-1002). Electron microscopic images of smMLCK cross-linked to F-actin show particles at variable distances (11-55 nm) from the filament, suggesting that a well-structured C-terminal segment of smMLCK is connected to the actin-binding domain by a long, flexible tether. We have used structural homology and molecular dynamics methods to construct various all-atom representation models of smMLCK and its two fragments. The theoretical sedimentation coefficients computed with HYDROPRO were compared with those determined by sedimentation velocity. We found agreement between the predicted and observed sedimentation coefficients for models in which the independently folded catalytic domain, Fn3, and Ig domains are aligned consecutively on the long axis of the molecule. The PEVK segment is modeled as an extensible linker that enables smMLCK to remain bound to F-actin and simultaneously activate the myosin heads of adjacent myosin filaments at a distance of >or=40 nm. The structural properties of smMLCK may contribute to the elasticity of smooth muscle cells.

Figure 1

Structural domains of smMLCK. Domain definition based on ref. (9, 10). The central segment marked by the arrows corresponds to the trypsin resistant fragment of smMLCK, which is equivalent to the recombinant 61k-MLCK used in this study. This fragment comprises the Ig2-Fn3 tandem and the catalytic/regulatory domain. The 77k-MLCK has the same N-terminus as the 61k fragment and extends to the C-terminus of smMLCK to include the Ig3 (telokin) region. The atomic models of the Ig2-Fn3 tandem and the catalytic/regulatory domain obtained by homology modeling are shown in ribbon representation (see text and Table 2 for details).

Figure 2

Purification of the full-length mammalian smMLCK and its C-terminal active fragments overexpressed in insect cells. Representative examples of electrophoresis on SDS-polyacrylamide gel (8%) of smMLCK samples at various steps of purification are shown: (a) - extract of soluble proteins from High Five cells expressing smMLCK, (b) - peak fraction from DEAE Sepharose CL 6B column, (c, d) - smMLCK eluted in EGTA from CaM -affinity column (two different loads are shown). The 77k-MLCK (e,f) and 61k-MLCK (g) fragments of smMLCK were obtained with similar yields. A molecular weight standard (Bio-Rad) is shown for each preparation (s).

Figure 3

Rotary shadowing images of smMLCK zero-length cross-linked to F-actin. Note particles that appear to be tethered to the filament. The distance between these particles and the actin filament is highly variable.

Figure 4

Contour length distribution of smMLCK cross-linked to F-actin. The length is measured from the edge of the actin filament to the most distant distinguishable parts of the tethered smMLCK. The distribution of distances is bimodal with a broad peak between 16-30 nm and a second peak at 40 nm. The width of the actin filament image is 15.9 ± 1.2 nm

Figure 5

Rotary shadowing images of smMLCK. A low magnification field (A) and ensembles of high magnification images of single molecules in the extended (B) and compact (C) conformation are shown. The measured contour length for the extended conformation is L_e=36.4±5.0 nm and for the compact conformation is L_c=23.9±3.2 nm. The corresponding widths are W_e=11.9±1.5 nm and W_c=14.8±2.4 nm.

Figure 6

Rotary shadowing images of the 77k fragment of smMLCK. The low (A) and high (B,C) magnification images are shown. The average contour lengths are L_e=25.0±2.9 nm and L_c=18.6±2.0 nm, for the extended (B) and compact (C) conformation, respectively. The corresponding widths are W_e=11.6±1.9 nm and W_c=15.6±1.8 nm.

Figure 7

Sedimentation velocity profiles of smMLCK and its fragments in the absence and presence of CaM. A - the full-length smMLCK at 0.13 mg/ml (solid lines) and at 10× dilution (dashed lines); B - 77k-MLCK; C - 61k-MLCK; D - calmodulin. The protein samples (0.13-0.2 mg/ml) were dialyzed against a solution containing 0.1 M NaCl, 1 mM MgCl₂, 20 mM MOPS pH 7.0 and 1 mM CaCl₂.

Figure 8

Models of the 61k fragment of smMLCK. Three different arrangements of the Ig2-Fn3 tandem with respect to the catalytic domain were tested. The domains are color coded: red - the catalytic/regulatory domain; blue - the Fn3 domain; magenta - the Ig(2) domain; gray - the linker region (linker-3) connecting the Fn3 domain with the catalytic domain. See Table 2 for the exact definition of the domains. Images of protein models were generated with the program PyMOL (85).

Figure 9

Structural features of the PEVK region of smMLCK. (A) - amino acid sequence showing the highly conserved repetitive nature of this segment. Note that each Glu residue is flanked on both sides with positively charged Lys side chains, which allow for the i, i±3 salt bridges in the poly-Pro helical configuration. (B) - Far UV circular dichroism spectra of a synthetic 24 residue peptide corresponding to the two 12-residue repeats marked by the rectangle in A. The negative peak at 198 nm is a characteristic of the poly-Pro helix type II. (C) - Example of the Lys-Glu-Lys salt bridges in the modeled structure of the PEVK region of smMLCK. The initial configuration (t=0) and after 50 ps molecular dynamics simulation are shown.

Figure 10

Modeling of the full-length smMLCK. The C-terminal part of the molecule starting at residue 327 modeled in the extended configuration corresponding to model 1 in Figure 8 is identical in all three models. The PEVK region (green) is subjected to MD simulation for 50ps, 200ps and 300ps, resulting in a progressive compaction of the polypeptide chain. The domains comprising the 61k-MLCK are colored in the same manner as in Figure 8. All linker regions are shown in gray, the N-terminal actin-binding domain - orange, the Ig1 domain - brown, and the C-terminal telokin region (Ig3) is shown in magenta.

Figure 11

Sedimentation coefficients of CaM and MLCK models computed from their atomic coordinates. The program HYDROPRO was used for the calculations. The effect of hydration on the calculated sedimentation coefficients is tested by varying the so-called atomic element radius (AER). The dashed line in each panel shows the experimentally determined value of s_(20,w) for each molecule. The abbreviations m1, m2 and m3 correspond to the three models of the 61k-MLCK shown in Fig. 8 and the respective models of the 77k-MLCK as described in the text. Note that only the most extended models of the 61k and 77k MLCK fragments are consistent with the observed sedimentation coefficients. For the full-length MLCK an extended C-terminal part combined with a moderately compacted PEVK region yields the s values consistent with the experiment.

Figure 12

A hypothetical mechanism of myosin phosphorylation in mammalian smooth muscle. smMLCK is pictured as a flexible, extensible molecule bound to actin filaments and capable of bridging thin and thick filaments. Those myosin heads that are in contact with actin filaments are phosphorylated preferentially, and thus become capable of producing force. The number of activated myosin heads depends on filament overlap. Dissociation of the catalytic domain of smMLCK from phosphorylated myosin heads enables filament sliding. smMLCK might contribute to the passive tension under resting conditions by bridging the thick and thin filaments.