A long helix from the central region of smooth muscle caldesmon

Abstract

The central region of smooth muscle caldesmon is predicted to form alpha-helices on the basis of its primary structure. We have isolated a fragment (CT54) that contains this region. The hydrodynamic properties and the electron microscopic images suggest that CT54 is an elongated (35 nm), monomeric molecule. The circular dichroic spectrum yields an overall alpha-helical content of 55-58%. These results are consistent with the model that the middle portion of CT54 forms a long stretch of single-stranded alpha-helix. Such a structure, if it in fact exists, is thought to be stabilized by numerous salt bridges between charged residues at positions i and i + 4. The structural characteristics of this fragment not only represent an unusual protein configuration but also provide information about the functional role of caldesmon in smooth muscle contraction.

Fig. 1. The 54-kDa fragment of caldesmon obtained from chymotryptic digestion.

To purify CT54, the chymotrypsin digestion mixture (weight ratio of chymotrypsin to caldesmon = 1:700; t = 10 min) was chromatographed on a Waters HPLC using a Pharmacia Mono Q column with an exponential gradient to 500 mm NaCl (A). The major peak was concentrated and chromatographed on the same column using a linear gradient of NaCl from 185 to 500 mm (B). C, the time course of chymotrypsin digestion of caldesmon. Lanes 1 and 10, molecular mass standards (66, 45, 36, 29, 24, and 20.4 kDa); lane 2, caldesmon alone; lanes 3–8, caldesmon (2.6 mg/ml) treated with chymotrypsin (1:400 by weight), t = 0, 3, 6, 10, 30, 60 min, noting the transient appearance of the 40-kDa fragment and the relative stable bands around 54 kDa; lane 9, the HPLC-purified CT54.

Fig. 2. Determination of molecular weight of CT54 by sedimentation equilibrium measurements.

High speed equilibrium ultracentrifugation runs were performed as described previously (31). Plot shown are the number average molecular weight (M_n, open circles), the weight average molecular weight (M_r, closed circles), and the z average molecular weight (M_z, open squares) versus local cell concentration.

Fig. 3. Repeating sequence in the central region of smooth muscle caldesmon.

Shown here is the sequence in one-letter code from residues 166 to 450 with the repetitive units aligned in the middle. The sequence in parenthesis (numbered 3′ and underlined) is the extra unit found in the longer isoform by Hayashi et al. (14). The residues in italic are variable parts in these repeats (see text for discussion). The leading unit (numbered 0) deviates significantly from the consensus sequence and may represent a degenerated motif. Alteration is also seen in the eighth unit which ends with an 11-residue insert.

Fig. 4. Secondary structures of gizzard caldesmon predicted by Chou-Fasman (56) (A) and Gibrat et al. (57) (B) algorithms.

The latter is a newer version of the Garnier-Osguthorpe-Bobson method (58). In the first method a string of 6 residues is considered, whereas in the second method, 17 residues are analyzed each time. The calculated conformational indeces (in arbitrary units) for α-helices (solid lines), β-sheets (broken lines), and random coils (dotted lines) are plotted. The beginning and the end of CT54 are indicated by arrows. Note that both methods predict that the central repeating region, residues from ~245 to ~400, has a high tendency to form α-helices, but a low tendency to form either β-sheets or random coils.

Fig. 5. Circular dichroism of CT54.

Panel A, CD spectra of CT54 in 2.5 mm potassium phosphate buffer, 0.04 mm EDTA, pH 7.6, 20 °C. Panel B, thermal unfolding measured at 222 nm of CT54 in 50 mm NaCl, 20 mm sodium phosphate buffer at pH 7.0 (closed circles), pH 2.02 (open circles), and pH 11.45 (crosses). At pH 9.5 the melting curve is identical to that at pH 7.0. Concentration of CT54, 0.047 mg/ml. All measurements were carried out in a 1-cm path length cell.

Fig. 6. Electron microscopic images of CT54.

CT54 (10–20 μg/ml) was sprayed onto the mica surface and processed for rotary shadowing by a platinum-rich mixture (platinum/tungsten = 3:1); bar indicates 100 nm; magnification, ×150.000. Inset, a typical histogram shows the distribution of the contour length of the images. The observed longer images are consistent with end-to-end oligomers.

Fig. 7. Postulated salt bridges that may be responsible for stabilizing the helical structure of CT54.

Peptide segment from Lys³²⁰ to Glu³³⁷ is plotted in a helical wheel. Salt bridges are indicated by curved lines. Residues involved in salt bridge formation are labeled with numbers. Note that all salt bridges are formed between residues at positions i and i+4: Lys³²⁰ to Glu³²⁴, Arg³²⁵ to Glu³²⁹, Lys³²⁶ to Glu³³⁰, etc. Glu³²² is to interact with Arg³¹⁸ which is not shown. Similar charge interactions are thought to operate in other repeating units (see Fig. 3).