Design considerations in coiled-coil fusion constructs for the structural determination of a problematic region of the human cardiac myosin rod

Abstract

X-ray structural determination of segments of the myosin rod has proved difficult because of the strong salt-dependent aggregation properties and repeating pattern of charges on the surface of the coiled-coil that lead to the formation of paracrystals. This problem has been resolved in part through the use of globular assembly domains that improve protein folding and prevent aggregation. The primary consideration now in designing coiled-coil fusion constructs for myosin is deciding where to truncate the coiled-coil and which amino acid residues to include from the folding domain. This is especially important for myosin that contains numerous regions of low predicted coiled-coil propensity. Here we describe the strategy adopted to determine the structure of the region that extends from Arg1677 - Leu1797 that included two areas that do not show a strong sequence signature of a conventional left-handed coiled coil or canonical heptad repeat. This demonstrates again that, with careful choice of fusion constructs, overlapping structures exhibit very similar conformations for the myosin rod fragments in the canonical regions. However, conformational variability is seen around Leu1706 which is a hot spot for cardiomyopathy mutations suggesting that this might be important for function.

Keywords: Cardiac myosin; Coiled-coils; Fusion proteins; Light meromyosin; Protein structure; X-ray structural determination.

Figure 1.. Predicted coiled-coil propensity for the cardiac myosin rod.

(A) Myosin rod coiled-coil prediction using a 28-amino acid window in COILS (blue) and Marcoil (Red). The COILS algorithm compares a sequence to a database of known parallel two-stranded coiled-coils and derives a similarity score (Lupas, 1996a; Lupas et al., 1991). The 28-amino acid window for coils smooths out most of the local variations in coiled coil propensity except for around the Skip residues which introduce a discontinuity. The Marcoil algorithm is based on a window-less Hidden Markov Model (Delorenzi and Speed, 2002). The propensities relate to the probability that a group of amino acids will adopt the structure of a coiled-coil, but do not indicate stability or structure of the resultant oligomeric state. (B) COILS prediction from myosin rod amino acids 1680–1810. This region contains 13 pathogenic mutation sites, as well as regions of poorly predicted coiled-coil.

Figure 2.. Structures and comparison of the fusion proteins covering Arg1677 - Ala1758.

(A) Structures of cardiac myosin containing residues 1677–1758 using both Xrcc4 and Gp7/Eb1 fusion domains. The Eb1 domain in the Gp7-1677-1758-Eb1 structure was not resolved. (B) The three structural conformations overlapped from residues 1677–1691. When aligned with this segment, the three conformations diverge most strongly around residue L1706, highlighting the flexible properties of this region of myosin. Figures 2–5 were prepared with Pymol (DeLano, 2002).

Figure 3.. Mismatch of the coiled-coil registration in the fusion protein design yields a tetrameric assembly.

(A) A coiled-coil mismatch in the fusion between Gp7 and myosin rod resulted in a tetrameric artifact of anti-parallel coiled-coil. (B) The core residues are highlighted and shown in red and blue for the respective dimers. The predicted coiled-coil register for the intact myosin rod is shown along with the amino acid labels. A predicted deviation from a canonical coiled-coil is underlined between 1662–1673. However, in this structure the M1664 is in the d position, not g as predicted by COILS. (C) Stereo view of the four-helix bundle along the hydrophobic core.

Figure 4.. Structures and comparison of the dimeric fusion proteins covering

Asp1733 - Leu1797. (A) Structure of the coiled-coil domain of myosin rod from amino acids 1733–1797 fused to Xrcc4 fusion domain. (B) Structure of myosin rod from amino acids 1733–1797 fused to Gp7 fusion domain. (C) Aligned structure of the coiled-coil domains of the Xrcc4-1733-1797 structure and Gp7-1733-1797 structure. The two conformations of myosin were aligned from amino acids 1733–1746, and the deviation in coiled-coil pitch is clearly visible between the two conformations.

Figure 5.. Composite preliminary models for the myosin rod extending from Arg1677 – Gly1807.

Composite model showing six conformations of myosin rod between amino acids 1677–1807. Models were overlapped from residues 1677–1691. Blue chains represent chains A and B of Xrcc4-1677-1758. Cyan chains represent chains C and D of Xrcc4-1677-1758. Yellow chains represent Gp7-1677-1755-Eb1. Orange chains represent Xrcc4-1733-1797. Magenta chains represent Gp7-1733-1797. Grey chains represent residues 1798–1807 of the published structure up to the skip 4 residue (PDB ID: 4XA6).