Location of glycine mutations within a bacterial collagen protein affects degree of disruption of triple-helix folding and conformation

Abstract

The hereditary bone disorder osteogenesis imperfecta is often caused by missense mutations in type I collagen that change one Gly residue to a larger residue and that break the typical (Gly-Xaa-Yaa)(n) sequence pattern. Site-directed mutagenesis in a recombinant bacterial collagen system was used to explore the effects of the Gly mutation position and of the identity of the residue replacing Gly in a homogeneous collagen molecular population. Homotrimeric bacterial collagen proteins with a Gly-to-Arg or Gly-to-Ser replacement formed stable triple-helix molecules with a reproducible 2 °C decrease in stability. All Gly replacements led to a significant delay in triple-helix folding, but a more dramatic delay was observed when the mutation was located near the N terminus of the triple-helix domain. This highly disruptive mutation, close to the globular N-terminal trimerization domain where folding is initiated, is likely to interfere with triple-helix nucleation. A positional effect of mutations was also suggested by trypsin sensitivity for a Gly-to-Arg replacement close to the triple-helix N terminus but not for the same replacement near the center of the molecule. The significant impact of the location of a mutation on triple-helix folding and conformation could relate to the severe consequences of mutations located near the C terminus of type I and type III collagens, where trimerization occurs and triple-helix folding is initiated.

FIGURE 1.

Schematic illustration of the design of VCL constructs with Gly substitutions at different positions. The VCL amino acid sequence is shown below, with the trimerization V domain in italics, the triple-helix domain in boldface, and the Gly replacement locations underlined.

FIGURE 2.

SDS-PAGE of VCL proteins as purified and after trypsin digestion. The lower band after trypsin digestion shows that the V domains were cleaved by trypsin, leaving the CL triple-helix domain intact. The band of VCL(T108M,G109R) after trypsin digestion is lower than the bands of the other control and mutant proteins, indicating that the mutation allowed partial cleavage of the CL domain; mass spectroscopy indicated that cleavage occurred at Arg-114.

FIGURE 3.

Thermal transition of VCL proteins by CD (A and B) and DSC (C and D) showing that Gly-to-Arg and Gly-to-Ser mutations at different positions lead to small but significant decreases in stability (PBS at pH 7). A, CD thermal transitions for VCL (black), VCL(G199R) (red), and VCL(G199S) (blue) (0.35 mg/ml). B, CD thermal transitions for VCL(T108M) (black) and VCL(T108M,G109R) (red) (1.0 mg/ml). C, DSC profiles of VCL (black), VCL(G199R) (red), and VCL(G199S) (blue) (0.35 mg/ml). D, DSC profiles of VCL(T108M) (black) and VCL(T108M,G109R) (red) (0.5 mg/ml). deg, degrees.

FIGURE 4.

CD thermal transitions of the collagen CL domains of control and mutant proteins. The sharp cooperative transitions observed with increasing temperature (right arrow) show the same small decrease in T_m values as a result of mutations as seen for VCL proteins. Cooling at the same rate (left arrow) led to only a small linear increase in ellipticity, suggesting no correct refolding in the absence of the V domain. Squares, CL; circles, CL(G199R); triangles, CL(G199S). (PBS at pH 7.0, 0.35 mg/ml). The observed raw ellipticity values are plotted versus temperature, rather than mean residue ellipticity (MRE), because of the difficulty in obtaining a reliable value of the concentration for the collagen domains, which contain no aromatic residues. Various estimates of the concentration through weighing and absorbance at 214 nm are consistent with estimates obtained by subtracting the V domain from the VCL spectrum and suggest that MRE₂₂₀ ∼ 7500 degrees cm² dmol⁻¹ for the CL domains. mdeg, millidegrees.

FIGURE 5.

Monitoring the refolding of VCL proteins by CD, plotted as MRE₂₂₀versus time. A, VCL (black), VCL(G199R) (red), and VCL(G199S) (blue) (PBS at pH 7.0, 0.35 mg/ml). B, VCL(T108M) (black) and VCL(T108M,G109R) (red) (PBS at pH 7.0, 0.5 mg/ml). Samples were denatured at 55 °C for 20 min and then rapidly cooled to 0 °C, at which the CD signal at 220 nm was measured. The half-time of refolding (t½) is defined as the time for the fraction folded to reach 0.5. The data are expressed as MRE₂₂₀ values versus time rather than as fraction folded because the MRE₂₂₀ value is a combination of decreasing values during the rapid refolding of the α-helix V domain and increasing values during the slower refolding of the triple helix, which presumably occurs after the V domain is folded. deg, degrees.

FIGURE 6.

DSC profiles of samples in 0.1 m acetic acid showing decreased thermal stability and multiple thermal transitions at acid pH. A, VCL; B, VCL(G199R); C, VCL(G199S); D, VCL(T108M,G109R).