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The amino-terminal region of group A streptococcal M protein determines its molecular state of assembly and function
K M Khandke1, T Fairwell, E H Braswell
1Rockefeller University, New York, New York 10021.
Abstract:
Group A streptococcal M protein, a major virulence factor, is an alpha-helical coiled-coil dimer on the surface of the bacteria. Limited proteolysis of type 57 streptococcus with pepsin released two fragments of the M57 molecule, with apparent molecular weights of 32,000 and 27,000 on SDS-PAGE. However, on gel filtration under nondenaturing conditions, each of these proteins eluted as two distinct molecular forms. The two forms corresponded to their dimeric and monomeric state as compared to the gel filtration characteristics of known dimeric coiled-coil proteins. The results of sedimentation equilibrium measurements were consistent with this, but further indicated that the "dimeric form" consisted of a dimer in rapid equilibrium with its monomer, whereas the "monomeric form" does not dimerize. The monomeric form was the predominant species for the 27 kD species, whereas the dimeric form predominated for the 32 kD species. Sequence analysis revealed the 27 kD species to be a truncated derivative of the 32 kD PepM57 species, lacking the N-terminal nonheptad region of the M57 molecule. These data strongly suggested that the N-terminal nonheptad region of PepM57 is important in determining the molecular state of the molecule. Consistent with this, PepM49, another nephritis-associated serotype, which lacks the nonheptad N-terminal region, also eluted as a monomer on gel filtration under nondenaturing conditions. Furthermore, removal of the N-terminal nonheptad segment of the dimeric PepM6 protein converted it into a monomeric form. The dimeric molecular form of both the 32 kD PepM57 and the 27 kD PepM57 did not represent a stable state of assembly, and were susceptible to conversion to the corresponding monomeric molecular forms by simple treatments, such as lyophilization. The 27 kD PepM57 exhibited a greater propensity than the 32 kD species to exist in the monomeric form. The 32 kD species contained the opsonic epitope of the M57 molecule, whereas the 27 kD species lacked the same. This is consistent with the previous reports on the importance of the N-terminal region of M protein for its opsonic activity. Together, these results strongly suggest that, in addition to its importance for the biological function, the N-terminal region of the M protein plays a dominant role in determining the molecular state of the M molecule, as well as its stability.
Insights
The N-terminal region of Group A streptococcal M protein is crucial for its molecular state and stability. This region influences whether the M protein exists as a dimer or monomer, impacting its biological function and opsonic activity.
Area of Science:
- Microbiology and Immunology
- Protein Biochemistry
Background:
- Group A streptococcal M protein is a key virulence factor, typically existing as an alpha-helical coiled-coil dimer on the bacterial surface.
- Previous studies highlight the importance of the M protein's N-terminal region for its opsonic activity.
Purpose of the Study:
- To investigate the role of the N-terminal nonheptad region of streptococcal M protein in determining its molecular state (dimeric vs. monomeric) and stability.
- To analyze the relationship between molecular state, N-terminal structure, and opsonic epitope presence in M protein fragments.
Main Methods:
- Limited proteolysis of M57 protein using pepsin to generate fragments.
- Analysis of protein fragments using SDS-PAGE and gel filtration chromatography under nondenaturing conditions.
- Sedimentation equilibrium measurements to determine molecular states and equilibria.
- Sequence analysis of protein fragments and comparison with other M protein serotypes (PepM49, PepM6).
Main Results:
- Pepsin digestion of M57 yielded 32 kDa and 27 kDa fragments, each existing in both monomeric and dimeric forms.
- The 27 kDa fragment, lacking the N-terminal nonheptad region, predominantly existed as a monomer, unlike the 32 kDa fragment.
- Removal of the N-terminal nonheptad region from PepM6 converted it to a monomeric form, and M57 fragments showed instability and conversion to monomers.
- The 32 kDa fragment contained the opsonic epitope, while the 27 kDa fragment lacked it.
Conclusions:
- The N-terminal nonheptad region of streptococcal M protein is critical for maintaining its dimeric state and stability.
- The molecular state of M protein, influenced by its N-terminal region, directly affects the presence of opsonic epitopes and biological function.
- The N-terminal region plays a dual role in M protein: mediating biological function and dictating molecular assembly and stability.