Intramolecular Isopeptide Bonds Give Thermodynamic and Proteolytic Stability to the Major Pilin Protein of Streptococcus pyogenes

摘要

The pili expressed by Streptococcus pyogenes and certain other Gram-positive bacterial pathogens are based on a polymeric backbone in which individual pilin subunits are joined end-to-end by covalent isopeptide bonds through the action of sortase enzymes. The crystal structure of the major pilin of S. pyogenes, Spy0128, revealed that each domain of the two domain protein contained an intramolecular isopeptide bond cross-link joining a Lys side chain to an Asn side chain. In the present work, mutagenesis was used to create mutant proteins that lacked either one isopeptide bond (E117A, N168A, and E258A mutants) or both isopeptide bonds (E117A/E258A). Both the thermal stability and proteolytic stability of Spy0128 were severely compromised by loss of the isopeptide bonds. Unfolding experiments, monitored by circular dichroism, revealed a transition temperature Tm of 85 °C for the wild type protein. In contrast, mutants with only one isopeptide bond showed biphasic unfolding, with the domain lacking an isopeptide bond having a Tm that was ∼30 °C lower than the unaltered domain. High resolution crystal structures of the E117A and N168A mutants showed that the loss of an isopeptide bond did not change the overall pilin structure but caused local disturbance of the protein core that was greater for E117A than for N168A. These effects on stability appear also to be important for pilus assembly. The pili expressed by Streptococcus pyogenes and certain other Gram-positive bacterial pathogens are based on a polymeric backbone in which individual pilin subunits are joined end-to-end by covalent isopeptide bonds through the action of sortase enzymes. The crystal structure of the major pilin of S. pyogenes, Spy0128, revealed that each domain of the two domain protein contained an intramolecular isopeptide bond cross-link joining a Lys side chain to an Asn side chain. In the present work, mutagenesis was used to create mutant proteins that lacked either one isopeptide bond (E117A, N168A, and E258A mutants) or both isopeptide bonds (E117A/E258A). Both the thermal stability and proteolytic stability of Spy0128 were severely compromised by loss of the isopeptide bonds. Unfolding experiments, monitored by circular dichroism, revealed a transition temperature Tm of 85 °C for the wild type protein. In contrast, mutants with only one isopeptide bond showed biphasic unfolding, with the domain lacking an isopeptide bond having a Tm that was ∼30 °C lower than the unaltered domain. High resolution crystal structures of the E117A and N168A mutants showed that the loss of an isopeptide bond did not change the overall pilin structure but caused local disturbance of the protein core that was greater for E117A than for N168A. These effects on stability appear also to be important for pilus assembly. The stability of a globular protein is in general a fine balance between large numbers of weak, noncovalent stabilizing forces (hydrophobic interactions, hydrogen bonds, and ion pairs) and the destabilizing loss of conformational entropy (1Pace C.N. Shirley B.A. McNutt M. Gajiwala K. FASEB J. 1996; 10: 75-83Crossref PubMed Scopus (558) Google Scholar). Stability can be greatly enhanced, however, by the presence of a small number of strategically placed covalent bonds, for example those provided by disulfide bonds between Cys residues or by a bound metal ion that bonds to several amino acid side chains. Indeed, some striking examples have been reported in which the introduction of disulfide bonds that link sequentially distant parts of a polypeptide chain can result in large increases in protein stability (2Matsumura M. Becktel W.J. Levitt M. Matthews B.W. Proc. Natl. Acad. Sci. U.S.A. 1989; 86: 6562-6566Crossref PubMed Scopus (256) Google Scholar). Our recent discovery of cross-linking isopeptide bonds within the protein subunits of the pili expressed by the Gram-positive organism Streptococcus pyogenes (3Kang H.J. Coulibaly F. Clow F. Proft T. Baker E.N. Scienc