title: Intramolecular isopeptide bonds give thermodynamic and proteolytic stability to the major pilin protein of Streptococcus pyogenes.
abstract: 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 T(m) of 85 degrees 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 T(m) that was approximately 30 degrees 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.
authors.affiliation: School of Biological Sciences, University of Auckland, Auckland 1142, New Zealand.
authors.forename: Hae Joo
authors.initials: HJ
authors.lastname: Kang
authors.forename: Edward N
authors.initials: EN
authors.lastname: Baker
mesh_terms: D019943:Amino Acid Substitution; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D036781:Fimbriae Proteins / Q000737:chemistry*; D050505:Mutant Proteins / Q000737:chemistry; D010455:Peptides / Q000737:chemistry*; D011489:Protein Denaturation; D011499:Protein Processing, Post-Translational; D055550:Protein Stability; D017433:Protein Structure, Secondary; D021241:Spectrometry, Mass, Electrospray Ionization; D055672:Static Electricity; D013297:Streptococcus pyogenes / Q000737:chemistry*; D013816:Thermodynamics
chemical_list: D050505:Mutant Proteins; D010455:Peptides; D036781:Fimbriae Proteins