Observed in the crystal structure as discussed above. The recent structures

Observed in the crystal structure as discussed above. The recent structures of the S. pneumoniae pilin RrgB give an explanation of the function of the N-domain cleft and what dictates its isopeptide bond formation. When RrgB was crystallized in a form devoid of the get Vitamin D2 sorting motif, an isopeptide bond in the N-terminal domain was not formed [10]. However, when a longer RrgB form was crystallized, its sorting motif was shown to interact with the pilin lysine of a neighboring pilin leading to a conformational change in the N-terminal domain and the subsequent formation of the covalent isopeptide bond [11]. In concurrence with the RrgB structures we propose a similar role for the FimP N-terminal groove; that the cleft and mobile loop (residues 56?4) function as recognition and interaction sites for the sortase-FimP complex during pili polymerization and that a preformed isopeptide bond (juxtaposed to the pilin Lys-182) results in a protein too rigid to properly present the pilin lysine to the sortase. However, during pili polymerization more strain may be imposed on the flexible hinge between the N- and M-domains and Asn-183 and Lys-52 may come close enough to form a bond. Indeed, in the BcpA pilin the N-domain isopeptide bond is observed only after BcpA polymerization [15].FimP Structure and Sequence AnalysesFigure 5. The pilin motif forms a groove. Stereo representation of the pilin motif residues lining a cleft that runs through the N-domain. Lys-182, involved in polymerization of FimP subunits, is localized at the rim of the cleft. The domain is presented as a semi-transparent electrostatic surface, colored in red and blue according to negative and positive electrostatic potential, respectively. The residues in the pilin motif are shown as stick models. The groove is highlighted with a dashed line. doi:10.1371/journal.pone.0048364.gare consistent with the 166518-60-1 site differential functional roles of the two pili. The evolution and adaptation of type-1 (FimP) and type-2 (FimA) pili to different intraoral niches and tropisms, suggest their pilin proteins to be organized and function more differently than assumed from their co-presence in the Actinomyces genus. The metal binding loop, proline-rich motifs or differential N-domain structures could accordingly participate in various intergeneric bacteria-bacteria adhesion (co-aggregation) or host-bacteria adhesion partnerships. Notably, Actinomyces has multiple co-aggregation partners and hosts, therefore their pilin proteins are expected to possess multiple binding activities.segment unique for FimA. In addition, FimA from A. oris (n = 14) and A. naeslundii (n = 17) were highly related (64 identity/76 similarity) with fully conserved isopeptide bond triads, position of cysteines, pilin and LPLTG motifs as well as proline residues in the proline-rich segment. The sequence alignment of FimA from A. oris is presented in Fig. 6b.Materials and Methods Cloning, Purification and CrystallizationFimP31?91was cloned from A. oris strain T14V, expressed and crystallized as described [31]. In short, N-terminal 6His-tagged FimP was purified by nickel-affinity chromatography followed by size-exclusion chromatography. The protein was concentrated to 92 mg/ml in 20 mM Tris-HCl, pH 8.0. Selenomethionine (SeMet)-substituted protein was obtained after mutating three isoleucines to methionines (FimP-3M) [31]. The protein was subjected to in situ proteolysis 1662274 with 1 (w/w) a-chymotrypsin immediately before crystallization set.Observed in the crystal structure as discussed above. The recent structures of the S. pneumoniae pilin RrgB give an explanation of the function of the N-domain cleft and what dictates its isopeptide bond formation. When RrgB was crystallized in a form devoid of the sorting motif, an isopeptide bond in the N-terminal domain was not formed [10]. However, when a longer RrgB form was crystallized, its sorting motif was shown to interact with the pilin lysine of a neighboring pilin leading to a conformational change in the N-terminal domain and the subsequent formation of the covalent isopeptide bond [11]. In concurrence with the RrgB structures we propose a similar role for the FimP N-terminal groove; that the cleft and mobile loop (residues 56?4) function as recognition and interaction sites for the sortase-FimP complex during pili polymerization and that a preformed isopeptide bond (juxtaposed to the pilin Lys-182) results in a protein too rigid to properly present the pilin lysine to the sortase. However, during pili polymerization more strain may be imposed on the flexible hinge between the N- and M-domains and Asn-183 and Lys-52 may come close enough to form a bond. Indeed, in the BcpA pilin the N-domain isopeptide bond is observed only after BcpA polymerization [15].FimP Structure and Sequence AnalysesFigure 5. The pilin motif forms a groove. Stereo representation of the pilin motif residues lining a cleft that runs through the N-domain. Lys-182, involved in polymerization of FimP subunits, is localized at the rim of the cleft. The domain is presented as a semi-transparent electrostatic surface, colored in red and blue according to negative and positive electrostatic potential, respectively. The residues in the pilin motif are shown as stick models. The groove is highlighted with a dashed line. doi:10.1371/journal.pone.0048364.gare consistent with the differential functional roles of the two pili. The evolution and adaptation of type-1 (FimP) and type-2 (FimA) pili to different intraoral niches and tropisms, suggest their pilin proteins to be organized and function more differently than assumed from their co-presence in the Actinomyces genus. The metal binding loop, proline-rich motifs or differential N-domain structures could accordingly participate in various intergeneric bacteria-bacteria adhesion (co-aggregation) or host-bacteria adhesion partnerships. Notably, Actinomyces has multiple co-aggregation partners and hosts, therefore their pilin proteins are expected to possess multiple binding activities.segment unique for FimA. In addition, FimA from A. oris (n = 14) and A. naeslundii (n = 17) were highly related (64 identity/76 similarity) with fully conserved isopeptide bond triads, position of cysteines, pilin and LPLTG motifs as well as proline residues in the proline-rich segment. The sequence alignment of FimA from A. oris is presented in Fig. 6b.Materials and Methods Cloning, Purification and CrystallizationFimP31?91was cloned from A. oris strain T14V, expressed and crystallized as described [31]. In short, N-terminal 6His-tagged FimP was purified by nickel-affinity chromatography followed by size-exclusion chromatography. The protein was concentrated to 92 mg/ml in 20 mM Tris-HCl, pH 8.0. Selenomethionine (SeMet)-substituted protein was obtained after mutating three isoleucines to methionines (FimP-3M) [31]. The protein was subjected to in situ proteolysis 1662274 with 1 (w/w) a-chymotrypsin immediately before crystallization set.