N 12104) was cut at the pilin motif lysine as well as in the C-domain (Lys-372). Mishra et al [5] reported that FimA from A. oris strain T14V is similarly cut at the pilin lysine. This difference in sensitivity to trypsin implies that FimP is more rigid than FimA in which the pilin motif is more exposed. A structural comparison between FimP31?91 and FimA (M- and C-domains) revealed that the major differences between the two structures are the loops connecting the b-strands. The most BTZ043 prominent difference is the segments that pack against the S4 sheet of their respective M-domains. In FimA this constitutes a prolinerich segment (332PPTPETPPTDPENPP347) that runs diagonally over half the sheet. In FimP the equivalent segment is a long, coiled region, located between b18 and b19, that folds over the whole S4 sheet like a clamp (Fig. 7). Another distinct structural difference is the FimP metal-binding loop that protrudes from the C-domain like a knob. This loop has no equivalent in FimA. Such a significant structural element is likely to be available for recognition by certain microbes of the biofilm. In analogy, the surface adhesin SspB of the oral bacteria Streptococcus gordonii exhibits a surface-exposed helix, stabilized by a metal ion [37]. This helix is solely Nafarelin recognized by the periodontal pathogen Porphyromonas gingivalis [38]. Likewise, the proline-rich segment in FimA may function as a recognition site for other bacteria-bacteria interactions. However, to further unravel the complex mechanisms that control colonization and formation of the oral biofilm, extensive structural and functional studies have to be performed. Sequence comparison of the FimP and FimA N-domains indicates different positions of the disulfide bridges. In FimP the first cysteine of the N-domain is located directly after the presumed isopeptide forming lysine and participate in a disulfide bond that unites the ends of a flexible loop. Also in FimA, the presumed disulfide bond is located near the putative isopeptide bond but with a different organization where its second cysteine is located directly before the catalytic glutamic acid. When the FimA sequence is applied on the FimP template, the first cysteine is located in the coil after the two helices A and B and the second in the S2 sheet, next to the isopeptide bond glutamic acid, as discussed. Assuming that a disulfide bridge is formed also in FimA, we suggest that its S2 sheet is more occluded due to tighter packing with the N-domain helices. The overall appearance of the Ndomains may therefore be quite different, which indeed is reflected by their different sensitivity to trypsin. The structural differencesThe Pilin Motif is Located in a GrooveIn FimP, the lysine of the pilin motif (WNYNVHVYPK) is exposed on the hinge that connects the N- and M-domains, and is thereby available for the sortase mediated pili polymerization reaction (Fig. 3a, 4a and 5). The pilin lysine, Lys-182, is located at the end of a mostly non-polar groove formed between helix A, the b-sandwich, and the mobile loop of the N-domain. The groove is lined by the pilin-motif conserved residues Trp-73, Val-177, Val179, and Pro-181 with the pilin lysine Lys-182 located at the rim of the channel. The conserved Tyr-180 stacks with His-51 and forms a hydrogen bond with the main chain amide of Asp-69, located close to the mobile loop. The isopeptide triad (Lys-52, Asn-183, Glu-145) is positioned within the groove however the isopeptide bond is not.N 12104) was cut at the pilin motif lysine as well as in the C-domain (Lys-372). Mishra et al [5] reported that FimA from A. oris strain T14V is similarly cut at the pilin lysine. This difference in sensitivity to trypsin implies that FimP is more rigid than FimA in which the pilin motif is more exposed. A structural comparison between FimP31?91 and FimA (M- and C-domains) revealed that the major differences between the two structures are the loops connecting the b-strands. The most prominent difference is the segments that pack against the S4 sheet of their respective M-domains. In FimA this constitutes a prolinerich segment (332PPTPETPPTDPENPP347) that runs diagonally over half the sheet. In FimP the equivalent segment is a long, coiled region, located between b18 and b19, that folds over the whole S4 sheet like a clamp (Fig. 7). Another distinct structural difference is the FimP metal-binding loop that protrudes from the C-domain like a knob. This loop has no equivalent in FimA. Such a significant structural element is likely to be available for recognition by certain microbes of the biofilm. In analogy, the surface adhesin SspB of the oral bacteria Streptococcus gordonii exhibits a surface-exposed helix, stabilized by a metal ion [37]. This helix is solely recognized by the periodontal pathogen Porphyromonas gingivalis [38]. Likewise, the proline-rich segment in FimA may function as a recognition site for other bacteria-bacteria interactions. However, to further unravel the complex mechanisms that control colonization and formation of the oral biofilm, extensive structural and functional studies have to be performed. Sequence comparison of the FimP and FimA N-domains indicates different positions of the disulfide bridges. In FimP the first cysteine of the N-domain is located directly after the presumed isopeptide forming lysine and participate in a disulfide bond that unites the ends of a flexible loop. Also in FimA, the presumed disulfide bond is located near the putative isopeptide bond but with a different organization where its second cysteine is located directly before the catalytic glutamic acid. When the FimA sequence is applied on the FimP template, the first cysteine is located in the coil after the two helices A and B and the second in the S2 sheet, next to the isopeptide bond glutamic acid, as discussed. Assuming that a disulfide bridge is formed also in FimA, we suggest that its S2 sheet is more occluded due to tighter packing with the N-domain helices. The overall appearance of the Ndomains may therefore be quite different, which indeed is reflected by their different sensitivity to trypsin. The structural differencesThe Pilin Motif is Located in a GrooveIn FimP, the lysine of the pilin motif (WNYNVHVYPK) is exposed on the hinge that connects the N- and M-domains, and is thereby available for the sortase mediated pili polymerization reaction (Fig. 3a, 4a and 5). The pilin lysine, Lys-182, is located at the end of a mostly non-polar groove formed between helix A, the b-sandwich, and the mobile loop of the N-domain. The groove is lined by the pilin-motif conserved residues Trp-73, Val-177, Val179, and Pro-181 with the pilin lysine Lys-182 located at the rim of the channel. The conserved Tyr-180 stacks with His-51 and forms a hydrogen bond with the main chain amide of Asp-69, located close to the mobile loop. The isopeptide triad (Lys-52, Asn-183, Glu-145) is positioned within the groove however the isopeptide bond is not.
