Med with the N-domain alone and with the M- and C-domains together gave the same hit. The N-domain search resulted in a MedChemExpress 38916-34-6 Z-score of 14.9 and an root meanTable 2. Mass determination of wild type and mutant FimP31?91 proteins.ProteinAverage mass (Da) Calculated from sequence Observed by MS 53033 53006aDifference (Da) calculated-observed 234 217FimP31?91 FimP31?91 D230A FimP31?91 E452Aa53067 53023MS, ESI-TOF mass spectrometry. doi:10.1371/journal.pone.0048364.tFimP Structure and Sequence Analyses?square deviation (RMSD) of 1.9 A on 115 aligned Ca residues compared to SpaA. When the M- and C-domains were used, 
the ?Z-score was 21.6 and the RMSD 3.3 A on 266 aligned Ca atoms. SpaA and and FimP31?91 are very similar in structure and share the same topology of the b-sheets. The SpaA and the FimP31?91 structures also share the positions of the isopeptide bonds in the M- and C-domains. The differences between the two structures are mainly the positions of the bound metal ions. Their respective structural metal ion is located in similar, but not identical, areas of the molecules close to the interface between the M- and Cdomains, but coordinated by loops from different domains. In FimP, the metal-coordinating loop belongs to the C-domain and protrudes from the core of the molecule, whereas the 1485-00-3 metalbinding loop in SpaA originates from the M-domain and is more involved in domain-domain interactions. Both FimP and SpaA have a disulfide bond in their respective C-domain, located in similar positions, however the presence of a disulfide bond in the N-domain is unique for FimP. Neither FimP nor SpaA has an isopeptide bond in the N-domain, although FimP has all the residues needed to form such a bond and SpaA does not. Other DALI hits, with somewhat lower Z-scores, were the C-terminal fragment of FimA from A. oris (PDB code 3QDH, Z-score, 19.9 [5]) that could be superposed onto FimP31?91 with an RMSD of ?2.8 A on 235 aligned Ca atoms. Similarly, the pilus protein GBS80 from S. agalactiae can be superimposed on 
the FimP with an ?RMSD of 3.2 A on 246 aligned Ca atoms (PDB code 3PF2, Zscore 15.9 [13]). These are all Gram-positive pilin proteins consisting of three IgG-like domain (only M- and C domains are present in the models of FimA and GBS80).In the quest for new drug candidates against pili-bearing Grampositive bacteria, the N-domain groove may constitute a target for the development of pili inhibiting peptides or chemicals, with the sorting signal as the original template.Structural Comparison with FimAActinomyces spp can express two different forms of pili, encoded by separate gene clusters. The respective shaft proteins, FimP and FimA, are similar in terms of size and sequence. FimP consists of 533 residues and FimA of 535. A sequence analysis reveals that the major features; the pilin motif, the number of cysteines, the residues involved in isopeptide bond formation and the LPXTG motif are strictly conserved. Thus FimP and FimA are expected to have very similar structures. On the other hand, some differences are expected to be found, e.g., in 16574785 the organization of loops and metal binding (Fig. 6). Limited proteolysis experiments digesting FimA and FimP with trypsin gave different results which indicate some differences in exposure of certain regions (data not shown). Trypsin treatment of FimP generated a cut at Lys-62, located in the mobile loop of the N-domain whereas FimA (from A. naeslundii strain 12104) was cut at the pilin motif lysine as well.Med with the N-domain alone and with the M- and C-domains together gave the same hit. The N-domain search resulted in a Z-score of 14.9 and an root meanTable 2. Mass determination of wild type and mutant FimP31?91 proteins.ProteinAverage mass (Da) Calculated from sequence Observed by MS 53033 53006aDifference (Da) calculated-observed 234 217FimP31?91 FimP31?91 D230A FimP31?91 E452Aa53067 53023MS, ESI-TOF mass spectrometry. doi:10.1371/journal.pone.0048364.tFimP Structure and Sequence Analyses?square deviation (RMSD) of 1.9 A on 115 aligned Ca residues compared to SpaA. When the M- and C-domains were used, the ?Z-score was 21.6 and the RMSD 3.3 A on 266 aligned Ca atoms. SpaA and and FimP31?91 are very similar in structure and share the same topology of the b-sheets. The SpaA and the FimP31?91 structures also share the positions of the isopeptide bonds in the M- and C-domains. The differences between the two structures are mainly the positions of the bound metal ions. Their respective structural metal ion is located in similar, but not identical, areas of the molecules close to the interface between the M- and Cdomains, but coordinated by loops from different domains. In FimP, the metal-coordinating loop belongs to the C-domain and protrudes from the core of the molecule, whereas the metalbinding loop in SpaA originates from the M-domain and is more involved in domain-domain interactions. Both FimP and SpaA have a disulfide bond in their respective C-domain, located in similar positions, however the presence of a disulfide bond in the N-domain is unique for FimP. Neither FimP nor SpaA has an isopeptide bond in the N-domain, although FimP has all the residues needed to form such a bond and SpaA does not. Other DALI hits, with somewhat lower Z-scores, were the C-terminal fragment of FimA from A. oris (PDB code 3QDH, Z-score, 19.9 [5]) that could be superposed onto FimP31?91 with an RMSD of ?2.8 A on 235 aligned Ca atoms. Similarly, the pilus protein GBS80 from S. agalactiae can be superimposed on the FimP with an ?RMSD of 3.2 A on 246 aligned Ca atoms (PDB code 3PF2, Zscore 15.9 [13]). These are all Gram-positive pilin proteins consisting of three IgG-like domain (only M- and C domains are present in the models of FimA and GBS80).In the quest for new drug candidates against pili-bearing Grampositive bacteria, the N-domain groove may constitute a target for the development of pili inhibiting peptides or chemicals, with the sorting signal as the original template.Structural Comparison with FimAActinomyces spp can express two different forms of pili, encoded by separate gene clusters. The respective shaft proteins, FimP and FimA, are similar in terms of size and sequence. FimP consists of 533 residues and FimA of 535. A sequence analysis reveals that the major features; the pilin motif, the number of cysteines, the residues involved in isopeptide bond formation and the LPXTG motif are strictly conserved. Thus FimP and FimA are expected to have very similar structures. On the other hand, some differences are expected to be found, e.g., in 16574785 the organization of loops and metal binding (Fig. 6). Limited proteolysis experiments digesting FimA and FimP with trypsin gave different results which indicate some differences in exposure of certain regions (data not shown). Trypsin treatment of FimP generated a cut at Lys-62, located in the mobile loop of the N-domain whereas FimA (from A. naeslundii strain 12104) was cut at the pilin motif lysine as well.
