Figure 1.
2D [15N,1H] HSQC spectra of lipoprotein YxeF.
(A) Spectrum recorded for the sample used for NMR structure determination at 750 MHz 1H resonance frequency. Resonance assignments are indicated using the one-letter amino acid code. Signals arising from side chains (Asn Hδ2/Nδ2, Gln Hε2/Nε2, Arg Hε/Nε and Trp Hε1/Nε1) are labeled with (*) and folded signals are designated with (†) next to the residue number. Signals arising from the His purification tag were not sequence specifically assigned. The spectral region indicated by dotted lines comprises most of the signals arising from the β-barrel (Figure 2) and is displayed for the spectra shown in (B). Those were recorded at different temperatures at 500 MHz 1H resonance frequency (see text).
Figure 2.
NMR structure of the soluble domain of lipoprotein YxeF.
Only residues 34–132 are shown because the terminal polypeptide segments are flexibly disordered in solution. The N-terminal residue Phe 34 and the C-terminal residue Ser 132 are labeled. Lines point at the Ω-type loop, which connects β-strands A and B and is poorly defined in the NMR structure. (A) Ribbon drawing of the first conformer of PDB entry 2JOZ with β-strands being depicted in cyan. (B) Superposition of the 20 conformers representing the NMR solution structure obtained after superposition of the backbone heavy atoms (N, Cα and C’) of the β-strands. (C) “Sausage” representation of backbone and best-defined side chains: a spline function was drawn through the mean positions of Cα atoms with the thickness being proportional to their mean global displacement in the 20 conformers after superposition as in B). The figure was generated using the program MOLMOL [59].
Table 1.
Statistics of YxeF(19–144) NMR Structure.
Figure 3.
Comparison of B. subtilis YxeF NMR structure and B. amyloliquefaciens A7ZAF5 homology model.
Surface electrostatic potential calculated for (A) the YxeF NMR structure (first conformer of ensemble deposited in the PDB) and (B) the homology model of A7ZAF5 by using the program GRASP [56] accessed through the protein function annotation server MarkUs [55]. The homology model was calculated using the SWISS-MODEL server in alignment mode [60], [61] and Verify3D [63], Procheck [64] and ProsaII [65] all atom z-scores (-1.12, −3.43 and −1.61, respectively) were obtained using the PSVS server [66] and are indicative of a good quality model. In (C) and (D), ribbon drawings are shown for the structures of YxeF and A7ZAF5 in the same orientation, that is, viewed on the open end of the β-barrels. The acidic residues giving rise to the negative potential inside the cavities are depicted in licorice representation and are labeled (black for YxeF, red for A7ZAF5). (E) Pfam multiple alignment of the sequences of all members of PF11631. Except for YxeF (P54945), the sequences are labeled with their UniProt [25] IDs (D4G3V0, E8VFY0, E0TYE6, D5MWC1, E3E109, A7ZAF5, E1UTS8). Amino acid background colors reflect average similarity inferred from the Blosum62 matrix, ranging from ‘most conserved’ (black) to ‘least conserved’ (white). YxeF and A7ZAF5 are highlighted in bold on the left and the region of the alignment used for building the comparative model of A7ZAF5 from the YxeF structure is enclosed by red boxes. The acidic residues labeled in (C) and (D) are marked with black (YxeF) and red (A7ZAF5) asterisks, respectively, above or below the alignment.
Figure 4.
Schematic representation of secondary structure element topologies.
(A) YxeF, (B) lipocalins and (C) fatty acid-binding proteins. β-strands are represented by arrows, α-helices by rectangles, and 310-helices by ellipses. N- and C-termini are indicated as N and C respectively, and the ‘Ω-type’ loop L1 shared by YxeF and lipocalins is labeled.
Figure 5.
Comparison of YxeF NMR structure (PDB ID 2JOZ, coded in blue) and Blc X-ray crystal structure (PDB ID 3MBT, orange).
(A) Structure-based sequence alignment between YxeF and Blc obtained with the program DALI [16]. The three structurally conserved regions (SCR1-3) typically found in lipocalins (see text) are boxed (continuous line for SCR1, which appears to be conserved in YxeF; dashed line for SCR2 and SCR3). Conserved residues being part of the calycin signature motif resulting in an interaction between Gly 36-X-Trp 38 in SCR1 and Arg 128 in SCR3 (see text) are highlighted using red boxes. Residues being part of the second hydrophobic core of Blc [see also (D] are highlighted using cyan boxes. (B) Superposition of the Trp and Arg residues being part of the calycin Gly-X-Trp and Arg motif in Blc (licorice representation, orange) and YxeF (line representation, all NMR conformers, blue). The superposition is obtained after superposition of the X-ray structure of Blc with each conformer of the NMR solution structure of YxeF (residues 32–132). (C) Structural superposition generated by the program DALI viewed from the open end of the β-barrels (for YxeF residues 32–132 were considered). In Blc, box 1 identifies the C-terminally located α-helix and box 2 the C-terminal β-strand, which are packed against the outside of the β-barrel and thereby form a second hydrophobic core (see D). (D) Ribbon drawing of the Blc structure with licorice representation of hydrophobic residues (in cyan) located in the C-terminal α-helix and on the outside of the β-barrel forming a second hydrophobic core [see also (C)].
Table 2.
Comparison of β-barrels occurring in selected structuresa yielding top DALI Z-scores after full structure alignment with YxeF.
Figure 6.
Ribbon drawings of β-barrels of avidin (PDB ID 1AVD, green) in (A) and, after rotation by 180°, in (D); bacterial lipocalin Blc from E. coli (PDB ID 3MBT, orange) in (B) and (E); YxeF in (C) and (F) (PDB ID 2JOZ, blue). For clarity, the disordered terminal polypeptide segments of YxeF, as well as the corresponding segments in avidin and Blc, are not shown. In (A)–(C), β-strands A and H are labeled, while in (D)–(F) β-strand D is indicated.