Figure 1.
Anomalous properties of darcin compared to central MUPs.
The peripheral MUP darcin (molecular weight 18893 Da) exhibits high mobility on SDS-PAGE compared to other urinary MUPs (molecular weight 18645–18708 Da). (A) Under non-reducing conditions, darcin (labelled) is readily resolvable from other urinary MUPs (major band). On reducing SDS-PAGE, darcin and other MUPs migrate more slowly, although the effect is much less pronounced for darcin. This is consistent with darcin retaining a more compact structure under reducing gel conditions. Urine samples were analysed from males of two mouse strains, C57BL/6, which express darcin, and BALB/c, which do not express darcin. (B) The same behaviour is evident for three recombinant MUPs, darcin, central MUP7 and central MUP11, with Darcin travelling further on the gel than other MUPs. (C) Darcin also exhibits anomalous behaviour under conditions of electrospray ionisation mass spectrometry. Compared to three central MUPs, darcin exhibits a different distribution of multiply protonated ions, tending to a lower charge state, despite having the same number of protonatable sites. This distribution of charge states may reflect the lower accessibility of some basic sites because darcin retains a degree of structure in the gas phase.
Figure 2.
Solution structure of darcin (left) and MUP11 (right).
For clarity 180° representations are shown. (A) The ensembles each comprise 20 lowest-energy models. (B) For each ensemble a representative closest-to-mean structure was selected and shown as a cartoon representation of the structural elements. Marked in asterisk is the conserved 310-helix between α1 and β9. (C) Alignment of the primary sequence of darcin (top) with MUP11 (bottom) with conserved residues highlighted in yellow. The structural schematic for darcin is coloured to correlate with the colouring on the cartoon representation shown in (B), from N to C terminus as blue to red. In (C), the S-S bridge between C64 and C157 is indicated as black lines linking the two residues.
Table 1.
Structural statistics for the refined NMR structures of darcin and MUP11.
Table 2.
Structural features of Darcin and MUP11(a).
Figure 3.
Schematic of MUP beta-barrel and inter-strand loop arrangement.
Top left: top-down view of the beta barrel. Loops at the top, N-terminal end of the barrel are highlighted in green and magenta. Top right: bottom-up (C-terminal end,) view of the beta barrel. Loops at the bottom, C terminal end of the barrel are highlighted in blue and tan. Bottom: Alignment of darcin and MUP11 sequences with paired loop residues used to measure inter-loop distances highlighted, each residue pairs are coloured green, magenta, tan and blue in accordance with the schematic views.
Table 3.
Closest opposing inter-loop distances (in Å) in Darcin and MUP11(a).
Figure 4.
Variation in surface amino acids between darcin and MUP11.
Darcin (mauve) (A) and MUP11 (orange) (B) are shown in the same orientations. Non-conserved surface exposed residue side-chains are shown as stick representations and shaded cyan (darcin) and red (MUP11). Only variations of residues that do not confer similar properties (polar, hydrophobic, charged, aromatic etc.) are shown, as Patches 1, 2 and 3 (see text). For clarity hydrogen atoms are omitted from the stick-representations of the residues shown.
Figure 5.
Comparison of binding cavities of darcin and MUP11.
Left: Overlay of binding residues of darcin (mauve) and MUP11 (orange), where the differing amino acid residues in both darcin/MUP11 are labelled. Right: schematic of residues highlighted as part of the SBT binding site, conserved residues between darcin and MUP11 are green and variable residues are coloured red with the darcin residue only indicated. Bottom: Aligned sequences with SBT binding residues highlighted using the same colour scheme as above (conserved = green; variable = red), secondary structure schematic is aligned below the sequences with identical colour scheme to Fig. 2.
Figure 6.
Isothermal titration calorimetry curves.
Plots showing 2-sec-butyl thiazole (SBT) binding to darcin and MUP11 in 25 mM PO43−, 25 mM NaCl, 298 K curve fit to a one-site model. (A) Darcin binds SBT with N (stoichiometry ratio) = 1.0, KD∼0.173 µM, ΔH = ∼ -13.1 kcal/mol and TΔS = ∼3.9 kcal/mol. (B) MUP11 binds SBT with N (stoichiometry ratio) = 1.0, KD ∼2.76 µM, ΔH ∼-9.8 kcal/mol and TΔS = ∼ 2.2 kcal/mol.
Figure 7.
A binding cavity consensus was determined based on the active ligands identified by LigPLOT [49] and PDBePISA [48] in over 50% of the complex structures and shown mapped (in yellow) onto darcin (2L9C) and MUP11 (2LB6) (top two sequences). All ligand:MUP complexes available in the PDB are analysed. Residues identified as part of the binding site are highlighted according to ligand type: aromatic/pyrazole ligand (blue), aliphatic/non-cyclic molecule (orange).
Figure 8.
Ligand binding analysis by NMR.
Histogram of chemical shift perturbations induced in darcin (blue) and MUP11 (orange) in the presence of at least five molar excess of SBT (A) K3 (B) and NPN (C). The secondary structure elements are represented by the schematic of darcin along the top of each plot, colour-coded as shown in Figure 2. All three ligands induced similar profiles of chemical shift changes with the most significant shift changes being observed for residues that form part of the pheromone-binding hydrophobic cavity. The exception being darcin with NPN which did not exhibit any combined backbone NH chemical shift changes above the cut-off threshold of Δδ = 0.15. Differences in the shift changes between the different complexes may be attributed to differences in affinities and/or residue composition of the binding cavity. The chemical shift perturbations (CSP) for non-overlapped resonances are calculated using the equation Δδ = {(ΔH)2 + (0.15ΔN)2}1/2 where ΔH and ΔN are, respectively, shift changes in the 1H and 15N dimensions.
Figure 9.
Distance between residues at the centre of the cavity.
Cartoon representation of darcin (mauve) and MUP11 (orange) with selected residues at the centre of the beta barrel. Closest distance (in Å) between non-hydrogen atoms is measured between amino acids L54 Cδ1 and I103 Cδ1 (A), L69 Cδ1 and E118 Cδ (B) and T82 Cγ1 and E118 Cδ (C) for darcin and L54 Cδ1 and A103 Cβ (A), M69 Cε and G118 Cα (B) and T82 Cγ1 and G118 Cα (C) for MUP11. The combined effect of these residues on the narrowness/restriction at the centre of the barrel is shown in (D).
Figure 10.
Urea denaturation of darcin and MUP11.
(Top) Schematic of secondary structures; loops (L), beta strands (B), and alpha helix (H) of darcin and MUP11. (Middle and Bottom) Plots of % of native backbone NH peaks observed at different urea concentrations for each secondary structure element in darcin (middle) and MUP11 (bottom); secondary structures are coloured coded as in the schematic. In darcin, the only region that is destabilised by urea is L8.