Figure 1.
Schematic Diagram Illustrating the Processes of DSC and DSE
In this diagram, β-strands and helices are represented as arrows and cylinders, respectively. At left, a topology diagram of a subunit bound to the chaperone is shown in pink. The Nte is shown as a disordered region at the N-terminus of strand A1. In the chaperone, only the G1 strand is shown in gray: it complements the fold of the subunit by inserting between strands A2 and F, providing in trans the missing G strand. Note that the complementing G1 strand runs parallel to strand F, thereby reconstituting an atypical Ig-fold for the subunit. The donor–strand-complemented subunit is referred to in the text as “dsc”. At right, the same subunit in pink is shown in the pilus. Its Nte is now inserted between strand A2 and F of the subunit in green. Its groove is now filled with the Nte of the subsequent subunit in dark blue. The subunit is referred to in the text as “dse”.
Figure 2.
Crystal Structure of PapD/(PapANtd1_G15N_T101L)2
Overall 3-D structure of the PapD/(PapANtd1_G15N_T101L)2 complex: PapD (yellow), dscPapA (purple), and dsePapA (orange) are shown in ribbon representation with β-strand indicated as arrows and α-helices indicated as cylinders. The β-strands of dsePapA are labeled, as are the PapD G1 strand and the dscPapA Nte. Figures 2–5 were made using PyMol (http://www.pymol.org).
Figure 3.
Comparison of dsePapA and dscPapA in the PapD/(PapANtd1_G15N_T101L)2 Structure
(A) Stereo ribbon diagram of dsePapA (orange) and dscPapA (purple). The N- and C-termini of both PapA molecules are indicated, as well as the “63–74” loop in dsePapA. In dscPapA, this loop is missing residues 70–73; thus, residues 69 and 74 are labeled.
(B) Surface representation of dsePapA (left) and dscPapA (right). Residues of the chaperone G1 strand as well as dscPapA Nte are shown in stick representation color-coded in red, dark blue, and cyan for oxygen, nitrogen, and carbon atoms, respectively. For clarity, only side chains of the residues interacting in the P1 to P4 (right) or P2 to P5 (left) pockets are shown.
Figure 4.
Comparison of PapD/PapANtd1_G15N and PapD/PapANtd1_G15N_T101L
(A) Surface representation of dscPapA in PapD/PapANtd1_G15N (left) and dsePapA in PapD/(PapANtd1_G15N_T101L)2 (right). The residues in the groove at the P4 position (F152 [purple and red in left and right panels, respectively]) and the one at the P5 position (T101 [purple] or L101 [red]) are indicated.
(B) Stereo view of a superimposition of the residues involved in DSE at the P4 and P5 positions. The green residues are N101 (PapD), F152, T101, and T99 (dscPapA in PapD/PapANtd1_G15N), whereas the gray residues are N15 (PapA Nte), F152, L101, and T99 (dsePapA in PapD/(PapANtd1_G15N_T101L)2).
Figure 5.
Comparison of dsePapA (in PapD/(PapANtd1_G15N_T101L)2) and dsePapE (in PapE/KNte)
(A) Surface representation of dsePapA (left) and dsePapE (right). Residues of PapKNte as well as dscPapA Nte are shown in stick representation color-coded in red, dark blue, and cyan for oxygen, nitrogen, and carbon atoms, respectively. For clarity, only side chains of the residues interacting in the P2 to P5 pockets are shown.
(B) Secondary structure representation of dsePapA (orange) and dsePapE (cyan) bound to KNte (green). The structures are in stereo ribbon representation. The N- and C-termini of both molecules are indicated, as well as the “63–74” loop in dsePapA.
Figure 6.
Gel Filtration Profiles and EM pictures of PapD/PapA Wild-Type (wt) and Mutant Complexes
Each gel filtration profile of a particular PapA construct is shown with a view of its corresponding EM picture on the left. The complex is labeled on the right. Only the gel filtration peaks corresponding to free PapD, PapD/(PapA)1 (1/1) or PapD/(PapA)2 (1/2) are labeled. Representative pili are labeled with arrows ([A–C]). Non-pilus polymerization of protein is labeled with arrowheads ([D] and [F]). The scale of all the EM pictures is represented by the black bar on the PapD/PapAwt picture and corresponds to 100 Å.