Structural aligment of ClfB, ClfA and SdrG.

<p>A. Sequence aligment of ClfB (amino acids 212–550), ClfA (amino acids 229–544) and SdrG (amino acids 117–441). Residues displaying 100% and 50% identity are shown in dark blue and light blue, respectively. F406 in ClfB is marked by red star. B. Ribbon representation of ClfB, with conserved residues colored from red to green following the order from highly conserved to less conserved. C. Superimposition of apo-ClfB and apo-SdrG, colored in orange and cyan, respectively. D. Superimposition of ClfB-Fg α, SdrG-Fg β and ClfA-Fg γ complexes. The SdrG-Fg β and ClfB-Fg α are colored as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002751#ppat-1002751-g003" target="_blank">Figure 3C</a>. The ClfA-Fg γ complex is colored in blue.</p

Dermokine is a potential ligand of ClfB.

<p>A. Left: Surface plasmon resonance shows the binding of different concentrations of ClfB_(197–542) to synthetic peptide 15 from Dermokine immobilized on a Proteon NLC Sensor Chip. Red, 700 µM; blue, 350 µM; green, 87.5 µM. K_D was found to be 2.37 µM. Right: kinetic and affinity binding values of the ClfB_(197–542) wildtype, S236A or W522A single mutants with Derm15 peptide. B. Comparative close-up view of CK10, Fg α and Derm15 binding to ClfB. The peptides CK10, Fg α and Derm15 are shown as sticks and colored in yellow, slate and green, respectively. The N and C termini are marked. The color schemes of the N2 and N3 domain of ClfB are the same as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002751#ppat-1002751-g001" target="_blank">Figure 1B</a>. C. Closer view of the superimposition of apo-ClfB and ligand-ClfB complexes. N238 and R529 are highlighted and shown as sticks. The apo-ClfB is colored in lime and the others are in the same color scheme as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002751#ppat-1002751-g001" target="_blank">Figure 1B</a>.</p

Detailed interactions between the ligand binding of ClfB in the ClfB-CK10/Fg α complexes.

<p>A. Detailed interactions between the ligand and ClfB in the ClfB-CK10 complex. The ClfB and CK10 peptides are shown as sticks, colored in magenta and yellow, respectively. The hydrogen bonds are indicated by red dashed lines. The amino acids of ClfB and CK10 are marked with black and red characters, respectively. B. Detailed interactions between the ligand and ClfB in the ClfB-Fg α complex. The ClfB and Fg α peptides are shown as sticks, colored in cyan and slate, respectively. The hydrogen bonds are indicated by red dashed lines. The amino acids of ClfB and Fg α are marked with black and red characters, respectively.</p

The ClfB-ligand binding is consistent with the DLL model.

<p>A. Ribbon representation of the superimposition of apo-ClfB, ClfB-Fg α and ClfB-CK10 complexes. The apo-ClfB is shown in limon. In the ClfB-Fg α complex, the protein and Fg α are shown in magenta and marine, respectively. In the ClfB-CK10 complex, ClfB and CK10 are shown in orange and yellow, respectively. B. Closer view of the interactions between the C-terminal G′ strand of N3 domain and N2 domain in the ClfB-Fg α complex. Residues involved in the interactions from both G′ strand and N2 domain are shown as sticks and marked by blue and black characters, respectively. The Fg α peptide is shown as sticks in yellow. C. Structural alignment of the ClfB-Fg α complex solved in this study and the one by V.Ganesh et al. (PDB entry: 3AT0) shown in stereo view, with RMSD 0.635 Å. The ClfB-Fg α complex in this study is shown in orange and the peptide is shown as sticks in the same color. The ClfB in the corresponding structure by V.Ganesh et al. is shown in cyan and Fg α is shown as sticks with its N and C termini marked. The C termini of ClfB in both structures and the G′ strand of ClfB in the current study are indicated. D. Structural alignment of the ClfB-CK10 complex solved in this study and the one by V.Ganesh et al. (PDB entry: 3ASW), with RMSD 0.479 Å. The ClfB-CK10 complex in this study is shown in marine and the peptide is shown as sticks with its N and C termini marked. The ClfB in the corresponding structure by V.Ganesh et al. is shown in lime and CK10 is shown as sticks. The C-termini of ClfB in both structures are indicated.</p

The SPR analysis of the interactions between ClfB and dermokine peptides.

<p>A. Panel of dermokine peptides. Peptide 1 corresponds to the 9-residue peptide derived from dermokine protein (253–261). The substituted peptides (Peptides 2–10) have individual amino acids replaced with Ala and peptide 11 is the six amino acid peptide. (B–L). The SPR analysis of the binding between ClfB and Peptides 1–11. Navy, 2000 µM; Magenta, 1000 µM; Dark cyan or dark yellow, 500 µM; Blue, 250 µM; Red, 125 µM; Green, 62.5 µM; Black, 31.25 µM. K_D values of individual binding assays are indicated below the sensorgrams.</p

Mechanisms of specifically recognizing repeat 5 of Fg α.

<p>A. Superimposition of the Fg α and CK10 peptides. The Fg α and CK10 peptides are shown as sticks, colored in yellow and slate, respectively. Residues highlighted within the boundaries of the red dashed line constitute the segment important for ClfB binding. The consensus amino acids are shown above the peptides. B. Sequence alignment of the repeat 2, 3, 4 and 5 of the Fg α, CK10 (type I cytokeratin 10, residues 473–485 and residues 499–511), K10 (Keratin 10, type I cytoskeletal 10 isoform-1 from <i>Pan troglodytes</i>, residues 501–513), Derm (Dermokine, residues 250–264), TCF20 (TCF20, residues 49–57), EN (Engrailed protein, residues 37–45) and the derived peptide 9. The conserved amino acids are shown in red and the consensus sequence is designated below the sequences. The repeat 2, 3 and 4 of the Fg α which have been proved cannot bind to ClfB are indicated in skyblue.</p

Statistics of data collection and structure refinement.

+<p>Values in parentheses are for the highest resolution shell.</p>*<p><i>R</i>_sym = Σ_hΣ_i|<i>I_h,i</i>−<i>I_h</i>|/Σ_hΣ_i<i>I_h,i</i>, where <i>I_h</i> is the mean intensity of the <i>i</i> observations of symmetry related reflections of <i>h</i>.</p>#<p><i>R</i> = Σ|<i>F_obs</i>−<i>F_calc</i>|/Σ<i>F_obs</i>, where <i>F_calc</i> is the calculated protein structure factor from the atomic model (R_free was calculated with 5% of the reflections).</p

Crystal structure of apo-ClfB_(197–542).

<p>A. Domain organization of ClfB. The numbers of the amino acid residues identifying the boundaries between adjacent domains are indicated below. S, signal sequence; N1-3, N-terminal fibrinogen binding region; R, serine-aspartate repeat region; W, wall-spanning domain; M, membrane anchor; C, cytoplasmic positively charged tail. The N2 and N3 domains were used in crystallization of the ClfB_(197–542)-peptide complexes. B. Ribbon representation of the structure of apo-ClfB_(197–542), with its N and C terminus indicated. The N2 and N3 domains are shown in orange and magenta, respectively. The strands and loops are marked. C. Ribbon representation of the two symmetry-related molecules in the unit cell, shown in orange and magenta, respectively. The N and C termini of both molecules are indicated. D. Closer view of the interaction between the two symmetry-related molecules. The N-terminus of one molecule (amino acids 196–201) is shown as sticks and the other one is colored in magenta as in (B). The amino acids from both molecules are marked in red and black characters, respectively. The hydrogen bonds are shown as red dashed lines.</p

Target Elucidation by Cocrystal Structures of NADH-Ubiquinone Oxidoreductase of <i>Plasmodium falciparum</i> (<i>Pf</i>NDH2) with Small Molecule To Eliminate Drug-Resistant Malaria

Drug-resistant malarial strains have been continuously emerging recently, which posts a great challenge for the global health. Therefore, new antimalarial drugs with novel targeting mechanisms are urgently needed for fighting drug-resistant malaria. NADH-ubiquinone oxidoreductase of <i>Plasmodium falciparum</i> (<i>Pf</i>NDH2) represents a viable target for antimalarial drug development. However, the absence of structural information on <i>Pf</i>NDH2 limited rational drug design and further development. Herein, we report high resolution crystal structures of the <i>Pf</i>NDH2 protein for the first time in Apo-, NADH-, and RYL-552 (a new inhibitor)-bound states. The <i>Pf</i>NDH2 inhibitor exhibits excellent potency against both drug-resistant strains in vitro and parasite-infected mice in vivo via a potential allosteric mechanism. Furthermore, it was found that the inhibitor can be used in combination with dihydroartemisinin (DHA) synergistically. These findings not only are important for malarial <i>Pf</i>NDH2 protein-based drug development but could also have broad implications for other NDH2-containing pathogenic microorganisms such as <i>Mycobacterium tuberculosis</i>