Complex 3D microfluidic architectures formed by mechanically guided compressive buckling.

Microfluidic technologies have wide-ranging applications in chemical analysis systems, drug delivery platforms, and artificial vascular networks. This latter area is particularly relevant to 3D cell cultures, engineered tissues, and artificial organs, where volumetric capabilities in fluid distribution are essential. Existing schemes for fabricating 3D microfluidic structures are constrained in realizing desired layout designs, producing physiologically relevant microvascular structures, and/or integrating active electronic/optoelectronic/microelectromechanical components for sensing and actuation. This paper presents a guided assembly approach that bypasses these limitations to yield complex 3D microvascular structures from 2D precursors that exploit the full sophistication of 2D fabrication methods. The capabilities extend to feature sizes <5 μm, in extended arrays and with various embedded sensors and actuators, across wide ranges of overall dimensions, in a parallel, high-throughput process. Examples include 3D microvascular networks with sophisticated layouts, deterministically designed and constructed to expand the geometries and operating features of artificial vascular networks

Crystal Structures Reveal the Multi-Ligand Binding Mechanism of Staphylococcus aureus ClfB

Staphylococcus aureus (S. aureus) pathogenesis is a complex process involving a diverse array of extracellular and cell wall components. ClfB, an MSCRAMM (Microbial Surface Components Recognizing Adhesive Matrix Molecules) family surface protein, described as a fibrinogen-binding clumping factor, is a key determinant of S. aureus nasal colonization, but the molecular basis for ClfB-ligand recognition remains unknown. In this study, we solved the crystal structures of apo-ClfB and its complexes with fibrinogen α (Fg α) and cytokeratin 10 (CK10) peptides. Structural comparison revealed a conserved glycine-serine-rich (GSR) ClfB binding motif (GSSGXGXXG) within the ligands, which was also found in other human proteins such as Engrailed protein, TCF20 and Dermokine proteins. Interaction between Dermokine and ClfB was confirmed by subsequent binding assays. The crystal structure of ClfB complexed with a 15-residue peptide derived from Dermokine revealed the same peptide binding mode of ClfB as identified in the crystal structures of ClfB-Fg α and ClfB-CK10. The results presented here highlight the multi-ligand binding property of ClfB, which is very distinct from other characterized MSCRAMMs to-date. The adherence of multiple peptides carrying the GSR motif into the same pocket in ClfB is reminiscent of MHC molecules. Our results provide a template for the identification of other molecules targeted by S. aureus during its colonization and infection. We propose that other MSCRAMMs like ClfA and SdrG also possess multi-ligand binding properties

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

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

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