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. 2015 Jun 8;209(5):739–758. doi: 10.1083/jcb.201411024

Figure 5.

Figure 5.

HHIP1 directly binds to HS through the N-terminal CRD. (A) Representative structural model of the HHIP1 C-terminal 30 residues shown as ribbons (left) and electrostatic potential (right). Four N-terminal arginines are highlighted (dotted circle). Electrostatic potential is calculated from −10 kbT/ec (red, acidic) to 10 kbT/ec (blue, basic). Selected residues are depicted in stick representation. (B, top) Cartoon depiction of HA::HHIP1. (bottom) Sequence analysis identifies a cluster of basic residues (blue) in the C-terminal 30 amino acids that were mutagenized to alanines (orange) to generate HHIP1C4R-4A. SP, signal peptide. (C–E) Heparin-agarose chromatography was used to measure heparin-binding affinities for HA::HHIP1 (C–E), HA::HHIP1ΔC30 (C) HA::HHIP1C4R->4A (D), and HA::HHIP1ΔCRD (E). NaCl concentrations (in millimolars) corresponding to elution peaks are indicated above each curve. The data shown are representative experiments from at least three replicates for each construct. (F–I) Representative SPR binding experiments demonstrating direct interactions between HHIP1(18–670) (F and H) or HHIP1(212–670) (F and H) with Heparin (F and G) and HS (H and I). (C–I) Cartoon depictions of each construct are presented above each dataset. Each SPR analysis is a representative experiment from at least three replicates per condition. (J) Representative model of the HHIP1 CRD. (left) Ribbon representation in rainbow coloring from blue (N terminus) to red (C terminus). Potential disulphide bridges are shown in Roman numerals. (right) Electrostatic potential from −10 kbT/ec (red, acidic) to 10 kbT/ec (blue, basic). The dotted box highlights the amino acids represented in K. (K) Close-up view of the potential HHIP1-CRD HS binding site shown in stick representation in atomic coloring (cyan, carbon; blue, nitrogen; red, oxygen; yellow, sulfur).