The E2 domains of APP and APLP1 have similar dimeric structures. (A) The domain structure of APP. The residue numbers shown above the diagram are according to the common APP isoform-751. (B) The structure of the E2 domain of APP in crystal form “B”. Helix αA is disordered in the crystal lattice. The two protein protomers are shown in different colors. Helices are shown as cylinders. The two subdomains (N, C) are also labeled. (C) The structure of the E2 domain of APLP1. In the crystal, helix αA is domain swapped. Shown here are the corresponding helices from neighboring molecules (the loop connecting αA to αB is represented by the dashed line). These illustrations and those in Fig. 3C and Fig. 5 were generated by PyMOL.

Differences at the dimeric interface. (A) Comparison between the two APP crystal forms. The new structure (black) is superimposed onto αD and αE of the original structure (red; PDB entry 1rw6). The helices that “moved” correspond to αB and αC of the second protomer across the dimeric interface. The Cα-traces are shown in stereo pairs. Grey circle, Leu-490*; black circle, Leu-515*; white circle, Met-387; green circle, Met-391; blue circle, Trp-394. αB and αC appear to pivot around Met-387. (B) Comparison between APP (red; the original structure 1rw6) and APLP1 (black). Grey circle, Leu-428*; black circle, Leu-453*; white circle, Ile-325; green circle, Met-329; blue circle, Trp-332. (C) A side view of the APLP1 dimer. The arrow indicates the “rotation” of αB/αC relative to αD/αE across the dimeric interface in APLP1 when compared to APP. The grey box indicates the region shown in A and B. (D) In this model, each subdomain is represented by a single oval-shaped object. Within each protomer, the two subdomains are connected at point “a” (grey circle), and can rotate about it (dashed lines). The dimer is held together by two joints (white circles; “b”). The two subdomains across the dimeric interface can only rotate about “b” (dashed lines).

The phosphate binding sites. (A) The P1 and P2 binding sites. The dashed lines represent hydrogen bonds. Red spheres represent water molecules. The two protein protomers are shown in different colors. (B) A similar view of the monomeric structure of C. elegans APL-1 in complex with sucrose octasulfate (25).

The heparin-binding mutants show decreased dimerization. (A) R369A. (B) R429A. (C) H433A. These experiments were conducted under exactly the same conditions as that for the wildtype protein (Fig. 4G). Filled circles, mutant protein alone; open circles, protein plus heparin.

Differences at the tertiary structural level. (A) Comparison between the two APP crystal forms. The new structure (black) is superimposed onto the C-terminal subdomain of the original APP structure (red; PDB entry 1rw6). The Cα-traces are shown in stereo pairs. (B) Comparison between APP (red; the original structure 1rw6) and APLP1 (blue and black). The two copies of APLP1 in the asymmetric unit are slightly different in their inter-subdomain angles. These images and those in Fig. 3A-B were generated by Molscript (48).

Heparin binding enhances E2 dimerization. (A) van Holde-Weischet g(s) graphs for the E2 domain of APLP1 for a range of protein concentrations (red, 3.2μM; green, 6.6μM; blue, 32μM; black, 66μM). When the protein concentration was raised from 3.2μM (0.31 OD at 230nm) to 66μM (0.76 OD at 280nm), the major peak gradually shifted from 2.2 S to 2.9 S. This result is due to the rapid exchange between monomers (24.9 kDa) and dimers (49.8 kDa), and was confirmed by fitting the data to a reversible monomer-dimer model (42, 43). This observation suggests that reversible dimerization is driven by mass action even in the absence of ligand. (B)-(F), Sedimentation velocity experiments of the E2 domains of APLP1 and APP in the presence and absence of heparin. Integral van Holde-Weischet s-value distributions (B: grey, APLP1; blue, APLP1+100μM heparin; red, APP; green, APP+100μM heparin) and genetic algorithm-Monte Carlo results (C: APLP1, D: APLP1+100μM heparin, E: APP, F: APP+100μM heparin) reveal enhanced dimerization when heparin is present. C-F: The y-axis shows the frictional ratio (f/f₀), which measures the globularity of the solute. An f/f₀ value of unity corresponds to a spherical molecule. The color gradients indicate optical density at 230nm. For clarity, only the monomer-dimer molecular weight range is shown in C-F. The full sedimentation range is shown in B. The protein loading concentration is approximately 3μM in all experiments shown in panels B-F. (G) Heparin binding causes a decrease of APLP1 E2 fluorescence intensity. Filled circles, protein alone (3μM); open circles, protein plus heparin (6μM). (H) A model where the heparin-induced dimer does not have a protein:protein interface. Additional ligand may dissociate the dimer by competing for the ligand binding site. Oval, protein; grey bar, ligand. (I) In this model the dimer is strengthened by protein:protein contact and by additional interactions between heparin (represented by the grey triangles) and the protein protomer across the dimeric interface (small indentation). Ligand may also change the conformation of the protein (oval to rectangle) to facilitate the formation of a protein:protein interface.

Mutational mapping of the heparin binding site. (A) A surface representation of the heparin binding site mapped by mutagenesis. The two protein protomers are shown in different colors. (B) The elution profiles of wild-type and mutant proteins from a heparin column. The dashed line represents salt gradient.