Crystal structure at 2.8 A of the DLLRKN-containing coiled-coil domain of huntingtin-interacting protein 1 (HIP1) reveals a surface suitable for clathrin light chain binding

Abstract

Huntingtin interacting protein 1 (HIP1) is a member of a family of proteins whose interaction with Huntingtin is critical to prevent cells from initiating apoptosis. HIP1, and related protein HIP12/1R, can also bind to clathrin and membrane phospholipids, and HIP12/1R links the CCV to the actin cytoskeleton. HIP1 and HIP12/1R interact with the clathrin light chain EED regulatory site and stimulate clathrin lattice assembly. Here, we report the X-ray structure of the coiled-coil domain of HIP1 (residues 482-586) that includes residues crucial for binding clathrin light chain. The dimeric HIP1 crystal structure is partially splayed open. The comparison of the HIP1 model with coiled-coil predictions revealed the heptad repeat in the dimeric trunk (S2 path) is offset relative to the register of the heptad repeat from the N-terminal portion (S1 path) of the molecule. Furthermore, surface analysis showed there is a third hydrophobic path (S3) running parallel with S1 and S2. We present structural evidence supporting a role for the S3 path as an interaction surface for clathrin light chain. Finally, comparative analysis suggests the mode of binding between sla2p and clathrin light chain may be different in yeast.

Figure 1

Topology of the human HIP1 482–586 sub-fragment and domain map of full-length HIP1 and HIP12/1R. a, Helix 1 of the “Y” shaped HIP1 482–586 homodimer shows side chains (magenta), while Helix 2 shows only the backbone (grey). The N and C designate the N- and C-termini of the parallel dimer model. Helix 1 and Helix 2 in the trunk region are coiled together but start to separate a 1/3 of the way towards the N-terminus. b, The left-handed coiled-coil twist is evident when the molecule is rotated 90 degrees from the view in a and shows 2 nodes or points of intersection ~70 Å apart, which is typical of coiled-coil proteins . Structural images were prepared using PyMol (http://www.pymol.org). c, Domain organization of HIP1 and related protein, HIP12/1R (numbering from human HIP1 and HIP12/1R is used). ANTH domain (residues 47–159 in HIP1; 39–150 in HIP12/1R) recognizes inositol phospholipids , , AP2 binding sites in HIP1 only (residues 262–266 and 358–360 3, 20), CLTD region (residues 332–336, LMDMD in HIP1 8) binds the N-terminal domain of clathrin, pDED is a pseudo-death effector domain that can bind HIPPI (residues 410–491 in HIP1; 393–470 in HIP12/1R 1, 21), LC is the clathrin light chain binding site (₄₈₄DLLRKN in HIP1; ₄₆₃ELLRKN in HIP12/1R 5), USH is an upstream regulatory helix, which modulates affinity for actin (residues 780–805 in both HIP1 and HIP12/1R 17), and an F-actin binding region (residues 813–1011 in HIP1 and HIP12/1R containing the actin-binding I/LWEQ motif 22). The Huntingtin binding site in HIP1 is indicated (residues 245–631) . The yellow bar indicates the position of the reported HIP1 482–586 crystal structure. Methods: HIP1 482–586 with an N-terminal GST tag was created by PCR mutagenesis starting with a construct of human HIP1 sub-fragment 371–645 inserted into pGEX-2T (pGST-HIP1h) (pGST-HIP1h was kindly provided by Valerie Legendre-Guillemin and Peter McPherson). Hip1 482–586 was over-expressed at 37°C in Rosetta 2 (DE3) pLysS bacterial cells (Novagen) in M9 minimal medium. After 10 hours of growth (OD₆₀₀ ~0.5), selenomethionine was added as previously described and IPTG (100 μg/ml, final concentration) was added to induce protein expression. Cells were then incubated for 15 hours at 30°C before being harvested and flash frozen for use. Cell pellets were thawed on ice for ~20 minutes, then gently resuspended in 45 ml of PBS buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH 7.3), supplemented with 0.25 ml DTT (1M stock), 0.25 ml protease inhibitor cocktail (Sigma), and 2 ml PMSF (17.4 mg/ml in 2-propanol). Before cells were sonicated, 2.5 ml of Triton X100 (20% stock) was added to facilitate lysis. The crude bacterial lysate was cleared by centrifugation and then mixed with ~5 ml glutathione Sepharose 4B (Amersham) resin suspended in PBS buffer. This slurry was rocked gently at room temperature for 2 hours before being transferred into a small column. The packed column was washed with 50 ml of PBS buffer until no more background protein was detected by Coomassie staining. The GST tag was removed by rocking the protein-charged resin overnight at room temperature with 30 units of sequencing grade thrombin (Novagen). The released HIP1 construct was eluted from the column with PBS buffer and 1/10^th volume of 0.5 M EDTA at pH 8.0 was added before exhaustively dialyzing the sample against 10 mM Tris, 10 mM 2-mercaptoethanol, pH 7.9 buffer at 4°C. The partially purified sample was concentrated and then passed through a strong anion exchange column (POROS 20 HQ) equilibrated in 10 mM HEPES, 2 mM TCEP, 1% glycerol buffer at pH 7.9 (Buffer A). The target protein was eluted off with a linear gradient of Buffer B (10 mM HEPES, 500 mM NaCl, 2 mM TCEP, 1% glycerol, pH 7.9). As a final polishing step, the protein was passed through a Superose 6 gel-filtration column equilibrated with Buffer A. We confirmed the single selenomethionine substitution by electrospray mass-spectroscopy. The protein was crystallized by the hanging drop vapor diffusion method in reservoir buffer containing 25% (v/v) PEG 3350, 0.2 M Li₂SO₄ and 0.1 M Bis-Tris at pH 6.5. The crystals grew in the tetragonal space group P4₂2₁2 (a = 54.2 Å, b = 54.2 Å, c = 152.2 Å), with one monomer in the asymmetric unit. The crystals were highly mosaic (>2 degrees) along the c-axis. Exhaustive screening for alternative crystallization conditions or small molecule additives to improve the crystals did not impact quality. However, we found diffraction improved dramatically when preexisting crystals were slowly dehydrated and annealed. After treatment, crystals became much less mosaic (~1.2 degrees) and yielded data to ~2.6 Å sufficient for structure determination. Data collection: The HIP 482–586 structure was solved by multi-wavelength anomalous dispersion (MAD). A 3-wavelength data set was obtained from a single crystal on beam line 4.2.2 at the Advanced Light Source, Lawrence Berkeley National Laboratory. The data were collected at 100K in 0.5 degree oscillations using a NOIR-1 CCD detector. Wavelengths λ₁ (0.97878Å) and λ₂ (0.97863Å) were at the peak and inflection, respectively, of the Kedge of selenium and λ₃ (0.96409Å), was a high energy remote. Each wavelength was collected in a single sweep with an optimized kappa angle to prevent overlaps in the long axis. Data were integrated and scaled using D*TREK. Peak data set: R_merge, 0.091 [0.435]; I/σ, 12.3 [4.6]; Completeness, 100%. Inflection: R_merge, 0.088 [0.400]; I/σ, 12.9 [4.8]; Completeness, 100%. High energy remote: R_merge, 0.133 [0.597]; I/σ, 9.2 [3.4]; Completeness, 100%. Statistics for the highest-resolution shell are given in brackets. Phasing and refinement: A Bayesian approach was used to phase the MAD data taking the high-energy remote (λ₃) as the ‘native’ wavelength and the other two as ‘derivative’ wavelengths. The single selenium site in each monomer was found by SOLVE (http://www.solve.lanl.gov) and the experimental map was improved using RESOLVE . Model building was carried out using O and the model was refined using CNS . Alternating rounds of positional, grouped B factor and simulated annealing were performed in reference to 2F_o–F_c and F_o–F_c maps and a bulk-solvent correction was applied near the end of refinement. The HIP1 482–586 structure refined against all the data from 30-2.8 Å with an R-factor of 28.6% and an R_free of 31.3% at 2.8 Å resolution. Ramachandran plot statistics: Most favored region, 97%; Additionally allowed regions, 1%; Generously allowed regions, 0%; Disallowed regions, 0%. Geometry statistics: B-values average, 77.4 Å; rmsd bond distances, 0.008 Å; rmsd bond angles, 0.9 degrees. The high working and free R-values reflect the limited quality of our best crystals. Structural analysis: Superpositions of Cα traces in Figure 3 were carried out using Lsqkab . The coiled-coil analysis was performed by the program COILS . d, Heptad repeat in the coiled-coil trunk domain 541–581. The F570 (a), L573 (d), E574 (e) and R577 (a) residues in Helix 2 form a pocket for L573 (d) protruding from Helix 1. The crystal structure validates the heptad repeat a-, d- and e-positions predicted by COILS in parentheses. The N and C label the N- and C-termini and the image was prepared using PyMol (http://www.pymol.org). e, The dimer splay site contains a hinge region. The region indicated by the purple bar from L532 and K539 and highlighted by the short ribbon in Helix 1 designate a flexible hinge. A corresponding hinge region is present in Helix 2, but is not shown for the sake of clarity. The residues with yellow dotted surface in Helices 1 and 2 are L532, V534 and L535 that cluster next to an acidic path that surrounds the beginning of the hinge at L532. There is an alternating pattern of oppositely charged residues that punctuates the end of the hinge region at K539 in Helix 1 and 2. K539 and R547 in this region are shown as sticks, while E541 and E548 are shown with red dotted surface.

Figure 1

Topology of the human HIP1 482–586 sub-fragment and domain map of full-length HIP1 and HIP12/1R. a, Helix 1 of the “Y” shaped HIP1 482–586 homodimer shows side chains (magenta), while Helix 2 shows only the backbone (grey). The N and C designate the N- and C-termini of the parallel dimer model. Helix 1 and Helix 2 in the trunk region are coiled together but start to separate a 1/3 of the way towards the N-terminus. b, The left-handed coiled-coil twist is evident when the molecule is rotated 90 degrees from the view in a and shows 2 nodes or points of intersection ~70 Å apart, which is typical of coiled-coil proteins . Structural images were prepared using PyMol (http://www.pymol.org). c, Domain organization of HIP1 and related protein, HIP12/1R (numbering from human HIP1 and HIP12/1R is used). ANTH domain (residues 47–159 in HIP1; 39–150 in HIP12/1R) recognizes inositol phospholipids , , AP2 binding sites in HIP1 only (residues 262–266 and 358–360 3, 20), CLTD region (residues 332–336, LMDMD in HIP1 8) binds the N-terminal domain of clathrin, pDED is a pseudo-death effector domain that can bind HIPPI (residues 410–491 in HIP1; 393–470 in HIP12/1R 1, 21), LC is the clathrin light chain binding site (₄₈₄DLLRKN in HIP1; ₄₆₃ELLRKN in HIP12/1R 5), USH is an upstream regulatory helix, which modulates affinity for actin (residues 780–805 in both HIP1 and HIP12/1R 17), and an F-actin binding region (residues 813–1011 in HIP1 and HIP12/1R containing the actin-binding I/LWEQ motif 22). The Huntingtin binding site in HIP1 is indicated (residues 245–631) . The yellow bar indicates the position of the reported HIP1 482–586 crystal structure. Methods: HIP1 482–586 with an N-terminal GST tag was created by PCR mutagenesis starting with a construct of human HIP1 sub-fragment 371–645 inserted into pGEX-2T (pGST-HIP1h) (pGST-HIP1h was kindly provided by Valerie Legendre-Guillemin and Peter McPherson). Hip1 482–586 was over-expressed at 37°C in Rosetta 2 (DE3) pLysS bacterial cells (Novagen) in M9 minimal medium. After 10 hours of growth (OD₆₀₀ ~0.5), selenomethionine was added as previously described and IPTG (100 μg/ml, final concentration) was added to induce protein expression. Cells were then incubated for 15 hours at 30°C before being harvested and flash frozen for use. Cell pellets were thawed on ice for ~20 minutes, then gently resuspended in 45 ml of PBS buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH 7.3), supplemented with 0.25 ml DTT (1M stock), 0.25 ml protease inhibitor cocktail (Sigma), and 2 ml PMSF (17.4 mg/ml in 2-propanol). Before cells were sonicated, 2.5 ml of Triton X100 (20% stock) was added to facilitate lysis. The crude bacterial lysate was cleared by centrifugation and then mixed with ~5 ml glutathione Sepharose 4B (Amersham) resin suspended in PBS buffer. This slurry was rocked gently at room temperature for 2 hours before being transferred into a small column. The packed column was washed with 50 ml of PBS buffer until no more background protein was detected by Coomassie staining. The GST tag was removed by rocking the protein-charged resin overnight at room temperature with 30 units of sequencing grade thrombin (Novagen). The released HIP1 construct was eluted from the column with PBS buffer and 1/10^th volume of 0.5 M EDTA at pH 8.0 was added before exhaustively dialyzing the sample against 10 mM Tris, 10 mM 2-mercaptoethanol, pH 7.9 buffer at 4°C. The partially purified sample was concentrated and then passed through a strong anion exchange column (POROS 20 HQ) equilibrated in 10 mM HEPES, 2 mM TCEP, 1% glycerol buffer at pH 7.9 (Buffer A). The target protein was eluted off with a linear gradient of Buffer B (10 mM HEPES, 500 mM NaCl, 2 mM TCEP, 1% glycerol, pH 7.9). As a final polishing step, the protein was passed through a Superose 6 gel-filtration column equilibrated with Buffer A. We confirmed the single selenomethionine substitution by electrospray mass-spectroscopy. The protein was crystallized by the hanging drop vapor diffusion method in reservoir buffer containing 25% (v/v) PEG 3350, 0.2 M Li₂SO₄ and 0.1 M Bis-Tris at pH 6.5. The crystals grew in the tetragonal space group P4₂2₁2 (a = 54.2 Å, b = 54.2 Å, c = 152.2 Å), with one monomer in the asymmetric unit. The crystals were highly mosaic (>2 degrees) along the c-axis. Exhaustive screening for alternative crystallization conditions or small molecule additives to improve the crystals did not impact quality. However, we found diffraction improved dramatically when preexisting crystals were slowly dehydrated and annealed. After treatment, crystals became much less mosaic (~1.2 degrees) and yielded data to ~2.6 Å sufficient for structure determination. Data collection: The HIP 482–586 structure was solved by multi-wavelength anomalous dispersion (MAD). A 3-wavelength data set was obtained from a single crystal on beam line 4.2.2 at the Advanced Light Source, Lawrence Berkeley National Laboratory. The data were collected at 100K in 0.5 degree oscillations using a NOIR-1 CCD detector. Wavelengths λ₁ (0.97878Å) and λ₂ (0.97863Å) were at the peak and inflection, respectively, of the Kedge of selenium and λ₃ (0.96409Å), was a high energy remote. Each wavelength was collected in a single sweep with an optimized kappa angle to prevent overlaps in the long axis. Data were integrated and scaled using D*TREK. Peak data set: R_merge, 0.091 [0.435]; I/σ, 12.3 [4.6]; Completeness, 100%. Inflection: R_merge, 0.088 [0.400]; I/σ, 12.9 [4.8]; Completeness, 100%. High energy remote: R_merge, 0.133 [0.597]; I/σ, 9.2 [3.4]; Completeness, 100%. Statistics for the highest-resolution shell are given in brackets. Phasing and refinement: A Bayesian approach was used to phase the MAD data taking the high-energy remote (λ₃) as the ‘native’ wavelength and the other two as ‘derivative’ wavelengths. The single selenium site in each monomer was found by SOLVE (http://www.solve.lanl.gov) and the experimental map was improved using RESOLVE . Model building was carried out using O and the model was refined using CNS . Alternating rounds of positional, grouped B factor and simulated annealing were performed in reference to 2F_o–F_c and F_o–F_c maps and a bulk-solvent correction was applied near the end of refinement. The HIP1 482–586 structure refined against all the data from 30-2.8 Å with an R-factor of 28.6% and an R_free of 31.3% at 2.8 Å resolution. Ramachandran plot statistics: Most favored region, 97%; Additionally allowed regions, 1%; Generously allowed regions, 0%; Disallowed regions, 0%. Geometry statistics: B-values average, 77.4 Å; rmsd bond distances, 0.008 Å; rmsd bond angles, 0.9 degrees. The high working and free R-values reflect the limited quality of our best crystals. Structural analysis: Superpositions of Cα traces in Figure 3 were carried out using Lsqkab . The coiled-coil analysis was performed by the program COILS . d, Heptad repeat in the coiled-coil trunk domain 541–581. The F570 (a), L573 (d), E574 (e) and R577 (a) residues in Helix 2 form a pocket for L573 (d) protruding from Helix 1. The crystal structure validates the heptad repeat a-, d- and e-positions predicted by COILS in parentheses. The N and C label the N- and C-termini and the image was prepared using PyMol (http://www.pymol.org). e, The dimer splay site contains a hinge region. The region indicated by the purple bar from L532 and K539 and highlighted by the short ribbon in Helix 1 designate a flexible hinge. A corresponding hinge region is present in Helix 2, but is not shown for the sake of clarity. The residues with yellow dotted surface in Helices 1 and 2 are L532, V534 and L535 that cluster next to an acidic path that surrounds the beginning of the hinge at L532. There is an alternating pattern of oppositely charged residues that punctuates the end of the hinge region at K539 in Helix 1 and 2. K539 and R547 in this region are shown as sticks, while E541 and E548 are shown with red dotted surface.

Figure 1

Topology of the human HIP1 482–586 sub-fragment and domain map of full-length HIP1 and HIP12/1R. a, Helix 1 of the “Y” shaped HIP1 482–586 homodimer shows side chains (magenta), while Helix 2 shows only the backbone (grey). The N and C designate the N- and C-termini of the parallel dimer model. Helix 1 and Helix 2 in the trunk region are coiled together but start to separate a 1/3 of the way towards the N-terminus. b, The left-handed coiled-coil twist is evident when the molecule is rotated 90 degrees from the view in a and shows 2 nodes or points of intersection ~70 Å apart, which is typical of coiled-coil proteins . Structural images were prepared using PyMol (http://www.pymol.org). c, Domain organization of HIP1 and related protein, HIP12/1R (numbering from human HIP1 and HIP12/1R is used). ANTH domain (residues 47–159 in HIP1; 39–150 in HIP12/1R) recognizes inositol phospholipids , , AP2 binding sites in HIP1 only (residues 262–266 and 358–360 3, 20), CLTD region (residues 332–336, LMDMD in HIP1 8) binds the N-terminal domain of clathrin, pDED is a pseudo-death effector domain that can bind HIPPI (residues 410–491 in HIP1; 393–470 in HIP12/1R 1, 21), LC is the clathrin light chain binding site (₄₈₄DLLRKN in HIP1; ₄₆₃ELLRKN in HIP12/1R 5), USH is an upstream regulatory helix, which modulates affinity for actin (residues 780–805 in both HIP1 and HIP12/1R 17), and an F-actin binding region (residues 813–1011 in HIP1 and HIP12/1R containing the actin-binding I/LWEQ motif 22). The Huntingtin binding site in HIP1 is indicated (residues 245–631) . The yellow bar indicates the position of the reported HIP1 482–586 crystal structure. Methods: HIP1 482–586 with an N-terminal GST tag was created by PCR mutagenesis starting with a construct of human HIP1 sub-fragment 371–645 inserted into pGEX-2T (pGST-HIP1h) (pGST-HIP1h was kindly provided by Valerie Legendre-Guillemin and Peter McPherson). Hip1 482–586 was over-expressed at 37°C in Rosetta 2 (DE3) pLysS bacterial cells (Novagen) in M9 minimal medium. After 10 hours of growth (OD₆₀₀ ~0.5), selenomethionine was added as previously described and IPTG (100 μg/ml, final concentration) was added to induce protein expression. Cells were then incubated for 15 hours at 30°C before being harvested and flash frozen for use. Cell pellets were thawed on ice for ~20 minutes, then gently resuspended in 45 ml of PBS buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH 7.3), supplemented with 0.25 ml DTT (1M stock), 0.25 ml protease inhibitor cocktail (Sigma), and 2 ml PMSF (17.4 mg/ml in 2-propanol). Before cells were sonicated, 2.5 ml of Triton X100 (20% stock) was added to facilitate lysis. The crude bacterial lysate was cleared by centrifugation and then mixed with ~5 ml glutathione Sepharose 4B (Amersham) resin suspended in PBS buffer. This slurry was rocked gently at room temperature for 2 hours before being transferred into a small column. The packed column was washed with 50 ml of PBS buffer until no more background protein was detected by Coomassie staining. The GST tag was removed by rocking the protein-charged resin overnight at room temperature with 30 units of sequencing grade thrombin (Novagen). The released HIP1 construct was eluted from the column with PBS buffer and 1/10^th volume of 0.5 M EDTA at pH 8.0 was added before exhaustively dialyzing the sample against 10 mM Tris, 10 mM 2-mercaptoethanol, pH 7.9 buffer at 4°C. The partially purified sample was concentrated and then passed through a strong anion exchange column (POROS 20 HQ) equilibrated in 10 mM HEPES, 2 mM TCEP, 1% glycerol buffer at pH 7.9 (Buffer A). The target protein was eluted off with a linear gradient of Buffer B (10 mM HEPES, 500 mM NaCl, 2 mM TCEP, 1% glycerol, pH 7.9). As a final polishing step, the protein was passed through a Superose 6 gel-filtration column equilibrated with Buffer A. We confirmed the single selenomethionine substitution by electrospray mass-spectroscopy. The protein was crystallized by the hanging drop vapor diffusion method in reservoir buffer containing 25% (v/v) PEG 3350, 0.2 M Li₂SO₄ and 0.1 M Bis-Tris at pH 6.5. The crystals grew in the tetragonal space group P4₂2₁2 (a = 54.2 Å, b = 54.2 Å, c = 152.2 Å), with one monomer in the asymmetric unit. The crystals were highly mosaic (>2 degrees) along the c-axis. Exhaustive screening for alternative crystallization conditions or small molecule additives to improve the crystals did not impact quality. However, we found diffraction improved dramatically when preexisting crystals were slowly dehydrated and annealed. After treatment, crystals became much less mosaic (~1.2 degrees) and yielded data to ~2.6 Å sufficient for structure determination. Data collection: The HIP 482–586 structure was solved by multi-wavelength anomalous dispersion (MAD). A 3-wavelength data set was obtained from a single crystal on beam line 4.2.2 at the Advanced Light Source, Lawrence Berkeley National Laboratory. The data were collected at 100K in 0.5 degree oscillations using a NOIR-1 CCD detector. Wavelengths λ₁ (0.97878Å) and λ₂ (0.97863Å) were at the peak and inflection, respectively, of the Kedge of selenium and λ₃ (0.96409Å), was a high energy remote. Each wavelength was collected in a single sweep with an optimized kappa angle to prevent overlaps in the long axis. Data were integrated and scaled using D*TREK. Peak data set: R_merge, 0.091 [0.435]; I/σ, 12.3 [4.6]; Completeness, 100%. Inflection: R_merge, 0.088 [0.400]; I/σ, 12.9 [4.8]; Completeness, 100%. High energy remote: R_merge, 0.133 [0.597]; I/σ, 9.2 [3.4]; Completeness, 100%. Statistics for the highest-resolution shell are given in brackets. Phasing and refinement: A Bayesian approach was used to phase the MAD data taking the high-energy remote (λ₃) as the ‘native’ wavelength and the other two as ‘derivative’ wavelengths. The single selenium site in each monomer was found by SOLVE (http://www.solve.lanl.gov) and the experimental map was improved using RESOLVE . Model building was carried out using O and the model was refined using CNS . Alternating rounds of positional, grouped B factor and simulated annealing were performed in reference to 2F_o–F_c and F_o–F_c maps and a bulk-solvent correction was applied near the end of refinement. The HIP1 482–586 structure refined against all the data from 30-2.8 Å with an R-factor of 28.6% and an R_free of 31.3% at 2.8 Å resolution. Ramachandran plot statistics: Most favored region, 97%; Additionally allowed regions, 1%; Generously allowed regions, 0%; Disallowed regions, 0%. Geometry statistics: B-values average, 77.4 Å; rmsd bond distances, 0.008 Å; rmsd bond angles, 0.9 degrees. The high working and free R-values reflect the limited quality of our best crystals. Structural analysis: Superpositions of Cα traces in Figure 3 were carried out using Lsqkab . The coiled-coil analysis was performed by the program COILS . d, Heptad repeat in the coiled-coil trunk domain 541–581. The F570 (a), L573 (d), E574 (e) and R577 (a) residues in Helix 2 form a pocket for L573 (d) protruding from Helix 1. The crystal structure validates the heptad repeat a-, d- and e-positions predicted by COILS in parentheses. The N and C label the N- and C-termini and the image was prepared using PyMol (http://www.pymol.org). e, The dimer splay site contains a hinge region. The region indicated by the purple bar from L532 and K539 and highlighted by the short ribbon in Helix 1 designate a flexible hinge. A corresponding hinge region is present in Helix 2, but is not shown for the sake of clarity. The residues with yellow dotted surface in Helices 1 and 2 are L532, V534 and L535 that cluster next to an acidic path that surrounds the beginning of the hinge at L532. There is an alternating pattern of oppositely charged residues that punctuates the end of the hinge region at K539 in Helix 1 and 2. K539 and R547 in this region are shown as sticks, while E541 and E548 are shown with red dotted surface.

Figure 2a

Multiple interaction interfaces exist in the open region of the HIP1 482–586 dimer. Top panel of the figure shows the Y shaped structure in the same orientation as in Fig. 1a with the N-termini of Helices 1 and 2 pointed to the left (labeled N). The bottom panel is a diagrammatic representation of Helix 1. Labeled circles indicate specific heptad positions that were predicted by COILS in the trunk region and validated by the crystal structure. In the trunk the heptad repeat d-positions, green side chains in Helix 1 in the top panel, are green circles labeled with “d”. The red circles labeled “a” are the heptad a-positions in Helix 1. Note that these a-position residues in the 2-D representation of Helix 1 are not shown in the model in the top panel for the sake of clarity. Instead we show the corresponding a-positions in the model that are in Helix 2. The amino acids corresponding to specific heptad repeat positions marked by color are indicated along the bottom edge of the diagram. The dashed line through the red and green circles represents the a-d coiled-coil interface between Helices 1 and 2 in the trunk. The box in the diagram shows the location of the hinge region (aa532–539). The continuation of the coiled-coil interface from the trunk into the open region is shown as dashed line labeled S2 passing through a- and d-positions indicated by grey and salmon colored circles. The anti-parallel crystal contact observed between neighboring HIP1 helices described in the text is shown as a dotted line labeled S1 and pass through d- and g-positions indicated by salmon and blue colored circles. The S1 residues in Helix 1 are also shown as blue colored side chains in the model in the top panel. The asterisk marks where S1 and S2 paths merge together (corresponds to point F in Fig. 2b). The S3 path described in the text is shown as a thin line going through a- and e-positions in the diagram. Note the yellow circles along S3 correspond to L486 (a), S497 (e) and V504 (e).

Figure 2b

Surface analysis of Helix 1 shows the S1 and S2 hydrophobic paths. The S1 path is shown in blue and traced in dashed line to guide the eye. The S1 path is in yellow and lies very close to S1. The S1 and S2 paths merge at point F. The S3 path is not visible in this view and is on the other side of the helix, opposite to S1 and S2 and exposed to water. The N- and C-termini are labeled N and C, respectively. The HIP1 surface was rendered using PyMol.

Figure 3

Surface analysis of the S3 path. a, N-terminal region of the HIP1 482–586 crystal structure containing ₄₈₄DLLRKN includes the hydrophobic S3 path (dotted trace) defined by the residues indicated in light yellow. The darker yellow surface (L486), critical for clathrin light chain binding , is part of the S3 path. The red and blue colored regions denote the location of acidic and basic amino acids, respectively. The basic pocket centered on K494 (may also involve R500) divides the S3 path in half. Amino acids in parentheses are those in s. cerevisiae (see text) and were assigned by aligning KDEQIKN (yeast) against ₄₈₃ADLLRKN. The N and C designate the N- and C-termini. The surface model was generated using PyMol. b, Surface potential shows two positively charged regions subdivide the S3 path. Model is oriented in the same as in (a) and the dotted tracing indicates the S3 path. Positive potential exists proximal to R487, a residue that is essential for clathrin light chain binding . As shown in Panel a, the middle of S3 is marked by a basic pocket that is centered on K494. The surface potentials (blue, positive and red, negative) were calculated using PyMol.