Summary
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 from 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 to S1 and S2. We present structural evidence supporting a role for 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.
Keywords: HIP1 clathrin light chain interaction, HIP1, HIP12/1R, endocytosis, Sla2p
Introduction
Huntingtin interacting protein 1 (HIP1) is an essential obligate binding partner for Huntingtin (htt). Loss of the HIP1/htt interaction triggers a cascade of events that results in the apoptosis of neuronal cells and the onset of Hungtinton’s disease 1. HIP1 and a related protein, HIP12/1R, have been found to participate in clathrin-mediated endocytosis 2, 3. HIP1 and HIP12/1R can exist as homodimers and there is evidence that HIP1 and HIP12/1R may also form heterodimers 4, 5. HIP1 and HIP12/1R interact with membrane phospholipids via an ANTH (AP180 N-terminal homology) domain at its N-terminus (see Fig. 1a) 3, 6. HIP1 and HIP12/1R also have a C-terminal THATCH domain, but only HIP12/1R has a C-terminal latch that activates its THATCH domain to tether CCVs to the cytoskeleton 7. HIP1 has AP2-binding FXDXF and DPF motifs (X denotes any amino acid) that are both absent in HIP12/1R and a clathrin box (332LMDMD) that recognizes the clathrin N-terminal domain 8. The clathrin light chain-binding site HIP1 was mapped to 484DLLRKN in HIP1 and 463ELLRKN in HIP12/1R 6. Apparently L486 and R487 are critical because substituting these residues with alanine blocked the ability of HIP proteins to bind clathrin light chain 5. Specifically, L486 and R487 interact with the regulatory 20EED sequence 9 in clathrin light chains a and b (LCa and LCb) 4, 5, 10. Although the L486A or R487A mutant was sufficient to destroy light chain binding 5, 10, the ability of widely spaced single mutations in LCb to inhibit binding to HIP12/1R 4 suggested to us that the interaction surface for clathrin light chain in the HIP proteins involved more than just the region around L486 and R487. To extend our understanding of the interaction between clathrin light chains and the coiled-coil domain of HIP1, we have determined the crystal structure of the coiled-coil domain of HIP1 that includes 484DLLRKN.
The HIP1 structure reported here to 2.8 Å is a partially coiled dimer that has a hinge region where the helical coils in the N-terminal region of the molecule separate from each other (Fig. 1). We found two closely spaced hydrophobic paths (S1 and S2) that can potentially be used to form a coiled-coil dimer interface. We also identified a third hydrophobic path (S3) that is on the solvent exposed side of each helix that may be involved in binding clathrin light chain.
Topology
Efforts to grow crystals of HIP1 constructs that contained the entire coiled-coil domain were disappointing, yielding only highly twinned crystals that diffracted poorly. We therefore made a series of shorter HIP1 sub-fragments that included 484DLLRKN, the sequence originally identified as being important for clathrin light chain binding, to see if resolution could be increased and twinning eliminated. We found that the HIP1 482–586 fragment (denoted by the yellow bar in the domain map in Fig. 1a, Panel c) gave large three-dimensional crystals, but these forms turned out to be very mosaic (~2 degrees) along the crystallographic c-axis, indicating there was a high degree of disordering in that direction. We tested about two hundred small molecule and detergent additives, but none increased crystal quality. We found, however, that dehydrating preexisting crystals improved the data sufficiently for MAD phasing. The refinement statistics (R=29%, Rfree=31%) are higher because of the limited quality of the final dehydrated crystals. Nevertheless, the HIP1 crystal structure yields new information about the coiled-coil domain that includes 484DLLRKN. The numbering scheme from human HIP1 is used throughout (accession number NP 005329), but the first two residues in the HIP1 model are from the GST tag that was removed by thrombin. The last 5 residues (582–586) could not be built because of weak electron density. Note that we refer to the dimeric crystal structure as HIP1 482–586 even though the first two residues are not from HIP1 and the refined model ends at residue 581.
Figure 1a shows the parallel HIP1 482–586 dimer is Y shaped (~145 Å long) with a left-handed coiled-coil trunk region spanning residues 541–581 that is ~54 Å long. The splaying of Helix 1 from Helix 2 is restricted to one plane because rotating the open HIP1 dimer by 90 degrees around the long axis (Fig. 1a, Panel b) yields a model that appears entirely closed, with a ~70 Å node-to-node distance typical of coiled-coil proteins 11. Amino acid sequences giving rise to coiled-coils have a seven amino acid repeating unit (denoted a-g) 12, 13. Residues at the a- and d-positions are mostly apolar, while those at e- and g-positions are usually charged 14, 15. The a-b-c-d-e-f-g heptad positions in the trunk of the model validated our COILS analysis of this region of the HIP1 482–586 sub-fragment. Fig. 1d shows that F570 (a), L573 (d), E574 (e) and R577 (a), in Helix 2 together form a pocket for L573 (d), protruding from Helix 1 (heptad positions predicted by COILS are in parenthesis). This observed heptad repeat is part of the a-b-c-d-e-f-g pattern that COILS predicts goes to the end of the coiled-coil domain at position 643.
A Cα backbone template (aa547–581) from the trunk region was employed to evaluate if N-terminal segments of Helix 1 and 2 assumed normal left-handed coiled-coil topology. The superpositions revealed that most of the sections N-terminal to where the helices begin to separate were topologically very similar to the trunk domain of the HIP1 482–586 sub-fragment. The exception was a short segment between L532 and K539 (indicated by bar over the short ribbon in Fig. 1e). We have operationally defined this 532–539 stretch as the molecular hinge that opens the HIP1 structure. There is a cluster of hydrophobic residues (L532, V534 and L535) next to E530, E533 and E536 at one end of this 8-residue hinge segment. The juxtaposition of hydrophobic and acid clusters at the beginning of the designated hinge suggests any conformational change caused by repulsions between E530, E533 and E536 in Helix 1 and in Helix 2 would be mitigated by the natural tendency of L532, V534 and L535 from Helix 1 to pack against the same residues in the other helix to avoid contact with water. Any destabilizing impact of the hinge region (L532–K539) may also be sterically restricted by a cluster of charged residues at the end of the hinge. Fig. 1e shows the pattern of alternating positively and negatively charged residues (K539, E541, R547 and E548) punctuating the C-terminal end of the hinge. Specifically, K539 in Helix 2 lies almost directly across E541 in Helix 1 and R547 in Helix 2 is oriented opposite E548 in Helix 1.
Identifying parallel hydrophobic paths
The packing of HIP1 482–586 molecules in the crystal requires the partially splayed structure because the space between separated helices is used to build up a vertical stack of HIP1 molecules. Each HIP1 column is related to the next over by 2-fold symmetry to produce a lattice of alternating up/down (anti-parallel) columns. The molecular contact between anti-parallel HIP1 molecules that connect neighboring columns is defined by the S1 path shown in Helix 1 as a dotted line through H482, N489, V496, Q503 and K510 (Fig. 2a). An identical S1 path exists in Helix 2 (not shown). According to COILS, the S1 path is part of the heptad repeat that starts at the beginning of the HIP1 coiled-coil domain at position 389. When the trajectory of S1 was projected into the trunk region of the crystal structure, we observed an offset between S1 and the heptad repeat pattern shown in Fig. 1d. We therefore defined the S2 path in Fig. 2a and 2b. This path was drawn by continuing the register of the heptad repeat observed in the trunk into the open area (dashed line in Fig. 2a going through L485, V492, A499, L506 and L513). We emphasize the S2 path represents the heptad repeat of the trunk projected into the open region. We found that the observed S1 path and projected S2 path merge just before the designated hinge described above (merge point marked by an asterisk in the diagram in Fig. 2a and is labeled F in Fig. 2b). The S1 and S2 paths overlap to become a single path at point F. This single heptad repeat (S2 path) passes through the trunk region of our structure and continues on. The merging together of S1 and S2 raises the possibility that the single heptad repeat detected by COILS starting at position 389 (coincident with S1) gives rise to S2 at a branching point located in structure that is before our HIP1 model. In this scenario an upstream heptad discontinuity creates S2 off of S1, producing the double hydrophobic path in the region of HIP1 that contains 484DLLRKN. The two paths coalesce at point F (Fig. 2b) and the resulting single S2 path continues on to the end of the entire coiled-coil domain. We point out that the S1/S2 offset may be functionally significant because heptad repeat discontinuities drive coiled-coils to under-or over-wind to produce loose or tightened coiled-coil structures 16.
The coiled-coil domain of HIP12/1R is straight and completely closed, but is sufficiently flexible to bend occasionally into a U shaped structure 17. We anticipate the HIP1 482–586 sub-fragment in the full-length molecule will also be in a closed state because COILS predicts the sequence preceding our structure contains a strong heptad repeat. The proximity of S1 to S2 (Fig. 2a and 2b) may allow both of these hydrophobic paths to contribute to the dimerization interface between Helix 1 and Helix 2 in a closed conformation. This creates the possibility for movement between Helix 1 and Helix 2 because there would be more total surface area between the contacting helices for sliding against each other. There are three possible scenarios how the S1 and S2 paths in Helix 1 can align with the S1 and S2 paths in Helix 2. The S1 path of Helix 1 may interact with S1 of Helix 2, or the S2 path of Helix 1 can rest against S2 of Helix 2, or S1 of Helix 1 can contact S2 of Helix 2. We suggest that these different scenarios could control access to important elements in the S3 path, which may determine the binding of clathrin light chain. For example, it may be sufficient to regulate access to L or R in 484DLLRKN to trigger the binding or release of clathrin light chain. The molecular hinge between position 532 and 539 that opens the structure would be a critical feature in any movement involving Helix 1 and Helix 2. We are currently generating point mutations in the hinge region to investigate if the interaction between HIP1 and clathrin light chain is regulated through molecular flexibility in the vicinity of the 484DLLRKN region.
Surface suitable for clathrin light chain binding
We identified a path (S3) in the region of HIP1 that involves the 484DLLRKN sequence (Fig. 3). Most of the S3 path is hydrophobic (L486, A483, A490, S497 and V504) and completely exposed to water and is therefore a potential surface for protein-protein interactions. The S3 path is short (~31 Å long) compared to S1 or S2, with an estimated surface area of ~150 Å2. We stress that the S3 path does not have a heptad pattern. The S3 path is on the helix face opposite where S1 and S2 are located and is flanked by R487 (Fig. 3a). The presence of L486 and R487 at the beginning of S3 is significant because both L486 and R487 are essential for binding clathrin light chain 5. Surface analysis in Fig. 3 reveals the S3 path is interrupted by K494, which is conserved in human and mouse HIP1 and HIP12/1R. Thus, S3 is subdivided by 2 positively charged regions, one close to the beginning involving R487, and the other in the middle, centered on K494.
HIP1 deletion studies conducted to find the clathrin light chain- binding region in HIP1 5 provide information about the possible function of the S3 path. The binding of LCb to a HIP1 deletion construct missing V504 was slightly reduced (the 336–463 construct reported by Legendre-Guillemin et al. 5 is numbered differently, but corresponds to 370–497 in our structure). However, deleting S497 and K494 (336–456 deletion construct) significantly weakened LCb binding 5. These mutagenesis data suggest the hydrophobic region between S497 and V504 is involved in binding light chain. Moreover, the basic pocket in the crystal structure (Fig. 3b) may also participate in binding clathrin light chain because LCb binding was significantly decreased when K494 was removed 5. Our HIP1 crystal structure also offers an explanation for why mutating L485 does not affect clathrin light chain binding 5. L485 is located on the opposite face of S3 and is part of the S1 path that is shown in Fig. 2a. Therefore L485 would be in the hydrophobic core of a fully closed HIP1 dimeric molecule and would not contribute to binding light chain.
The determinants essential for binding HIP12/1R, and presumably HIP1, were found to depend on a variety of residues in the conserved region of LCa and LCb 4. This work revealed that E20V, Q31R and I38M mutants weakened the binding of LCb to HIP12/1R 4. Conversely, D22V, I35A and D39A mutants were found to completely inhibit the interaction 4. Apparently E32A, S33A and E34A mutants have the opposite effect, enhancing the binding of LCb to HIP12/1R 4. The fact that the S3 path is very hydrophobic may explain why changing E32, S33 or E34 to alanine significantly increases binding 4.
The two positively charged regions present in S3 (Fig. 3b) suggest R487 and K494 may be placed close to D22 and D39 when LCb (or LCa) complexes with HIP1 or HIP12/1R. There is evidence that D22, in the regulatory region of LCb 9, interacts with R487 to stimulate clathrin assembly 4, 5. Since there are no other basic residues in the immediate area, a possible contact model would position D39 in light chain proximal to K494 in HIP1 (or the corresponding lysine in HIP12/1R). We further assessed the likelihood of the D22/R487, D39/K494 contact model by calculating the Cα-to-Cα distance (~27 Å) between D22 and D39 in LCb, assuming helical conformation. This distance is 10 Å more than the distance between the η1 and η2 nitrogens of R487 and the ζ nitrogen of K494 (~17 Å), but the difference could be less if the oxygens from the γ carbons in the side chains of D22 and D39 point toward each other. However, we cannot rule out the possibility that the disagreement in the estimated distances is due to the assumption that the light chain is helical between D22 and D39. There is a proline at position 23 in both LCa and LCb immediately following the 20EED regulatory region, which could discourage this part of light chain from folding into a helix.
Finally the HIP1 482–586 structure may be useful in thinking about how the yeast homolog of HIP1, sla2p, interacts with yeast clathrin light chain. In vivo data indicate that the association between sla2p and yeast clathrin light chain is absolutely required for invaginating the plasma membrane to form transport vesicles, but this interaction does not require the clathrin heavy chain 18. In s. cerevisiae, s. pombe and y. lipolytica the KD(E)E(D)Q(L)I(L)K(T, E)N(A,S) sequence corresponds to 483ADLLRKN (HIP1). The underlined amino acids (KDEQIKN) are from s. cerevisiae and those in parentheses are the changes in pombe or lipolytica. We determined where yeast (s. cerevisiae) residues (in parentheses in Fig. 3a) might be positioned in the S3-containg region of the crystal structure by aligning KDEQIKN against 483ADLLRKN. In yeast, leucine also occupies position 486 (Fig. 3a), but R487 is not conserved (R->T). We also found that A483 is replaced by aspartic acid in yeast, which would make this region negatively charged. Lastly, a cluster of acidic residues in yeast replace the basic pocket centered on K494 (see Fig. 3a), suggesting the yeast clathrin light chain itself would have to be significantly substituted in order to compensate for the flipped electrostatic charges. This is consistent with the fact that the 22-amino acid region (aa20–41, LCb numbering) in LCa and LCb that is 100% conserved in all vertebrates and highly conserved in light chains from non-vertebrates 19, is not conserved in yeast light chain. Only three residues (underlined) in the 20EEDPAAAFL sub-region (from LCb) are identical in the corresponding yeast KKDDDTDFL light chain sequence. Thus, the mode of interaction between sla2p and yeast clathrin light chain may be different.
Conclusions
Mutational studies demonstrated that L486 and R487 in HIP1 are crucial for the binding of clathrin light chain 5. These two HIP1 residues are revealed in our crystal structure and are part of the 484DLLRKN region in HIP1 (ELLRKN in HIP12/1R) that was initially thought to interact with clathrin light chain 4. We report here that the heptad repeat in the coiled-coil trunk region of our Y shaped HIP1 molecule is shifted from the heptad repeat coming from the N-terminus of the protein to produce two hydrophobic paths, S1 and S2, that ultimately merge together. The S3 path, solvent exposed and much shorter than S1 and S2, may function as a clathrin light chain-binding surface. Finally, the comparison of surface features suggests the interaction between sla2p and yeast clathrin light chain may be unique.
Protein Data Bank accession code
The coordinates have been deposited in the RCSB Protein Data Bank with accession code 2NO2. The coordinates will be released on October 24, 2007.
Acknowledgments
We thank Valerie Legendre-Guillemin and Peter McPherson for the pGST-Hip1h construct; Ed Westbrook at beamline 4.2.2 of the Molecular Biology Consortium at the Advanced Light Source for early data collection runs; Samantha Perez-Miller for comments on the manuscript. This work was supported by NIH grant RO1 GM064387 to J.A.Y.
Footnotes
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Contributor Information
Joel A. Ybe, Department of Biology, Indiana University, Bloomington, IN 47405
Sanjay Mishra, Department of Biology, Indiana University, Bloomington, IN 47405.
Stephen Helms, Department of Biology, Indiana University, Bloomington, IN 47405.
Jay Nix, Molecular Biology Consortium, Advanced Light Source, Lawrence Berkeley National Laboratory, Berkeley, CA 94720.
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