Journal of Molecular Biology
Volume 375, Issue 5, 1 February 2008, Pages 1197-1205
Journal home page for Journal of Molecular Biology

Communication
Crystal Structure at 2.8 Å of Huntingtin-Interacting Protein 1 (HIP1) Coiled-Coil Domain Reveals a Charged Surface Suitable for HIP1 Protein Interactor (HIPPI)

https://doi.org/10.1016/j.jmb.2007.11.036Get rights and content

Abstract

Huntington's disease is a genetic neurological disorder that is triggered by the dissociation of the huntingtin protein (htt) from its obligate interaction partner Huntingtin-interacting protein 1 (HIP1). The release of the huntingtin protein permits HIP1 protein interactor (HIPPI) to bind to its recognition site on HIP1 to form a HIPPI/HIP1 complex that recruits procaspase-8 to begin the process of apoptosis. The interaction module between HIPPI and HIP1 was predicted to resemble a death-effector domain. Our 2.8-Å crystal structure of the HIP1 371–481 subfragment that includes F432 and K474, which is important for HIPPI binding, is not a death-effector domain but is a partially opened coiled coil. The HIP1 371–481 model reveals a basic surface that we hypothesize to be suitable for binding HIPPI. There is an opened region next to the putative HIPPI site that is highly negatively charged. The acidic residues in this region are highly conserved in HIP1 and a related protein, HIP1R, from different organisms but are not conserved in the yeast homologue of HIP1, sla2p. We have modeled ∼ 85% of the coiled-coil domain by joining our new HIP1 371–481 structure to the HIP1 482–586 model (Protein Data Bank code: 2NO2). Finally, the middle of this coiled-coil domain may be intrinsically flexible and suggests a new interaction model where HIPPI binds to a U-shaped HIP1 molecule.

Section snippets

Structure

The HIP1 371–481 subfragment produced tetragonal crystals (P43) that diffracted to a maximum resolution of ∼ 2.7 Å at the Advanced Light Source (Molecular Biology Consortium beamline 4.2.2). These crystals were highly mosaic (∼ 2°) due to the disordered packing of protomers in the unit cell. A search for different conditions yielded very thin two-dimensional needles that diffracted poorly. We tested about 100 small-molecule and detergent additives to improve the original P43 crystals, but

Possible HIPPI binding surface

Two amino acids in HIP1 have been implicated in HIP1-induced cell toxicity.3, 15 When F432 is replaced with glycine, cell toxicity is dramatically reduced, but a conservative F-to-Y substitution does not alter HIP1-induced apoptosis.15 Past work suggested that K474 that is in our HIP1 371–481 subfragment was important for recruiting HIPPI.3 Gervais et al. observed that mutating K409 in the pDED of HIPPI to leucine (K474/L409 combination) decreased the HIP1/HIPPI interaction, but they found that

Coiled-coil plasticity and implication for HIP1/HIPPI binding

The flexibility suggested by the new HIP1 371–481 structure reported here and by our HIP1 482–586 model24 has new implications for how HIPPI interacts with HIP1. Gervais et al. transiently transfected a variety of HIP1 and HIPPI deletion constructs in 293 cells and assessed the HIP1/HIPPI interaction by coimmunoprecipitation.3 They could not reduce the binding of HIPI to HIPPI when the pDED of HIP1 was deleted, but the binding between HIP1 and HIPPI decreased slightly when the HIPPI pDED was

Conclusions

In summary, we have discovered that HIP1 371–481 is not folded into the DED but is a coiled coil. The C-terminus of HIP1 371–481 is splayed open, which is a continued structural feature that we first observed in the N-terminus of HIP1 482–586.24 Together, our HIP1 crystal structures raise the possibility that the center of the coiled-coil domain is highly flexible. The proposed flexibility of HIP1 is supported by the presence of destabilizing residues at a- and d-positions in the opened region

PDB accession code

The coordinates have been deposited in the Research Collaboratory for Structural Bioinformatics PDB with accession code 2QA7. The coordinates will be released upon publication.

Acknowledgements

We thank Michael Hayden and Peter McPherson for the pGST-HIP1h construct, Sanjay Mishra for generating constructs and protein purification, and Jay Nix at beamline 4.2.2 of the Molecular Biology Consortium at the Advanced Light Source for assistance in collecting MAD data. This work was supported by National Institutes of Health grant RO1 GM064387 to J.A.Y.

References (35)

  • S.C. Kwok et al.

    Stabilizing and destabilizing clusters in the hydrophobic core of long two-stranded a-helical coiled-coils

    J. Biol. Chem.

    (2004)
  • E.E. Wanker et al.

    HIP-I: a huntingtin interacting protein isolated by the yeast two-hybrid system

    Hum. Mol. Genet.

    (1997)
  • F.G. Gervais et al.

    Recruitment and activation of caspase-8 by the huntingtin-interacting protein HIP-1 and a novel partner HIPPI

    Nat. Cell Biol.

    (2002)
  • Y. Sun et al.

    Interaction of Sla2p's ANTH domain with PtdIns(4,5)P2 is important for actin-dependent endocytic internalization

    Mol. Biol. Cell

    (2005)
  • M.A. Senetar et al.

    Intrasteric inhibition mediates the interaction of the I/LWEQ module proteins Talin1, Talin2, Hip1, and Hip12 with actin

    Biochemistry

    (2004)
  • R.O. McCann et al.

    The I/LWEQ module: a conserved sequence that signifies F-actin binding in functionally diverse proteins from yeast to mammals

    Proc. Natl Acad. Sci. USA

    (1997)
  • T.J. Brett et al.

    Structural definition of the F-actin-binding THATCH domain from HIP1R

    Nat. Struct. Mol. Biol.

    (2006)
  • Cited by (15)

    • Nuclear localization of clathrin involves a labile helix outside the trimerization domain

      2013, FEBS Letters
      Citation Excerpt :

      In vertebrates and invertebrates there are two types of light chain subunits (LCa and LCb [7]), but yeast clathrin has only one kind of light chain [8]. The light chain subunit is proposed to modify the flexibility of the knee joint to regulate lattice formation [4], mediates the binding of Huntingtin-Interacting Proteins (HIPs) [9,10], and stabilizes the global three-legged clathrin structure by making contact with the trimerization domain [11,12]. It has become clear that clathrin function extends beyond conventional endocytosis.

    • Therapeutic approaches to preventing cell death in Huntington disease

      2012, Progress in Neurobiology
      Citation Excerpt :

      The ANTH domain can mediate binding to the 3-phosphoinositides, such as phosphatidylinositol-3,4-bisphosphate and phosphatidylinositol-3,5-bisphosphate, thus allowing the protein to dock to the plasma membrane (Hyun et al., 2004). The coiled-coiled domain spans the mid-portion of the HIP1 protein and is the site where the majority of protein partners interact with HIP1; some of these are clathrin light and heavy chains (Legendre-Guillemin et al., 2005; Waelter et al., 2001), polyglutamine-track-containing androgen receptor (Mills et al., 2005), HIP1 protein interactor (HIPPI) (Gervais et al., 2002; Niu and Ybe, 2008), NMDA receptor (Metzler et al., 2007), and of course huntingtin protein (Hackam et al., 2000; Kalchman et al., 1997). The THATCH domain can interact and bind to filamentous actin (F-actin) in the cytoskeleton (Wilbur et al., 2008).

    • Replacement of charged and polar residues in the coiled-coiled interface of huntingtin-interacting protein 1 (HIP1) causes aggregation and cell death

      2012, FEBS Letters
      Citation Excerpt :

      Residues at the a-position are frequently leucine, isoleucine or alanine and the d-position is usually leucine or alanine [4,5]. Crystal structures from our laboratory reveal the CC region of a HIP1 sub-fragment is splayed opened, suggesting this coiled-coil region is plastic [6–8]. The HIP1 parallel CC has a number of a- and d-residues that are polar or charged.

    • Essential role of coiled coils for aggregation and activity of Q/N-rich prions and PolyQ proteins

      2010, Cell
      Citation Excerpt :

      The ubiquitin ligase CHIP, the chaperone Hsp104, the polyQ tract-binding protein-1 (PQBP-1), and the Htt-interacting protein (HIP-1) interact with Q/N-rich or polyQ proteins modulating their aggregation (Orr and Zoghbi, 2007; Wickner et al., 2007). In search of shared features among them, we discovered the common occurrence of CC domains, as previously found in three of them (e.g., Niu and Ybe, 2008), and as predicted with high probability for PQBP-1 (Figure 1A). We extended our analysis to other interactors of the Q/N-rich prion Ure2 and the polyQ protein Htt listed in the BioGrid database, and found that 54% and 63% of Ure2 and Htt interactors, respectively, have or are predicted to have CC domains (Figure 1B).

    View all citing articles on Scopus
    View full text