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. 2017 Apr 28;292(17):7077-7086.
doi: 10.1074/jbc.M116.769778. Epub 2017 Mar 15.

Structural and functional characterization of Caenorhabditis elegans α-catenin reveals constitutive binding to β-catenin and F-actin

Hyunook Kang  1 , Injin Bang  1 , Kyeong Sik Jin  2 , Boyun Lee  3 , Junho Lee  1   3   4 , Xiangqiang Shao  5 , Jonathon A Heier  6 , Adam V Kwiatkowski  6 , W James Nelson  7   8 , Jeff Hardin  5 , William I Weis  8   9 , Hee-Jung Choi  10
Affiliations

Structural and functional characterization of Caenorhabditis elegans α-catenin reveals constitutive binding to β-catenin and F-actin

Hyunook Kang et al. J Biol Chem. .

Abstract

Intercellular epithelial junctions formed by classical cadherins, β-catenin, and the actin-binding protein α-catenin link the actin cytoskeletons of adjacent cells into a structural continuum. These assemblies transmit forces through the tissue and respond to intracellular and extracellular signals. However, the mechanisms of junctional assembly and regulation are poorly understood. Studies of cadherin-catenin assembly in a number of metazoans have revealed both similarities and unexpected differences in the biochemical properties of the cadherin·catenin complex that likely reflect the developmental and environmental requirements of different tissues and organisms. Here, we report the structural and biochemical characterization of HMP-1, the Caenorhabditis elegans α-catenin homolog, and compare it with mammalian α-catenin. HMP-1 shares overall similarity in structure and actin-binding properties, but displayed differences in conformational flexibility and allosteric regulation from mammalian α-catenin. HMP-1 bound filamentous actin with an affinity in the single micromolar range, even when complexed with the β-catenin homolog HMP-2 or when present in a complex of HMP-2 and the cadherin homolog HMR-1, indicating that HMP-1 binding to F-actin is not allosterically regulated by the HMP-2·HMR-1 complex. The middle (i.e. M) domain of HMP-1 appeared to be less conformationally flexible than mammalian α-catenin, which may underlie the dampened effect of HMP-2 binding on HMP-1 actin-binding activity compared with that of the mammalian homolog. In conclusion, our data indicate that HMP-1 constitutively binds β-catenin and F-actin, and although the overall structure and function of HMP-1 and related α-catenins are similar, the vertebrate proteins appear to be under more complex conformational regulation.

Keywords: Caenorhabditis elegans (C. elegans); HMP-1; HMP-2; X-ray crystallography; actin; cell adhesion; four helix bundle; small-angle X-ray scattering (SAXS); α-catenin (a-catenin).

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
Overall domain structure of HMP-1 and crystal structure of the HMP-1 M domain. A, domain structures of HMP-1, mouse αE-catenin, and chicken vinculin. Both HMP-1 and αE-catenin have similar domain structures, including a β-catenin-binding domain, M domain, and ABD. Domain boundaries are labeled with residue numbers. B, crystal structure of the HMP-1 M domain shown in two orientations. There are two molecules in an asymmetric unit. In one molecule, MI is colored green, MII yellow, and MIII cyan, as shown in A. In the other molecule, MI to MIII are colored from light gray to dark gray. C, non-polar interactions between MII and MIII bundles. Interacting residues are labeled and their side chains are shown as sticks. Cα atom of Gly is represented as a sphere. Non-polar interactions within 3.8 Å distance are shown with solid lines. D, polar interaction network among three 4-helix bundles. Interacting atoms within 3.4 Å distance are connected with dashed lines and each residue is labeled and its side chain is shown as sticks.
Figure 2.
Figure 2.
Structural comparisons of HMP-1 M with the corresponding regions of αE-catenin and vinculin. Structural alignment of the HMP-1 M domain with the αE-catenin M domain (A) and the corresponding region of vinculin (B). An additional 4-helix bundle domain denoted as D2 in vinculin shown as a surface model. αE-catenin and vinculin are colored orange (A) and pink (B), respectively.
Figure 3.
Figure 3.
Sequence alignment between HMP-1 M and αE-catenin M. A, structure-based sequence alignment of the HMP-1 M domain with mouse αE-catenin M domain. Conserved residues involved in forming salt bridges are labeled in red; the italicized residues are not visible in the structure. The colored rectangular box on top of the sequence represents the helical region. B, residues in the HMP-1 M domain structure homologous to those from αE-catenin that form the six salt bridges that stabilize the αE-catenin M domain. Polar interactions are shown as dashed lines in the four conserved salt bridges. Non-conserved residues are circled and corresponding residues of αE-catenin are colored orange and labeled in parentheses.
Figure 4.
Figure 4.
Intramolecular interaction of HMP-1 M domain. A, a detailed view of HMP-1 M domain intramolecular interaction. The MI and MII bundles stabilize one another through polar and hydrophobic interactions. Each interacting residue is labeled and represented as sticks. Polar and non-polar interactions are shown as dashed and solid lines, respectively. B, intramolecular interactions between the second and third helices in the MI domains of HMP-1 and αE-catenin compared. Residues involved in extensive polar interactions in HMP-1 are shown as sticks and interacting atoms are connected with dashed lines. The corresponding residues of αE-catenin are shown and labeled in parentheses. Glu298 and Phe296 of αE-catenin do not structurally align well with Arg292 and Tyr290 of HMP-1, respectively.
Figure 5.
Figure 5.
Model of full-length HMP-1. A, a HMP-1 full-length model constructed within an averaged and filtered envelope from SAXS data analysis is shown together with the SAXS envelope of the HMP-1 head in two different orientations. The HMP-1 M domain and crystal structures of the N and C domains of αN-catenin (PDB codes 4P9T and 4K1O) were fitted within the molecular envelope using the head domain of αE-catenin as a guide (PDB code 4IGG). The N and C domains are colored blue and red, respectively. B, structural alignment of the HMP-1 full-length model with one protomer of the dimeric structure of αE-catenin (PDB code 4IGG). The aligned head domain of αE-catenin is omitted and the differently positioned tail domain of αE-catenin is shown and colored salmon.
Figure 6.
Figure 6.
Actin pelleting assays. A, F-actin binding assays were carried out with HMP-1 full-length (HMP-1(FL)), HMP-1·HMP-2 binary complex (HMP-1(FL) + HMP-2(13–678)), tailless HMP-1 (HMP-1(12–646)), and two different constructs of the ABD (HMP-1(672–927) and HMP-1(672–863)) in the presence and absence of F-actin (labeled as +F-actin and −F-actin, respectively). Equal amounts of supernatant (S) and pellet (P) fractions after high speed centrifugation were loaded onto an SDS-PAGE gel and stained with Coomassie Blue. Each protein band and actin are marked with arrows. B, F-actin pelleting assays for HMP-1(FL), a binary complex, HMP-1(FL)·HMP-2(13–678), and a ternary complex, HMP-1·HMP-2(13–678)·pHMR-1(cyto80). 16 of the pellet was loaded for 0.5, 1, 3, and 6 μm samples of HMP-1, whereas 112 of the pellet was loaded for 9, 12, and 15 μm samples. The sample loading amount was the same for both binary and ternary complexes, except for 6 μm, for which only 112 of the pellet was used. Concentration of bound HMP-1 was plotted against free HMP-1 concentration using GraphPad to obtain Kd. For each protein, at least three different experiments were performed and a representative SDS-PAGE gel and saturation curve are shown.
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