Research Article: Structure and Binding Interface of the Cytosolic Tails of αXβ2 Integrin

Date Published: July 26, 2012

Publisher: Public Library of Science

Author(s): Geok-Lin Chua, Xiao-Yan Tang, Alok Tanala Patra, Suet-Mien Tan, Surajit Bhattacharjya, Nils Cordes. http://doi.org/10.1371/journal.pone.0041924

Abstract

Integrins are signal transducer proteins involved in a number of vital physiological processes including cell adhesion, proliferation and migration. Integrin molecules are hetero-dimers composed of two distinct subunits, α and β. In humans, 18 α and 8 β subunits are combined into 24 different integrin molecules. Each of the subunit comprises a large extracellular domain, a single pass transmembrane segment and a cytosolic tail (CT). The CTs of integrins are vital for bidirectional signal transduction and in maintaining the resting state of the receptors. A large number of intracellular proteins have been found to interact with the CTs of integrins linking integrins to the cytoskeleton.

In this work, we have investigated structure and interactions of CTs of the leukocyte specific integrin αXβ2. We determined the atomic resolution structure of a myristoylated CT of αX in perdeuterated dodecylphosphocholine (DPC) by NMR spectroscopy. Our results reveal that the 35-residue long CT of αX adopts an α-helical conformation for residues F4-N17 at the N-terminal region. The remaining residues located at the C-terminal segment of αX delineate a long loop of irregular conformations. A segment of the loop maintains packing interactions with the helical structure by an extended non-polar surface of the αX CT. Interactions between αX and β2 CTs are demonstrated by 15N-1H HSQC NMR experiments. We find that residues constituting the polar face of the helical conformation of αX are involved in interactions with the N-terminal residues of β2 CT. A docked structure of the CT complex indicates that a network of polar and/or salt-bridge interactions may sustain the heteromeric interactions.

The current study provides important insights into the conservation of interactions and structures among different CTs of integrins.

Partial Text

Integrins are heterodimeric cell surface receptors that mediate cell attachment and migration, and they modulate cell growth, proliferation and differentiation [1], [2]. In humans, there are 24 integrin heterodimers that are categorized into subfamilies based either on the specific-pairing of the α and β subunits or their ligands. Each integrin subunit has a large ectodomain and a transmembrane domain followed by a cytoplasmic tail (CT). Integrin ligand-binding is mediated by its ectodomain while its cytoplasmic tail allows docking of cytosolic proteins, many of which have been shown to regulate integrin ligand-binding via long-ranged allostery or to induce integrin-derived cellular signaling [2]. The β2 integrins are expressed exclusively in leukocytes and there are four members in this subfamily, namely αLβ2 (LFA-1, CD11aCD18), αMβ2 (Mac-1, CR3, CD11bCD18), αXβ2 (p150, 95, CR4, CD11cCD18) and αDβ2 (CD11dCD18) [3]. Integrin αXβ2 is expressed primarily in myeloid cells, dendritic cells and NK cells. Integrin αXβ2 has many ligands that overlap with that of integrin αMβ2, including iC3b, fibrinogen, and denatured proteins [3]. Notably, integrin αXβ2 has been shown to bind exposed negatively charged residues in decayed proteins, suggesting that it plays a role in neutrophil migration and pericellular degradation of extracellular matrix [4]. High-fat diet induced less adipose tissue inflammation in integrin αX−/− knockout mice compared with wild-type mice [5]. Further, double-knockout mice (integrin αX−/− and apoE−/−), but not apoE−/− mice, showed less accumulation of macrophages in atherlosclerotic lesions [6]. These observations are in line with integrin αXβ2 as a phagocytic receptor and its role in monocyte adhesion to endothelium [7]. Integrin αXβ2 also serves as a marker to distinguish between two populations of HLA-DR+ human peripheral blood dendritic cells [8]. Although integrins do not possess enzymatic activity, they can trigger intracellular signaling by recruiting cytosolic proteins to their cytoplasmic tails aforementioned. Except for their juxtamembrane regions, the integrin α CTs are divergent in lengths and sequences [3]. There are many lines of evidence that suggest the α CTs in mediating integrin signaling specificity [9]–[12]. However, structural information of these integrin α CTs is needed to define the underlying mechanisms. Previously, we have reported the solution structures of integrin αL and αM CTs [13], [14]. Here we report for the first time the structure of integrin αX CT. Considering that the structure of the entire integrin αXβ2 ectodomain has been recently solved [15], our data will allow better understanding of integrin αXβ2 function and regulation as a whole.

The CTs of integrins are involved in bidirectional signaling by interacting with cytoplasmic proteins [1]–[3]. Most of the integrin β-subunits have CTs that are well conserved with sequence motifs NPXX(Y/F) binding to talin, kindlin and DOK proteins. By contrast, integrin α-subunits have CTs that are less conserved, except for the membrane proximal region (Figure S1). Notably, different α CTs exhibit specific interactions with cytoplasmic proteins namely α5 with nischarin [20], α4 with paxillin [21], αIIb with calcein integrin binding protein [22], and αL with CD45 cytoplasmic domain [23]. Conceivably, interactions between α CTs and the cytosolic binding partners may dictate specific function of integrins. Thus, structural elucidation of various α CTs could be useful not only to gain insights into integrin regulation but also for the development of specific anti-integrin drugs [24], [25]. To-date, structures are known for three α CTs namely αIIb/β3, αLβ2 and αMβ2 integrins [13], [14], [16] (Figure 8, top panel). The current study defined the 3-D structure of the myristoylated αX CT in DPC micelles. The αX CT demonstrates a folded structure characterized by the N-terminal amphipathic helix and a distal loop akin to the CTs of αM and αIIb integrins. However, there are striking differences between the structures of the αX CT and the αIIb CT in terms of the length of the N-terminal helix and molecular contacts of the distal loop with the helix. The NMR structure of the myristoylated integrin αIIb CT which is 20-residue long, is characterized by a short helix (V990-R997) and an acidic loop (E1001EDDEEGE1008) (Figure 8A). The acidic loop binds to metal ions and folds back onto the helix by salt-bridge interactions [16]. By contrast, the N-terminal helix of αX CT is considerably longer involving residues F4-N14, and it is also amphipathic (Figure 5). The hydrophilic or polar face of the amphipathic helix of αX CT is well characterized by an array of possible salt-bridge, (residues R7-E11, K10-E14) and hydrogen bond (residues E14-Q19), interactions. The membrane proximal helix of the αIIb CT is rather hydrophobic with fewer polar interactions. In addition, the acidic loop residues of the αIIb CT acquires a tighter packing with the helix by ionic interactions. On the other hand, the long distal loop, residues I21-K37, of the αX CT experiences a higher degree of conformational variations. Only the first few residues of loop of αX CT is involved in packing interactions with the hydrophobic face of the N-terminal helix. The other residues of the loop of αX CT remains extended. Recently, we have determined NMR structure of the myristoylated αM CT of integrin αMβ2 in DPC micelles [14]. The N-terminal region of the 24-residue long αM CT adopted an amphipathic helix, residue F4-E15, followed by a short, residues G15-Q23, fold back loop [14] (Figure 8B). By contrast, the 3-D structure of 57-residue long αL CT was defined by mutual packing of three helices with inter-connecting loops (Figure 8C). The folded conformation of αL CT, sustained by salt bridges and/or hydrogen bonds, display a large negatively charged surface that is involved in binding to metal ions [13]. Recently, NMR structures have been solved for the TM domain of αIIb either with the full-length or C-terminal truncated CT under different solution conditions [26], [27]. A helical conformation for the membrane proximal region has been deduced for the full-length CT in the context of the TM domain in a membrane mimetic organic solvent-water mixture [26]. By contrast, NMR structure, in lipid bicelles, of the TM with truncated CT of αIIb revealed a bent or reverse turn conformation for the membrane proximal segment packing with the TM helix [27]. The structural disparity of the membrane proximal region of the αIIb CT noted in these studies may either simply result from the differences in constructs used or an indicative of favored mode of the structural intermediates. Further, NMR derived structure of the full-length TM and CT of α1 integrin shows helical structure for the membrane proximal region of CT [Lai C et. al. unpublished results, pdb accession number 2L8S]. Aforementioned studies including our current results, therefore, suggest that the membrane proximal region of the full-length α CTs of various integrins assumes a conserved helical structure. However, the C-terminal regions of α CTs appear to show a marked variability in their conformations. It is tempting to speculate that such conformational disparity may allow binding of α CT specific cytosolic proteins resulting in various signaling outcomes.

Source:

http://doi.org/10.1371/journal.pone.0041924