Dramatic and concerted conformational changes enable rhodocetin to block α2β1 integrin selectively.

The collagen binding integrin α2β1 plays a crucial role in hemostasis, fibrosis, and cancer progression amongst others. It is specifically inhibited by rhodocetin (RC), a C-type lectin-related protein (CLRP) found in Malayan pit viper (Calloselasma rhodostoma) venom. The structure of RC alone reveals a heterotetramer arranged as an αβ and γδ subunit in a cruciform shape. RC specifically binds to the collagen binding A-domain of the integrin α2 subunit, thereby blocking collagen-induced platelet aggregation. However, until now, the molecular basis for this interaction has remained unclear. Here, we present the molecular structure of the RCγδ-α2A complex solved to 3.0 Å resolution. Our findings show that RC undergoes a dramatic structural reorganization upon binding to α2β1 integrin. Besides the release of the nonbinding RCαβ tandem, the RCγ subunit interacts with loop 2 of the α2A domain as result of a dramatic conformational change. The RCδ subunit contacts the integrin α2A domain in the "closed" conformation through its helix C. Combined with epitope-mapped antibodies, conformationally locked α2A domain mutants, point mutations within the α2A loop 2, and chemical modifications of the purified toxin protein, this molecular structure of RCγδ-α2A complex explains the inhibitory mechanism and specificity of RC for α2β1 integrin

Correction: Dramatic and concerted conformational changes enable rhodocetin to block α2β1 integrin selectively.

[This corrects the article DOI: 10.1371/journal.pbio.2001492.]

Data and refinement statistics of the RCγδ-α2A crystal structure.

<p>Data and refinement statistics of the RCγδ-α2A crystal structure.</p

Molecular mechanism of the RCγδ-α2A interaction.

<p>As RCαβγδ binds to α2A in its “closed” conformation, it induces the conformational change of α2A from its “open” to “closed” conformation and thus shifts the conformational equilibrium (1). This interaction is mediated via the conformationally robust RCδ interaction site within helix C, which is only present in the “closed” conformation of α2A. Subsequently, the index finger loop of RCγ changes its conformation, which is accompanied by a global movement of both rhodocetin (RC) core domains towards each other and by a release of the RCαβ subunit (2). As the RCαβ subunit diffuses away, this step is likely irreversible in nature. The global shape change of RCγδ forms a new bay region that embraces α2A and locally leads to the repositioning of RCγ key residues, which forms another binding-competent interacting site in RCγ for the α2A loop 2 (3).</p

An overview of the RCγ and RCδ binding residues, depicting the local conformational changes that occur upon α2A binding.

<p>(<b>A</b>) A comparison of the RCγ subunit binding site (L66/R109/W110) between the RCαβγδ (purple) and RCγδ-α2A complex (blue) structures. Due to the global movements within the index finger swapping domain that accompany the formation of the RCγδ-α2A complex, a local repositioning of the key α2A interacting residues within RCγ takes place such that they adopt an orientation that is compatible for α2A binding. (<b>B</b>) A comparison between the 2 RCδ subunit binding sites (K59/Y60/K101 and R92/Y94/K114) between the RCαβγδ (yellow) and RCγδ-α2A complex (orange) structures. In contrast to RCγ, all the RCδ residues involved in α2A binding would be in an α2A-competent orientation in both the RCαβγδ (yellow) and RCγδ-α2A complex (orange) structures, with the exception of R92, which forms an internal salt bridge with D74γ in the RCαβγδ tetramer but interacts with D219 of α2A in the RCγδ-α2A complex.</p

Loop 2 of the α2A domain is the interaction site for the RCγ subunit.

<p>(<b>A</b>) Loop 2 of α2A is an additional binding site for rhodocetin (RC). It contains the epitope for the monoclonal antibody (mAb) JA202, which inhibits binding of RC to immobilized α2A. Bound RC was quantified by ELISA, and values were normalized to noninhibited controls. One set of inhibition curves out of 3 independent experiments with each measurement made in triplicate and the means ± SD for each data point are shown. (<b>B</b>) The α2A loop 2 sequence was replaced with the homologous sequence VGRGGRQ of integrin α1 (α2A L2^α1 mutant). The binding-irrelevant antibody JA218 was immobilized to capture wild-type (wt) α2A and α2A L2^α1. They were titrated with RC, and bound RC was quantified as in (<b>A</b>). One set of titration curves out of 4 independent experiments, each done in triplicates, is shown with the means ± SD indicated. The α2A L2^α1 mutant (light gray ■) significantly reduced affinity for RC compared to the wt (●) (<i>p</i> = 0.0013, two-tailed <i>t</i> test) (<b>C</b>) Stereo view of the α2A loop 2 sequence in contact with the RCγ contact site. The Sigma-A weighted 2Fo-Fc map is shown at 1.5σ contour level. The 2 glycine residues, G217 and G218, form the bottom of a shallow dimple, which is flanked on either side by the side chains of Y216 and D219, in addition to residue N154 of loop 1 (not shown). The indole side chain of W110γ stacks directly above this dimple and interacts with the main chain of the 2 glycine residues. (<b>D</b>) Point mutation analysis of the α2A loop 2 sequence S²¹⁴QYGGD²¹⁹. The binding activity of these mutants for RC was tested as in (<b>B</b>). Binding signals taken from at least 7 independent titration curves for each mutant were normalized to the saturation signal of wild type α2A. Means ± SEM are shown for the mutants (◆ of different colors) in comparison to wt (●) and the α2A L2^α1 mutant (light gray ■). This analysis showed that the 2 glycines at position 217 and 218 were key to the RCγδ-α2A interaction, as only mutations abrogated α2A binding. (<b>E</b>) The K_d values of the loop 2 point mutations for binding to RC as derived from (<b>D</b>). At least 7 titration curves were evaluated for each mutant. The K_d values were pairwise compared to the K_d value of the wild type α2A domain in a two-tailed Student <i>t</i> test. Significant difference (<i>p</i> < 0.02) is asterisked (*). (<b>F</b>) Modification of tryptophan residues of RCγδ with 2-nitrophenyl sulfenylchloride (NPS-Cl) showed that W110γ is required for α2A domain binding. The wells of a microtiter plate were coated with 10 μg/ml α2A domain and titrated with RCαβγδ (●), with nonmodified RCγδ (green ▲) and with RCγδ with chemically modified W110γ (W-NPS, red ▼) One representative out 3 independent titration experiments done in duplicate is shown with the means ± SD indicated. The data of plots (<b>A</b>), (<b>B</b>), (<b>D</b>), (<b>E</b>), and (<b>F</b>) are summarized in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001492#pbio.2001492.s006" target="_blank">S1 Data</a>.</p

The monoclonal antibody IIIG5 recognizes its epitope within the RCγ subunit in the RCγδ-α2A complex but not in the tetrameric RCαβγδ.

<p>(<b>A</b>) The monoclonal antibody IIIG5 recognized an epitope of the RCγ subunit, which is fully accessible in the RCγδ subunit (○), partially accessible in the RCγδ-α2A complex (light gray ▲), and completely covered in the RCαβγδ tetramer (dark gray ■). IIIG5 was immobilized on microtiter plates and titrated with RCαβγδ, RCγδ-α2A complex, or RCγδ subunit. Bound rhodocetin (RC) components were fixed and detected using rabbit RC antiserum with ELISA at 405 nm. The data presented here are taken from 3 independent experiments with each measurement done in duplicate. Means ± SD are shown. The data are summarized in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001492#pbio.2001492.s006" target="_blank">S1 Data</a>. (<b>B</b>) Molecular structure of the C-type lectin-related protein (CLRP)-fold typical of all 4 RC chains. Both the γ and δ subunits of RC are very similar (Cα-RMSD 0.8Å) and feature a core structure with 2 α-helices (H1 and H2) flanked by 2 antiparallel β-sheets (S1–S2–S6 and S3–S4–S5). The amino acid residues V94–R109 of the IIIG5 epitope of RCγ are highlighted.</p

PCR primers for cloning the α2A loop2 mutants.

<p>PCR primers for cloning the α2A loop2 mutants.</p

Isolation of the rhodocetin γδ-α2A complex on Ni Sepharose column.

<p><b>(A)</b> Elution profile of the Ni Sepharose affinity chromatography column. The RCγδ-α2A complex was formed on a Ni Sepharose column by subsequently loading the oligo His-tagged α2A domain and RCαβγδ. RCαβ and the RCγδ-α2A complex were eluted with EGTA and an imidazole gradient, respectively. (<b>B</b>) SDS-PAGE of eluate fractions (lanes “EGTA eluate” and “imidazole eluate”), in comparison to isolated control proteins (lanes “α2A domain” and “rhodocetin γδ”), under nonreducing and reducing conditions and stained with silver. Note that the trypsin-trimmed RCγδ-α2A complex showed a slightly reduced size of the α2A domain due to the proteolytic removal of the His₆-tag. The physical contact of co-eluted rhodocetin (RC) γδ and α2A domain was analytically proven by cross-linkage with 0.5 mM BS³ (lane “CL-imidazole eluate”).</p

A comparison of the RCγδ-α2A and EMS16αβ-α2A binding interfaces.

<p>(<b>A, B</b>) The C-type lectin-related protein (CLRP) folds of both homologous subunits of RCγδ (<b>A</b>) and EMS16αβ (<b>B</b>) are highly homologous with many of the residues involved in the α2A binding conserved between the 2 proteins. These residues have been mapped onto the CLRP fold and colorcoded for rhodocetin (RC) (blue and orange for the γ and δ subunits, respectively, in [<b>A</b>]) and for EMS16 (light blue and magenta for the α and β subunits, respectively, in [<b>B</b>]). The partnering residues of the α2A domain contacted by RC and EMS16 are color coded in white and yellow, respectively. The same colorcoding scheme is used throughout the figure. (<b>C, D</b>) A superposition of the key residues from RCγ/EMS16α at the loop 2 binding site (<b>C</b>) and of RCδ/EMS16β at the helix C binding site (<b>D</b>), respectively, on α2A. The contact sites are largely conserved between RCγδ/EMS16αβ and α2A, although there are a couple of notable differences. For example, L66 of RCγ contacts Y216 of α2A in addition to the N154 of loop 1 observed for the corresponding I66 of EMS16α. In addition, K59 of RCδ forms a salt bridge to D292 of α2A, whereas, in EMSβ, the corresponding K59 points towards helix C.</p