Disclosing the Interaction of Carbonic Anhydrase IX with Cullin

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DISCLOSING THE INTERACTION OF CARBONIC ANHYDRASE IX WITH CULLIN-ASSOCIATED NEDD8-DISSOCIATED PROTEIN 1 BY MOLECULAR MODELING AND INTEGRATED BINDING MEASUREMENTS Martina Buonanno, Emma Langella, Nicola Zambrano, Mariangela Succoio, Emanuele Sasso, Vincenzo Alterio, Anna Di Fiore, Annamaria Sandomenico, Claudiu T Supuran, Andrea Scaloni, Simona Maria Monti, and Giuseppina De Simone ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00055 • Publication Date (Web): 07 Apr 2017 Downloaded from http://pubs.acs.org on April 11, 2017

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ACS Chemical Biology

DISCLOSING THE INTERACTION OF CARBONIC ANHYDRASE IX WITH CULLINASSOCIATED NEDD8-DISSOCIATED PROTEIN 1 BY MOLECULAR MODELING AND INTEGRATED BINDING MEASUREMENTS Martina Buonanno,†§ Emma Langella,†§ Nicola Zambrano,± Mariangela Succoio,± Emanuele Sasso,± Vincenzo Alterio,† Anna Di Fiore,† Annamaria Sandomenico,† Claudiu T. Supuran,≠ Andrea Scaloni,‡ Simona Maria Monti,†* and Giuseppina De Simone†* †

Istituto di Biostrutture e Bioimagini-CNR, Naples, Italy.

±

Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università degli Studi di Napoli

Federico II and CEINGE Biotecnologie Avanzate SCaRL, Naples, Italy ≠

Neurofarba Department, Section of Pharmaceutical and Nutriceutical Sciences, Università degli

Studi di Firenze, Sesto Fiorentino, Florence, Italy. ‡

Proteomics and Mass Spectrometry Laboratory, ISPAAM, CNR, Naples, Italy.

§

These authors contributed equally to this work

*Corresponding authors. Phone: +39-081-2534583, e-mail: [email protected] (SMM); Phone: +39-081-2534579, e-mail: [email protected] (GDS).

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ABSTRACT: Human Carbonic Anhydrase (hCA) IX is a membrane-associated member of the CA enzyme family, involved in solid tumor acidification. This enzyme is a marker of tumor hypoxia and a prognostic factor for several human cancers. In a recent paper we showed that CA IX interacts with cullin-associated NEDD8-dissociated protein 1 (CAND1), a nuclear protein involved in gene transcription and assembly of SCF ubiquitin ligase complexes. A functional role for this interaction was also identified, since lower CA IX levels were observed in cells with decreased CAND1 expression via shRNA-mediated interference. In this paper, we describe the identification of the structural determinants responsible of the CA IX/CAND1 interaction by means of a multidisciplinary approach, consisting of binding assay measurements, molecular docking and sitedirected mutagenesis. These data open a novel scenario in the design of anticancer drugs targeting CA IX. Indeed, the knowledge of the structural determinants responsible for the CAND1/CA IX interaction provides the molecular basis to design molecules able to destabilize it. Due to the proposed function of CAND1 in stabilizing CA IX, these molecules could represent an efficient tool to lower the amount of CA IX in hypoxic cancer cells, thus limiting its action in survival and metastatic spread of tumors.


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Human Carbonic Anhydrases (hCAs, EC 4.2.1.1) are ubiquitous zinc-enzymes, which catalyze the reversible hydration of carbon dioxide to bicarbonate ion and proton (CO2 + H2O ⇆ HCO3−+ H+).1 To date fifteen isoforms have been described, which differ for cellular localization, tissue distribution and catalytic properties.2 Human CA IX is one of the most interesting members of the CA family; indeed, it has been recognized as a tumor-associated protein due to its limited presence in normal tissues and high overexpression in a large number of solid tumors.3 Notably, the presence of CA IX in tumors has been related to i) a hypoxic phenotype mediated by the transcription factor HIF-14, ii) survival and metastatic spread of tumors2 and iii) poor responsiveness to the classical radio- and chemo-therapy.5-7 Due to these features, CA IX has been the object of recent research studies aimed at determining its biochemical and structural features for drug design purposes4 and at clarifying its role in tumor biology.6 hCA IX is a multi-domain protein consisting of an intracytoplasmic (IC) tail, a transmembrane (TM) segment and an extracellular region, which in turn comprises an N-terminal proteoglycan-like (PG) domain and a CA catalytic domain.4,

8

Biochemical studies revealed that this enzyme is

dimeric and has a very high catalytic activity,9 whereas structural studies identified some peculiar characteristics of the protein, such as the dimeric interface and the localization of the PG domain on the border of the active site.10 Finally, functional studies allowed to define in detail the role of each CA IX domain in the survival, migration and invasion of tumor cells.4 In particular, the CA catalytic domain was identified as the main player in growth and survival of tumor cells. The reaction catalyzed by the extracellular catalytic domain is, indeed, fundamental to the maintenance of the neutral intracellular pH necessary for cell survival.11-13 The PG domain is involved in cell-cell adhesion,14 in mediating tumor cell interactions with microenvironment15 and in supporting CAdomain mediated catalysis at the acidic pH values of the solid tumors, where the enzyme is generally overexpressed.10,

16

Finally, the IC tail has a number of different functions. It contains

three phosphorylation sites, namely Thr443, Ser448 and Tyr449 (numbering refers to the full-length protein including signal peptide); the first two modulate CA IX catalytic activity,17 while the latter is involved in EGFR-induced signal transduction to PI3/Akt kinase pathway.18 Moreover, the integrity of such tail is fundamental to ensure the correct functioning of hCA IX; it has indeed been shown that mutations in the juxtamembrane region cause the suppression of both cell adhesion and extracellular acidification capability.19 Given the well-established role of hCA IX in hypoxic tumors,3, 4, 6 in recent years a lot of work has been dedicated to the development of inhibitors able to modulate its catalytic activity, thus interfering with tumor survival and progression. In this context, plenty of molecules have been investigated, some of which giving very interesting results.1 However, the majority of such



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molecules lacks of selectivity with respect to the other human isoforms, thus limiting their potential use as drugs.1 For this reason, alternative strategies able to inhibit CA IX functions in tumors were considered by our group. In particular, we have undertaken an interactomic study aimed at identifying CA IX cellular partners, which support its functions in tumor cells.20 Surprisingly, most of the CA IX-binding proteins identified in this study belongs to the nuclear transport machinery, opening a novel scenario on the possible roles of this multifaceted protein and positioning it among the cell-surface signal transducers, which undergo nuclear translocation. In agreement with these data, CA IX was shown to interact with cullin-associated NEDD8-dissociated protein 1 (CAND1), a nuclear protein involved in gene transcription and assembly of SCF ubiquitin ligase complexes.20 A functional role for the interaction of CA IX with CAND1 was also identified. Indeed, lower CA IX levels were observed in cells with decreased CAND1 expression via shRNA-mediated interference. Thus, it was suggested that this interaction could be necessary for CA IX stabilization.20 The minimal portion of CA IX protein necessary for the interaction with CAND1 was also identified.20 Such region corresponds to the sequence Leu418-Ala459 of CA IX, phosphorylated on either Thr443 or Tyr449 and comprises both the TM segment and the IC tail.20 In this paper, we report the identification of the molecular determinants responsible of the CA IX/CAND1 interaction by means of a multidisciplinary approach. In particular, we firstly verified by means of fluorescence and SPR experiments the occurrence of a direct CA IX/CAND1 interaction. By using a docking approach, we then modeled the CA IX/CAND1 complex and such model was finally validated by means of site-directed mutagenesis and pull-down experiments. As a first step of our investigation, we decided to verify whether the previously reported20 interaction between CA IX and CAND1 was direct. To this aim, the CA IX sequence previously identified as responsible of the interaction with CAND1, namely Leu418-Ala459,20 phosphorylated at Thr443, was synthesized (peptide P1 in Figure 1A) and CAND1 protein was expressed in E. coli and purified as described in SI. Light scattering and circular dichroism experiments demonstrated that the recombinant CAND1, in agreement with structural data previously reported,21 was a monodisperse monomeric protein with an hydrodynamic radius (Rh) of 5.1 ± 0.9 nm (Figures S1A and S1B) and a 70% content of alpha helical secondary structure (Figure S1C). Two different techniques were used to get information on CAND1/P1 binding affinity. Firstly, intrinsic fluorescence binding assays were set up, taking advance of the absence of tryptophan in the sequence of peptide P1. To avoid the fluorescence contribution of tyrosines, excitation was set at 295 nm and emission of fluorescence monitored at 350 nm. KD and stoichiometry were calculated by direct fitting of the data using a non-linear least squares fitting algorithm.22 From this analysis, a KD value of 1.7 ± 0.5 µM and a stoichiometric ratio of 1:1 were obtained (Figures 1B and 1C).


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SPR analysis was used to further support direct interaction data. However, due to the low solubility of the peptide P1 in SPR aqueous buffer, a new peptide (hereafter indicated as P1-TAG) was used. This peptide, which consisted of P1 plus a small tag at the C-terminus to improve the solubility (see SI for details), was already available, since it had been used for previously reported pull-down assays.20 CAND1 was immobilized on the sensor chip and P1-TAG used as analyte. In agreement with fluorescence assays, a direct binding was evident, which corresponded to a saturation curve associated with a KD value of 2.7 ± 0.3 µM (Figures 1D and 1E); accordingly, the sequence containing only the TAG showed no binding (Figure S2). Altogether, these data proved for the first time that the CA IX C-terminal region interacts directly with CAND1 with a weak binding affinity, but did not provide any information regarding the molecular basis responsible for the binding. Since this kind of information is crucial for the development of therapeutic protein–protein-interaction inhibitors, structural studies on the CAND1/P1 adduct were undertaken. In particular, since and all crystallization attempts performed on the complex were unsuccessful, a docking approach was carried out. For docking studies the structure of CAND1, extrapolated from the crystallographic complex CAND1-Cul1-Roc1 (PDB code: 1U6G)21 and refined by adding the missing loop regions with MODLOOP web server (see SI for details),23 was used. This structure consists of 27 HEAT repeats, which form a highly sinuous super-helical structure (Figure 2A).21 In agreement with what reported by Goldemberg and coworkers,21 the overall three-dimensional arrangement of CAND1 resembles a U-shaped belt with two arms twisted in opposite directions. Three arches have been identified in the structure: the first one forming the base of the belt and the two terminal ones corresponding to the curved arms (Figure 2A).21 No structural information was instead available on peptide P1, whose 3D model was obtained using the QUARK ab initio server (Figure 2B),24 which proved to be effective also for modeling transmembrane protein sequences.25 The obtained model has a very high confidence of prediction (TM-score = 0.5019) and shows a helix-loop-helix fold, with the two helices, hereafter indicated as helix 1 and helix 2, encompassing residues 419-441 and 451-457, respectively. The U-shaped three-dimensional arrangement is stabilized by van der Waals contacts between residues located at the interface of the two helices, as well as by the high solvent accessible surface area of charged residues belonging to the helices. Interestingly, this model is in agreement with the predictions made by HHPred26 and PSIPRED27 servers, and is also consistent with CD spectrum that indicated for P1 a high tendency to assume a helical conformation (see Figures S3 and S4 of SI). Three different docking web servers were used to build the CAND1/P1 complex model: ClusPro,28 GRAMM-X29 and SwarmDock.30 The top-10 scored clusters of structures, obtained by each of the



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three servers, are shown in Figure S5. Most of these solutions are located in the region between the N- and C-terminal arches of CAND1. It is worth noting that the three docking approaches provided only one common binding mode (Figure 2C). To better characterize this “consensus” binding mode, the three corresponding docking solutions were further analyzed by Firedock server,31 allowing to identify the lowest-energy solution, hereafter indicated as consensus model (Figure 2C). Analysis of the consensus model suggests that helix 1 of P1 plays a key role into the interaction with CAND1. In particular, the hydrophobic N-terminal region of this helix is involved in several stabilizing van der Waals interactions with the protein (Figure 2D), while its C-terminal region, which is characterized by the presence of several positively charged arginine residues, interacts with CAND1 mainly through electrostatic interactions (Figure 2E). Interestingly, even if the P1 phosphorylated residue in position 443 (mimicked by Glu443) (see SI for details) does not interact directly with CAND1, it plays a key role into the binding, opportunely orienting Arg441 toward Glu289 of CAND1 through electrostatic interactions (Figure 2E). A large number of hydrogen bonds further contributes to stabilize the binding (Table S1). Support to these results comes from the observation that the position of P1 in the model is compatible with the presence of the large CA IX extracellular domain, as schematically shown in Figure 3. Indeed, the position occupied in the model by the N-terminal end of P1 allows the positioning of the remaining CA IX protein both inside or outside the super-helical structure of CAND1. To obtain an experimental validation of the obtained CAND1/P1 model, several mutants of the CA IX full-length were designed, prepared and subsequently used in pull-down assays. Mutants were aimed at disrupting either the van der Waals or the electrostatic interactions responsible for the interaction with CAND1. In detail, residues Leu423 and Thr427, which are involved in the formation of a strong hydrophobic core (Figure 2D), were mutated in arginine. In the same way, residues Arg436, Arg440 and Arg441, which are involved in strong electrostatic interactions (Figure 2E), were mutated in alanine or glutamic acid (Figure 4A). The wild-type CA IX and its mutants (MUT1-MUT5) were therefore expressed in HEK-293 cells with a C-terminal Strep-tag for capture of their complexes on Strep-Tactin beads. The pull-down analysis showed that each of the selected mutations results in the loss of interaction with native CAND1 (Figure 4B); as expected, the wild-type protein keeps its ability to bind to CAND1.20 Thus, these data provide a full validation of the proposed model. In conclusion, data here reported, exploiting different binding approaches integrated with molecular modeling and site directed mutagenesis, provided evidence that the interaction between CAND1 and CA IX is direct and in the micromolar range, and identify the structural determinants


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responsible for this interaction, thus opening a novel scenario in the design of anticancer drugs targeting CA IX. Indeed, the knowledge of the structural determinants responsible for the CAND1/CA IX interaction provides the molecular basis to design molecules able to destabilize it. Due to the proposed function of CAND1 in stabilizing CA IX,20 these molecules could represent an efficient tool to lower the amount of CA IX in hypoxic cancer cells, thus limiting its action in survival and metastatic spread of tumors. Studies are currently underway in our labs to test this hypothesis.

METHODS Details of experimental procedures are provided in the Supporting Information.

ASSOCIATED CONTENT Supporting Information The Supporting Information, containing Methods and supporting figures, is available free of charge on the ACS Publications website at DOI:XXX.

NOTES The authors declare no competing financial interest.

ACKNOWLEDGEMENTS This work was supported by a grant from CNR-DSB ProgettoBandiera "InterOmics”, by Associazione Culturale DiSciMuS RFC (Progetto “Biologia dei tumori ipossici”) and by MIURPRIN to NZ. The Authors thank C. Marciano for the help in mutagenesis of CA IX cDNAs.



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FIGURE LEGENDS

Figure 1.

CAND1/P1 binding assays. (A) Sequence of the P1 peptide, corresponding to the CA IX C- terminal sequence Leu418-Ala459. The phosphorylated Thr443 is starred. The TM region is underlined, while the IC tail is highlighted in black. (B) Intrinsic fluorescence spectrum of CAND1 in response to increasing concentrations of peptide P1. (C) Binding curve of CAND1/P1 as monitored by changes in intrinsic fluorescence of CAND1. All data are the mean from 3 different assays (errors in the range of ± 5 % of the reported values). (D) SPR data for the binding of P1-TAG to immobilized

CAND1.

Sensorgrams

were

recorded

at

increasing

peptide

concentrations: 0.05 µM (1), 0.5 µM (2), 1 µM (3), 2 µM (4), 4 µM (5). (E) Plot of RUmax from each binding as a function of P1-TAG concentration,32 data are fit by non-linear regression algorithm (GraphPad Prism 4). Figure 2.

CAND1/P1 molecular docking studies. (A) CAND1 overall fold. (B) Structural model of peptide P1 as obtained by QUARK ab initio server. Residue side chains are displayed as sticks. Positively and negatively charged residues are colored in red and blue, respectively. (C) Common binding mode for CAND1/P1 complex among the docking solutions obtained by ClusPro (magenta), Gramm-X (green) and SwarmDock (blue) programs. The final consensus model, corresponding to the lowest energy solution according to FireDock energetic calculations, was the ClusPro solution; global binding energies computed for the ClusPro, SwarmDock and Gramm-X models were -100, -87 and -45, respectively. (D) Main van der Waals interactions involved in the stabilization of the CAND1/P1 consensus model. (E) Main polar interactions involved in the stabilization of the CAND1/P1 consensus model.

Figure 3.

Schematic drawing of the putative positioning of the CA IX extracellular domain, according to the CAND1/P1 consensus docking model. The CA IX catalytic domain (green cartoon) is bound to the N-terminal end of P1 (magenta cartoon) through a 23residue linker (orange dotted line). Some possible orientations of CA IX catalytic domain are schematically represented by dotted black circles.

Figure 4.

Pull-down assays. (A) Designed CA IX mutants. (B) Coprecipitation of Strep-tagged, wild-type and mutants of CA IX proteins (MUT1-5) with CAND1. HEK-293 cells were transfected with plasmids driving the expression of Strep-tagged wild-type or mutants of CA IX proteins, or with an empty vector (Mock), as indicated. For pulldown analysis, cell lysates were challenged to Strep-tactin resin and probed with the


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indicated antibodies in western blot experiments, to detect the endogenous CAND1 (upper panel). The protein lysate from mock-transfected cells was used as a migration control for CAND1. Overexpression of CAIX-WT or MUT1, MUT2, MUT3, MUT4 and MUT5 proteins was checked by Western blotting with an antibody against CA IX (lower panel).



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CA IX/P1 binding assays. (A) Sequence of the P1 peptide. The phosphorylated Thr443 is starred. The TM region is underlined, while the IC tail is highlighted in black. (B) Intrinsic fluorescence spectrum of CAND1 in response to increasing concentrations of peptide P1. (C) Binding curve of CAND1/P1 as monitored by changes in intrinsic fluorescence of CAND1. All data are the mean from 3 different assays (errors in the range of ± 5 % of the reported values). (D) SPR data for the binding of P1-TAG to immobilized CAND1. Sensorgrams were recorded at increasing peptide concentrations: 0.05 µM (1), 0.5 µM (2), 1 µM (3), 2 µM (4), 4 µM (5). (E) Plot of RUmax from each binding as a function of P1-TAG concentration; data are fit by non-linear regression algorithm (GraphPad Prism 4). 96x77mm (600 x 600 DPI)


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CAND1/P1 molecular docking studies. (A) CAND1 overall fold. (B) Structural model of peptide P1 as obtained by QUARK ab initio server. Residue side chains are displayed as sticks. Positively and negatively charged residues are colored in red and blue, respectively. (C) Common binding mode for CAND1/P1 complex among the docking solutions obtained by ClusPro (magenta), Gramm-X (green) and SwarmDock (blue) programs. The final consensus model, corresponding to the lowest energy solution according to FireDock energetic calculations, was the ClusPro solution; global binding energies computed for the ClusPro, SwarmDock and Gramm-X models were -100, -87 and -45, respectively. (D) Main van der Waals interactions involved in the stabilization of the CAND1/P1 consensus model. (E) Main polar interactions involved in the stabilization of the CAND1/P1 consensus model. 128x103mm (300 x 300 DPI)



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Schematic drawing of the putative positioning of the CA IX extracellular domain, according to the CAND1/P1 consensus docking model. The CA IX catalytic domain (green cartoon) is bound to the N-terminal end of P1 (magenta cartoon) through a 23-residue linker (orange dotted line). Some possible orientations of CA IX catalytic domain are schematically represented by dotted black circles. 119x180mm (300 x 300 DPI)


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Pull-down assays. (A) Designed CA IX mutants. (B) Coprecipitation of Strep-tagged, wild-type and mutants of CA IX proteins (MUT1-5) with CAND1. HEK-293 cells were transfected with plasmids driving the expression of Strep-tagged wild-type or mutants of CA IX proteins, or with an empty vector (Mock), as indicated. For pull-down analysis, cell lysates were challenged to Strep-tactin resin and probed with the indicated antibodies in western blot experiments, to detect the endogenous CAND1 (upper panel). The protein lysate from mock-transfected cells was used as a migration control for CAND1. Overexpression of CAIX-WT or MUT1, MUT2, MUT3, MUT4 and MUT5 proteins was checked by Western blotting with an antibody against CA IX (lower panel). 71x64mm (600 x 600 DPI)



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TOC graphic 30x11mm (300 x 300 DPI)


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Disclosing the Interaction of Carbonic Anhydrase IX with Cullin

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