Biochemical and Structural Studies of the Interaction between ARAP1

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Biochemical and structural studies of the interaction between ARAP1 and CIN85 Qingxia LI, Wanfa YANG, Yue WANG, and WEI LIU Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00057 • Publication Date (Web): 20 Mar 2018 Downloaded from http://pubs.acs.org on March 26, 2018

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Biochemistry

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Biochemical and structural studies of the interaction between ARAP1

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and CIN85

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Qingxia Li1, Wanfa Yang2,3, Yue Wang1, Wei Liu1,2*

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1

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Shenzhen Peking University-The Hong Kong University of Science and Technology Medical Center,

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Shenzhen 518036, China

Shenzhen Key Laboratory for Neuronal Structural Biology, Biomedical Research Institute,

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2

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of Science and Technology

Division of Life Science, State Key Laboratory of Molecular Neuroscience, Hong Kong University

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3

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Study, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong

Center of Systems Biology and Human Health, School of Science and Institute for Advanced

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Running title: Biochemical and structural characterization of the ARAP1/CIN85 interaction

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*Correspondence address. Tel/Fax: +86-755-83923333-3615; E-mail:[email protected]

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ACS Paragon Plus Environment

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Abstract.

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Arf-GAP with Rho-GAP domain, ANK repeat and PH domain-containing protein 1

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(ARAP1), Cbl-interacting protein of 85 kDa (CIN85), and casitas B-lineage lymphoma

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(Cbl) play important roles in epidermal growth factor receptor (EGFR) internalization

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and recycling. In previous studies, ARAP1 was found to interact with CIN85, and their

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interaction attenuated the ubiquitination of EGFR. However, the molecular

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mechanism was still unclear. In this study, we first biochemically and structurally

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characterized the interaction between ARAP1 and CIN85, and found that the CIN85

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SH3B domain bound to the ARAP1 PXPXXRX (except P) XXR/H/K motif with high

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affinity and specificity. Based on this binding model, we further predicted other

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potential CIN85 binding partners and tested their interactions biochemically.

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Moreover, our swapping data and structure alignment analysis suggested that the

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β2-β3 loops of the CIN85 SH3 domains and the H87ARAP1/E132CIN85 interaction were

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critical for ARAP1 binding specificity. Finally, our competitive analytical gel-filtration

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chromatography and isothermal titration calorimetry (ITC) results showed that

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ARAP1 could compete with Cbl for CIN85 binding, which provides a biochemical basis

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for the regulatory roles of ARAP1 in the CIN85-mediated EGFR internalizing process.

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Key words: ARAP1, CIN85, Cbl, SH3 domain, Crystal structure

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Introduction. The ADP-ribosylation factor GTPase-activating proteins (Arf GAPs) are critical for


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Biochemistry

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the dynamic regulation of the actin cytoskeleton and membrane trafficking (1). Arf-

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GAP with Rho-GAP domain, ANK repeat and PH domain-containing proteins (ARAPs)

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belong to a special subfamily of Arf GAPs with similar domain organizations, each of

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which contains a SAM domain, a proline-rich (PR) domain, an Arf GAP domain, a

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Rho GAP domain, two ankyrin repeats, a Ras-associating domain, and five PH

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domains (Fig. 1B). The ARAPs have been shown as the point of convergence for Arf

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and Rho signaling and are involved in cytoskeleton reorganization and Golgi

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apparatus remodeling (2-7). In addition, as a member of the ARAP family, ARAP1

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has also been reported to be involved in the endocytic trafficking of membrane

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receptors such as epidermal growth factor receptor (EGFR) (8, 9) and TNF-related

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apoptosis-inducing ligand receptor 1 (TRAIL-R1) (10).


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Figure 1. Verification of the ARAP1/CIN85 interaction and mapping their minimal interaction regions A. Schematic diagram of the domain organization of CIN85. B. Domain organization of ARAP1. C. Previously identified CIN85 binding region in ARAP1 (aa 1-100) and proline-rich sequences in ARAP1 aa 1-100. D. Sequence alignment of the two matched segments in the ARAP1 1-100 region according to the previously reported binding model. E. A 1:1 stoichiometric mixture of ARAP1 1-100 and Trx-CIN85 SH3ABC proteins (100 µM each) had a significantly smaller elution volume than the individual proteins, indicating that the interaction was quite strong. F. Binding affinity between the ARAP1 1-100 and CIN85 SH3ABC tandem was measured by ITC assay. Kd = 0.86 ± 0.54 μM, N = 1.10 ± 0.02. G. Summary of the dissociation constants derived from the ITC experiments to deduce the minimal interaction region of the CIN85 SH3ABC tandem in the ARAP1 1-100 region. H. A 1:1 stoichiometric mixture of ARAP1 P2 (aa 80-90) and Trx-CIN85 SH3B proteins (100 µM each) had a significant elution volume shift compared to the individual proteins, showing that they strongly interacted with each other.


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Biochemistry

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I. Binding affinity of the interaction between ARAP1 P2 (aa 80-90) and the CIN85 SH3B domain was measured by ITC. Kd = 0.32 ± 0.03 μM, N = 0.98 ± 0.00. J. Summary of the dissociation constants derived from the ITC experiments to deduce the minimal interaction region of ARAP1 P2 (aa 80-90) in the CIN85 SH3ABC tandem. ND means not detectable.

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EGFR is a member of the erythroblastic leukemia viral oncogene homolog (ErbB)

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family of receptor tyrosine kinases (RTKs). The spatial and temporal distribution of

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EGFR is strictly controlled, and the degradation and recycling of EGFR play

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indispensable roles in this process (11). The Cbl-interacting protein of 85 kDa (CIN85)

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is an adapter protein that contains three Src homology 3 (SH3) domains, a proline-

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rich region, and a coiled-coil (Cc) domain (Fig. 1A). It has been reported that CIN85,

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through its SH3B domain, interacts with the proline-rich region of casitas B-lineage

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lymphoma (Cbl) (12, 13) and regulates the endocytosis and degradation of EGFR (14-

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16). Breaking the CIN85/Cbl interaction is sufficient to block EGF receptor

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internalization, delay receptor degradation, and enhance EGF-induced gene

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transcription (17).

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In previous studies, ARAP1 has been reported to regulate the intracellular

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trafficking of EGFR (8, 9). Down-regulation of ARAP1 promotes the internalization

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and subsequent degradation of EGFR (8). Using the yeast two-hybrid method, the 1-

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100 region of ARAP1 has been identified to interact with CIN85 and drive the exit of

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EGFR from the degradative pathway to the recycling pathway by competing with Cbl

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for the CIN85 interaction (18). However, the detailed biochemical and structural

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mechanisms remain unclear.


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In this study, we first biochemically characterized the interaction between

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ARAP1 and CIN85 using analytical gel-filtration chromatography and isothermal

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titration calorimetry (ITC). The results showed that ARAP1 aa 80-90 bound to the

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CIN85 SH3B domain with a 1:1 molar ratio, and their binding affinity was 0.32 ± 0.03

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μM. Then, X-ray crystallography was used to elucidate the molecular mechanism of

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this highly specific interaction, and a general binding model of the CIN85 SH3B

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domain was proposed. Following the binding model, other potential CIN85 binding

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partners were found, and their interactions with CIN85 were tested biochemically. In

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addition, the structure also allowed us to illustrate the molecular basis of the ARAP1

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binding specificities of the CIN85 SH3 domains. Finally, using competitive analytical

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gel-filtration chromatography and ITC assays, we determined the relationship

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between the ARAP1, CIN85, and Cbl, which showed that ARAP1 could compete with

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Cbl for CIN85 binding. This work provides a biochemical basis of the regulatory roles

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of ARAP1 in the CIN85-mediated EGFR internalizing process.

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Materials and Methods.

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Protein expression and purification

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The cDNA sequences encoding different boundaries of mouse ARAP1 (NCBI

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accession code: NM_001040111.1) and CIN85 (NCBI accession code: NM_021389.6)

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were PCR-amplified from a mouse cDNA library and cloned into a modified pET32a

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vector (19, 20). The plasmids encoding the recombinant proteins were transformed

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into E. coli BL21 (DE3). A single colony was inoculated in LB media containing 50


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Biochemistry

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µg/ml ampicillin. When A600 nM = 0.8, the cells were induced with 0.1 mM IPTG at

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16°C for 20 h. Then the collected cells were homogenized in a high-pressure

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homogenizer (Union-biotech, Shanghai), and the cell lysate was centrifuged at

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39,191 g for 30 min at 4°C. The recombinant proteins in the supernatant were

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purified by Ni2+ Sepharose™ 6 Fast Flow beads (GE Healthcare, USA), followed by a

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Superdex-200 prep grade size-exclusion (GE Healthcare) column in buffer of 50 mM

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Tris, pH 7.5, 100 mM NaCl, 1 mM EDTA, and 1 mM DTT. The ARAP1 aa 80-90 peptide

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was commercially synthesized by ChinaPeptides Co., Ltd.

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Analytical gel-filtration chromatography assay

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Analytical gel-filtration chromatography assays were carried out on an ÄKTA

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purifier system using a Superose 12 10/300 GL column (GE Healthcare). Protein

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samples (50 µM each, 100 µL) were injected into the column equilibrated with buffer

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of 50 mM Tris, pH 7.5, 100 mM NaCl, 1 mM EDTA, and 1 mM DTT.

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Isothermal titration calorimetry (ITC) assay

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ITC assays were carried out on a MicroCal ITC-200 calorimeter (Malvern, USA) at

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25°C in buffer of 50 mM Tris, pH 7.5, 100 mM NaCl, 1 mM EDTA, and 1 mM DTT. The

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protein concentrations in the syringe and the cell were around 200 μM and 20 μM,

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respectively. The titration data were analyzed using the Origin 8.0 program and fitted

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by a one-site binding model.

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Crystallography

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Crystals of the ARAP1 P2/CIN85 SH3B complex (in 50 mM Tris-HCl pH 7.5, 100

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mM NaCl, 1 mM EDTA, 1 mM DTT buffer) were obtained by the sitting drop vapor

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diffusion method at 16°C. The crystals were grown in buffer containing 0.2 M sodium

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acetate trihydrate, 0.1 M Tris hydrochloride pH 8.5, and 30% w/v polyethylene glycol

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4000. X-ray diffraction data were collected at the Shanghai Synchrotron Radiation

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Facility BL19U1 at 100K. Diffraction data were processed and scaled using HKL3000

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(21).

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The structure of the ARAP1 P2/CIN85 SH3B complex was solved by molecular

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replacement with the model of the second SH3 domain of CD2AP (PDB: 3U23) using

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PHASER (22). Further manual model building and refinement were completed

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iteratively using Coot (23) and PHENIX (24). The final model was validated by

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MolProbity (25). The final statistics of X-ray crystallographic data collection and

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model refinement are summarized in Table 1. All structure figures were prepared by

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PyMOL. The structure factors and the coordinates of the structure reported in this

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work have been deposited to Protein Data Bank (PDB) under the accession code:

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5XHZ.

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Table. 1 Statistics of X-ray crystallographic data collection and model refinement. Data Collection Datasets Space group Wavelength (Å) Unit cell parameters (Å) Resolution range (Å) No. of unique reflections Redundancy I/σ Completeness (%)

ARAP1_80-90/CIN85_SH3B P 43 0.9785 a=b=66.162, c=34.935 α=β=γ=90° 50-1.32 (1.34-1.32) 34450 (1749) 9.3 (8.7) 19.6 (2.0) 96.0 (97.4)


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Biochemistry

Rmergea (%) Structure Refinement Resolution (Å) Rcrystb /Rfreec (%) Rmsd bonds (Å)/angles (°) Average B factor(Å2)d No. of atoms Protein atoms Other molecules No. of reflections Working set Test set Ramachandran plot regions d Favored (%) Allowed (%) Outliers (%) 159 160 161 162 163 164 165 166 167

8.1 (69.8) 50-1.32 16.3/18.7 0.005/0.811 18.73 1219 269 34440 1766 98.50 1.50 0

Numbers in parentheses represent the value for the highest-resolution shell. Rmsd, root-mean-square deviation. a. Rmerge = Σ |Ii - | / ΣIi, where Ii is the intensity of measured reflection and is the mean intensity of all symmetry-related reflections. b. Rcryst =Σ||Fcalc| – |Fobs||/ΣFobs, where Fobs and Fcalc are observed and calculated structure factors. c. Rfree =ΣT||Fcalc| – |Fobs||/ΣFobs, where T is a test dataset of about 5% of the total unique reflections randomly chosen and set aside prior to refinement. d. B factors and Ramachandran plot statistics were calculated using MolProbity (25).

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Results and Discussion.

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ARAP1 aa 80-90 (P2) specifically bound to CIN85 SH3B with a 1:1 ratio

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Previously, the ARAP1 1-100 region was identified to interact with CIN85 in a

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yeast two-hybrid screen (18). We first verified the interaction by using purified

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ARAP1 1-100 and CIN85 1-333 (named as SH3ABC tandem) proteins. On an analytical

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gel-filtration column, a 1:1 mixture of ARAP1 1-100 and CIN85 SH3ABC tandem was

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eluted at a volume clearly smaller than each of the two individual proteins alone (Fig.

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1E), suggesting that the two proteins formed a stable complex. Then, ITC assays were

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used to quantify the interaction, and the result showed that the two proteins bound

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to each other with a 1:1 stoichiometry and the dissociation constant (Kd) was 0.86 ±


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0.54 μM (Fig. 1F).

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It has also been shown that the ARAP1 PXPXXRXXXR motif specifically binds to

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CIN85 SH3 domains (18). However, in the ARAP1 1-100 region, we found two

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matched segments (i.e. aa 70-80 and aa 80-90) (Fig. 1C and D). So we divided the

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ARAP1 1-100 region into four parts (SAM domain, P1 (aa 70-80), P2 (aa 80-90), aa 90-

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100) and tested their interactions with CIN85 SH3 domains, respectively (Fig. 1C). As

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a result, the SAM domain, P1, and aa 90-100 did not interact with the CIN85 SH3ABC

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tandem in either analytical gel-filtration chromatography or ITC assays (Fig. 1G and

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S1A1, A2, B1, B2, D1, and D2), while P2 bound to the CIN85 SH3ABC tandem in both

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the analytical gel-filtration chromatography and ITC assays, and their binding affinity

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was measured to be 1.10 ± 0.6 μΜ (Fig. 1G and Fig. S1C1, C2).

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In the next step, we examined which SH3 domain of CIN85 was responsible for

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binding to ARAP1 P2. In analytical gel-filtration chromatography and ITC experiments,

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CIN85 SH3AB tandem and SH3BC tandem showed strong binding to ARAP1 P2,

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indicating that their mutual part, the SH3B domain, might be the binding site of

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ARAP1 P2 (Fig. S2A1, A2, B1, and B2). To confirm this hypothesis, we further purified

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the isolated SH3 domains of the CIN85 SH3ABC tandem (namely SH3A, aa 1-77; SH3B,

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aa 96-160; and SH3C, aa 257-333). Interestingly, the P2 bound to both the SH3B

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domain and the SH3C domain with binding affinities of 0.32 ± 0.03 μM and 6.41 ±

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0.42 μM, respectively (Fig. 1H, I, J; and Fig. S2D1, D2). These results were

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contradictory to the 1:1 binding ratio in the previous ITC titration result (Fig. 1F). We

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then speculated that the linker region outside the SH3B and SH3C domains might


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Biochemistry

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also contribute to the binding. So we extended the boundary of the SH3C domain to

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linker2-SH3C (aa 161-333) (Fig. 1J and Fig. S2E1, E2) and the SH3B domain to linker1-

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SH3B-linker2 (aa 69-256) (Fig. 1J and Fig. S2F1, F2), respectively. In line with our

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expectations, we did not detect an interaction between ARAP1 P2 and linker2-SH3C,

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indicating that the SH3C domain was autoinhibited by linker2 (Fig. 1J and Fig. S2E1,

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E2). In contrast, linker1-SH3B-linker2 still bound to ARAP1 P2 with a similar strong

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binding affinity (0.32 ± 0.02 μΜ) (Fig. 1J and Fig. S2F1, F2), confirming that the SH3B

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domain was the binding site of ARAP1 P2.

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The crystal structure of the ARAP1 P2/CIN85 SH3B complex revealed the molecular basis of the binding specificity

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To understand the molecular basis of the ARAP1/CIN85 interaction, we solved

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the crystal structure of the ARAP1 P2/CIN85 SH3B protein complex at 1.32 Å

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resolution (Table 1). The overall structure showed that CIN85 SH3B is composed of

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four β-strands (β1-4) (Fig. 2A). Detailed structural analysis revealed that the PVP

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motif of the ARAP1 P2 peptide was inserted into the hydrophobic surface formed by

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F107, Y109, W135, P148, and F151 of the CIN85 SH3B domain (Fig. 2B). The side

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chains of the R86, H87, and R90 of ARAP1 formed charge-charge interactions with

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D115, E132, and E137 of CIN85, respectively. And the backbone amide hydrogen of

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H87ARAP1 and the side chain of E132CIN85 also formed a hydrogen-bond (Fig. 2C).

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Mutations of these critical amino acids greatly weakened the binding of ARAP1 P2 to

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CIN85 SH3B (Fig. 2D).


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Figure 2. Overall structure of the ARAP1 P2 (80-90)/CIN85 SH3B complex and the detailed interaction interface A. Ribbon diagram of the ARAP1 P2 (80-90)/CIN85 SH3B domain complex structure. The ARAP1 P2 (aa 80-90) and CIN85 SH3B domains are colored in magenta and green, respectively. B. Surface representation the CIN85 SH3B domain. The positively charged amino acids of the CIN85 SH3B domain are colored in blue, the negatively charged residues in red, the hydrophobic residues in yellow, and the others in white. The ARAP1 P2 (aa 80-90) peptide is displayed in a stick model and colored in magenta. The PVP motif of P2 is highlighted by the red circle. C. The residues involved in the critical charge-charge and hydrogen bonding interactions are shown in the stick model and their interactions are highlighted by red dashed lines. D. Summary of the ITC results showing that mutations of the critical residues in the interface weakened the binding. H87A, R90H, and R90K mutations did not significantly change the binding affinity. ND means not detectable.

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Since the P1 segment (PVPLPRPAPR) also fitted with the previously reported

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CIN85 SH3 domain binding model (PXPXXRXXXR) (18) (Fig. 1D), we were curious as to

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why it could not bind to the SH3B domain. We found that by forming a hydrogen

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bond with E132CIN85, the backbone hydrogen of H87ARAP1 played a vital role in


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Biochemistry

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interacting with the CIN85 SH3B domain. The H87P mutation, which lacks the

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backbone amide hydrogen, greatly decreased the ARAP1 P2/CIN85 SH3B interaction

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(Fig. 2D). Consistently, the counterpart of H87 in P1 is P77 (Fig. 1D), which perfectly

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explains why P1 could not interact with the CIN85 SH3B domain. In addition, we also

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predicted that any other amino acid except P at this position would be favorable to

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provide the backbone hydrogen to interact with E132CIN85. Consistent with our

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prediction, the H87A mutation showed a slightly weakened binding affinity compared

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to wild type P2, since it lost the charge-charge interaction between the side chains of

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H87ARAP1 and E132CIN85 (Fig. 2D). In addition, we proposed that ARAP1 R90 could also

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be replaced by H or K, which could still form charge-charge interactions or hydrogen

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bonding with E137 of CIN85. Our resulting R90H and R90K mutation data strongly

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supported this prediction (Fig. 2D). Therefore, according to our structural information

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and mutagenesis data, we generalized the binding motif of the CIN85 SH3B domain

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to be: PXPXXRX (except P) XXR/H/K.

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In the next step, we searched for more potential CIN85 interacting proteins

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based on our structure. Since H87 can provide hydrogen bonding and charge-charge

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interactions simultaneously, in order to find strong binders of the CIN85 SH3B

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domain, we used PXPXXRHXXR/H/K as the template to search the human protein

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sequence

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(https://prosite.expasy.org/scanprosite/). Five more potential CIN85 binding partners

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were found. Using the purified proteins and ITC experiments, we biochemically

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verified their interactions with the CIN85 SH3B domain (Table 2). Among them, a

database

using

the


ScanProsite

tool

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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downstream scaffold protein of EGF signaling, named Kinase suppressor of Ras 2

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(KSR2), bound to the CIN85 SH3B domain with a strong binding affinity of 1.74 ± 0.16

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μΜ. A previous simulation study has shown that KSR and CIN85 can collaborate with

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other proteins to fine-tune the sensitivity of EGFR endocytosis and ERK activation

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(26). So it is very likely that KSR is another binding partner of CIN85.

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Table 2. Prediction of the potential binding partners of the CIN85 SH3B domain. Name ARAP1＿80-90 WT KSR＿250-259 HCFC1＿28-37 MRC2＿8-17 PLD1＿140-149 DNN＿360-369

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Sequence PVPMKRH MKRHIF RHIFR IFR PSPRQRH RQRHAV RHAVR AVR PVPRPRH RPRHGH RHGHR GHR PAPWPRH WPRHLL RHLLR LLR PIPTRRH TRRHTF RHTFR TFR PAPRSRH RSRHHL RHHLK HLK

Kd (μM) 0.32±0.03 1.74±0.16 3.00±0.28 11.16±1.07 24.01±4.10 41.67±3.56

We predicted five proteins that contain a PXPXXRHXXR/H/K (highlight in yellow) motif by using the ScanProsite tool. The binding affinities between the predicted proteins and the CIN85 SH3B domain were measured by ITC.

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ARAP1 binding specificities of the three SH3 domains in CIN85

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Both the sequence and overall structure alignment showed that CIN85 SH3A,

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SH3B, and SH3C domains were highly similar (Fig. 3A and B). The three key charged

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residues (D115, E132, E137) which bound to ARAP1 P2 were also conserved in the

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SH3A, SH3B, and SH3C domains (red boxes in Fig. 3A). However, ARAP1 P2 only

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interacted with the isolated CIN85 SH3B and SH3C domains instead of the SH3A

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domain. We therefore examined the structural basis of their binding specificities.


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Biochemistry

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Figure 3. Structural basis of the CIN85 SH3 domain binding specificity A. Sequence alignment of human and mouse CIN85 SH3A, SH3B, and SH3C domains. Key residues involved in ARAP1 binding are highlighted in the red boxes. The β2-β3 linker regions are labeled with the blue box. Secondary structure diagrams of the CIN85 SH3A, SH3B, and SH3C domains derived from the ARAP1 P2 (aa 80-90)/CIN85 SH3B complex structure and the PDB database (SH3A: 2BZ8 (27), SH3C: 2YDL (28)) are shown above the sequence alignment and colored in purple, green, and orange, respectively. B. Structure alignment of the three SH3 domains of CIN85. The ARAP1 P2 (aa 80-90) peptide is colored in magenta and the CIN85 SH3A (PDB ID: 2BZ8 (27)), SH3B (PDB ID: 5XHZ), and SH3C (PDB ID: 2YDL (28)) domains are colored in purple, green, and orange, respectively. The β2-β3 linker regions are enlarged in the black box. The charge-charge and hydrogen bonding interactions between H87ARAP1 and E132SH3B are displayed by the red dashed lines. C. Summary of the binding affinities between ARAP1 P2 (aa 80-90) and the wild-type SH3B domain or chimeric CIN85 SH3 domains. “SH3A with B linker” means that the β2-β3 linker of the SH3A domain was replaced by that of the SH3B domain. Other chimeras followed this rule. D. Structure alignment of the CIN85 SH3B domain and SH3A domain (PDB ID: 2BZ8 (27)). The critical amino acids in the charge-charge and hydrogen bonding interaction interface are aligned and shown in D1, D2, and D3. The critical charge-charge and hydrogen bonding interactions between CIN85 SH3B and the ARAP1 P2 (aa 80-90) peptide are displayed by red dashed lines. E. Structure alignment of CIN85 SH3B and SH3C domain (PDB ID: 2YDL (28)). The critical amino


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

302 303 304

acids in the charge-charge and hydrogen bonding interaction interface are aligned and shown in E1, E2, and E3. The critical charge-charge and hydrogen bonding interactions between CIN85 SH3B and the ARAP1 P2 (aa 80-90) peptide are displayed by red dashed lines.

305

By carefully analyzing the sequence alignment, we found that the β2-β3 loop

306

was the most variable region between the three SH3 domains of CIN85 (blue box in

307

Fig. 3A and enlarged region in Fig. 3B). To test our hypothesis, we created chimeric

308

SH3 domains by swapping the β2-β3 loops between SH3B and SH3A/SH3C domains.

309

ITC results showed that the SH3A domain and the SH3C domain carrying the β2-β3

310

loop from the SH3B domain had significantly enhanced binding affinities to ARAP1

311

(Fig. 3C). In contrast, weakened interactions were detected between ARAP1 and the

312

SH3B domain carrying the β2-β3 loop either from the SH3A or SH3C domains (Fig.

313

3C).

314

In addition, in the structure alignment, we found that E38SH3A, D16 SH3A, and

315

D308SH3C, D284SH3C showed similar orientations compared to E137SH3B, D115SH3B (Fig.

316

3D1, D2, E1, and E2). However, the orientation of D33SH3A (the counterpart of

317

E132SH3B in the β2-β3 loop) was not favorable for its interaction with H87ARAP1 (Fig.

318

3D3). In contrast to D33SH3A, D303SH3C showed only a slightly different orientation

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from E132SH3B (Fig. 3E3). Thus, the structure alignment provided cues as to why the

320

SH3A domain could not bind to ARAP1, and why the isolated CIN85 SH3C domain

321

could still bind to ARAP1 P2 with moderate affinity (6.47 ± 0.42 μΜ) (Fig. S2D2).

322

Taken together, our swapping data and structural alignment suggested that the

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β2-β3 loops and the H87ARAP1/E132CIN85 interaction play important roles in the ARAP1

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binding specificities of the three SH3 domains in CIN85.

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Biochemistry

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ARAP1 competed with Cbl for association with the CIN85 SH3B domain

327

Previously, aa 825-835 (PERPPKPFPRR) of Cbl was reported to bind to the CIN85

328

SH3B domain (13). We confirmed their binding by analytical gel-filtration

329

chromatography and ITC experiments (Kd = 16.26 ± 2.08 μM) (Fig. 4A and C). Then we

330

performed competition assays to test the relationship between ARAP1, CIN85, and

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Cbl. Analytical gel-filtration chromatography data showed that by adding excessive

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ARAP1 P2 peptide (molar ratio = 1.2:1) into the protein mixture, the Trx-Cbl peptide

333

did not form a complex with the CIN85 SH3B domain anymore (Fig. 4B). Consistently,

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the ITC results showed that Trx-Cbl did not interact with a protein mixture of Trx-

335

ARAP1 P2 (aa 80-90) and the CIN85 SH3B domain (molar ratio = 1.2:1) (Fig. 4D). In

336

contrast, Trx-ARAP1 P2 (aa 80-90) strongly interacted with a protein mixture of Trx-

337

Cbl and the CIN85 SH3B domain (molar ratio = 1.2:1) (Fig. 4E), and the dissociation

338

constant (Kd = 0.29 ± 0.04 μM) was similar to that of the ARAP1 P2/CIN85 SH3B

339

interaction. Therefore, our results showed that the CIN85 SH3B domain preferred to

340

bind to Trx-ARAP1 P2 instead of the Trx-Cbl peptide, which provides direct

341

biochemical evidence that the presence of ARAP1 P2 breaks the complex formation

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between Cbl and the CIN85 SH3B domain.


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

343 344 345 346 347 348 349 350 351 352 353 354 355 356 357

Figure 4. ARAP1 P2 (aa 80-90) peptide competes with Cbl to interact with the CIN85 SH3B domain A. A 1:1 stoichiometric mixture of Trx-Cbl peptide (PERPPKPFPRR) and CIN85 SH3B domain (100 µM each) had a significantly smaller elution volume than the individual proteins, indicating that they interacted with each other in the analytical gel-filtration column. B. Competitive analytical gel-filtration experiments showed that by pre-mixing with slightly excessive (120 µM) synthetic ARAP1 P2 (aa 80-90) peptide (RPVPMKRHIFR), the Trx-CIN85 SH3B domain (100 µM) did not interact with the Trx-Cbl peptide (100 µM). C. ITC assay was used to measure the binding affinity between Trx-Cbl and the CIN85 SH3B domain. Kd = 16.26 ± 2.0 2μM. D. In ITC experiments, Trx-Cbl did not interact with the protein mixture of Trx-ARAP1 P2 (aa 80-90) and the CIN85 SH3B domain (molar ratio = 1.2:1). ND means not detectable. E. In ITC experiments, Trx-ARAP1 P2 (aa 80-90) interacted with the protein mixture of Trx-Cbl and the CIN85 SH3B domain (molar ratio = 1.2:1). Kd = 0.29 ± 0.04 μM.

358 359

Conclusions

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Previously, the ARAP1 1-100 region was found to interact with the CIN85 SH3

361

domains, and a “PXPXXRXXXR” binding model was provided (18). Based on this


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Biochemistry

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model, both the ARAP1 70-80 and 80-90 regions can be predicted to bind to CIN85.

363

However, in our study, we only found 1:1 binding between ARAP1 1-100 and CIN85

364

SH3ABC. By re-mapping the binding boundary and solving the high-resolution crystal

365

structure, we elucidated the molecular basis of the ARAP1/CIN85 interaction and

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generalized a new motif for the strong binding to the CIN85 SH3B domain: PXPXXRX

367

(except P) XXR/H/K. This new binding model explains well why ARAP1 70-80 could

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not bind to the CIN85 SH3B domain and elucidates the molecular mechanism of the

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highly specific interaction between the ARAP1 aa 80-90 and CIN85 SH3B domains. In

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addition, by using this model, we found five more potential binding partners of

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CIN85 (Table 2). Currently, we have biochemically verified their interactions with the

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CIN85 SH3B domain. Further investigations are needed to explore the biological

373

importance of these predicted interactions.

374

The three SH3 domains of CIN85 are highly similar in protein sequence and

375

overall structure. However, only the SH3B domain of CIN85 could interact with

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ARAP1 aa 80-90. By performing swapping studies and structural alignment analysis,

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we found that the β2-β3 loops and the H87ARAP1/E132CIN85 interaction played

378

important roles in the ARAP1 binding specificities of the three SH3 domains in CIN85.

379

Accidentally, when mapping the minimal ARAP1 P2 binding region in the CIN85

380

SH3ABC tandem, we found that P2 could bind to the isolated SH3C domain (aa 257-

381

333) but not with the longer version (linker2-SH3C, aa 161-333). This finding

382

indicated that the SH3C domain might be auto-inhibited by the long linker between

383

the SH3B and SH3C domains (Fig. 1A). The molecular mechanism of this auto-


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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inhibition will be an interesting subject for further exploration.

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Cbl, CIN85, and ARAP1 play important roles in the endosomal sorting and

386

degradation of EGFR (13, 14, 17, 27, 29). In this study, we biochemically explored the

387

relationship between ARAP1, CIN85, and Cbl. Competitive experimental results

388

showed that ARAP1 aa 80-90 peptide could break down the interaction between Cbl

389

and CIN85. In future work, we will test whether adding the ARAP1 aa 80-90 peptide

390

can significantly affect the surface expression levels of EGFR in EGFR high-expressing

391

cancer cells and animal models.

392 393 394 395

Accession Numbers The structure factors and coordinates of the CIN85 SH3B/ARAP1 80-90 structure have been deposited in the PDB under the accession code PDB: 5XHZ.

396 397

Supplementary Materials

398

The supplementary materials include three figures. Figure S1 and S2 support the

399

results of Fig 1G and J, showing the detailed analytical gel-filtration chromatography

400

and ITC results for mapping the shortest CIN85 SH3ABC tandem binding region in

401

ARAP1, and the shortest ARAP1 P2 binding region in the CIN85 SH3ABC tandem.

402

Figure S3 shows the control experiments of Figure 4.

403 404 405

Acknowledgments We thank the Shanghai Synchrotron Radiation Facility (SSRF) BL19U1 for X-ray


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Biochemistry

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beam times. This work was supported by the National Natural Science Foundation of

407

China (No.31400647 and 31670765), the National Basic Research Program of China

408

(973 Program) (2014CB910204), the National Key Research and Development

409

Program (2016YFA0501900), the Natural Science Foundation of Guangdong Province

410

(2016A030312016), and Shenzhen Basic Research Grants (JCYJ20160229153100269,

411

JCYJ20160427185712266 and JCYJ20170411090807530).

412


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For Table of Contents Use Only.

414 415

Biochemical and structural studies of the interaction between ARAP1

416

and CIN85

417

Qingxia Li1, Wanfa Yang2,3, Yue Wang1, Wei Liu1,2*

418 419 420 421


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Biochemistry

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References:

428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464

1

Randazzo PA, Hirsch DS. Arf GAPs: multifunctional proteins that regulate membrane traffic and

actin remodelling. CELL SIGNAL 2004, 16: 401-413 2

I ST, Nie Z, Stewart A, Najdovska M, Hall NE, He H, Randazzo PA, et al.. ARAP3 is transiently

tyrosine phosphorylated in cells attaching to fibronectin and inhibits cell spreading in a RhoGAPdependent manner. J CELL SCI 2004, 117: 6071-6084 3

Miura K, Jacques KM, Stauffer S, Kubosaki A, Zhu K, Hirsch DS, Resau J, et al.. ARAP1: a

point of convergence for Arf and Rho signaling. MOL CELL 2002, 9: 109-119 4

Krugmann S, Anderson KE, Ridley SH, Risso N, McGregor A, Coadwell J, Davidson K, et al..

Identification of ARAP3, a novel PI3K effector regulating both Arf and Rho GTPases, by selective capture on phosphoinositide affinity matrices. MOL CELL 2002, 9: 95-108 5

Chen PW, Jian X, Yoon HY, Randazzo PA. ARAP2 signals through Arf6 and Rac1 to control

focal adhesion morphology. J BIOL CHEM 2013, 288: 5849-5860 6

Hasegawa J, Tsujita K, Takenawa T, Itoh T. ARAP1 regulates the ring size of circular dorsal

ruffles through Arf1 and Arf5. MOL BIOL CELL 2012, 23: 2481-2489 7

Yoon HY, Miura K, Cuthbert EJ, Davis KK, Ahvazi B, Casanova JE, Randazzo PA. ARAP2

effects on the actin cytoskeleton are dependent on Arf6-specific GTPase-activating-protein activity and binding to RhoA-GTP. J CELL SCI 2006, 119: 4650-4666 8

Yoon H, Lee J, Randazzo PA. ARAP1 Regulates Endocytosis of EGFR. TRAFFIC 2008, 9: 2236-

2252 9

Daniele T, Di Tullio G, Santoro M, Turacchio G, De Matteis MA. ARAP1 regulates EGF receptor

trafficking and signalling. TRAFFIC 2008, 9: 2221-2235 10

Simova S, Klima M, Cermak L, Sourkova V, Andera L. Arf and Rho GAP adapter protein

ARAP1 participates in the mobilization of TRAIL-R1/DR4 to the plasma membrane. APOPTOSIS 2008, 13: 423-436 11

Miaczynska M. Effects of membrane trafficking on signaling by receptor tyrosine kinases. Cold

Spring Harb Perspect Biol 2013, 5: a9035 12

Haglund K, Schmidt MH, Wong ES, Guy GR, Dikic I. Sprouty2 acts at the Cbl/CIN85 interface

to inhibit epidermal growth factor receptor downregulation. EMBO REP 2005, 6: 635-641 13

Kowanetz K, Szymkiewicz I, Haglund K, Kowanetz M, Husnjak K, Taylor JD, Soubeyran P, et

al.. Identification of a novel proline-arginine motif involved in CIN85-dependent clustering of Cbl and down-regulation of epidermal growth factor receptors. J BIOL CHEM 2003, 278: 39735-39746 14

Ronning SB, Pedersen NM, Madshus IH, Stang E. CIN85 regulates ubiquitination and

degradative endosomal sorting of the EGF receptor. EXP CELL RES 2011, 317: 1804-1816 15

Take H, Watanabe S, Takeda K, Yu ZX, Iwata N, Kajigaya S. Cloning and characterization of a

novel adaptor protein, CIN85, that interacts with c-Cbl. Biochem Biophys Res Commun 2000, 268: 321-328


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497

16

Dikic I. CIN85/CMS family of adaptor molecules. FEBS LETT 2002, 529: 110-115

17

Soubeyran P, Kowanetz K, Szymkiewicz I, Langdon WY, Dikic I. Cbl-CIN85-endophilin

complex mediates ligand-induced downregulation of EGF receptors. NATURE 2002, 416: 183-187 18

Yoon HY, Kales SC, Luo R, Lipkowitz S, Randazzo PA. ARAP1 association with CIN85 affects

epidermal growth factor receptor endocytic trafficking. BIOL CELL 2011, 103: 171-184 19

Liu W, Wen W, Wei Z, Yu J, Ye F, Liu CH, Hardie RC, et al.. The INAD scaffold is a dynamic,

redox-regulated modulator of signaling in the Drosophila eye. CELL 2011, 145: 1088-1101 20

Wang Y, Li Q, Zheng Y, Li G, Liu W. Systematic biochemical characterization of the SAM

domains in Eph receptor family from Mus Musculus. Biochem Biophys Res Commun 2016, 473: 1281-1287 21

Otwinowski Z, Minor W. Processing of X-ray diffraction data collected in oscillation mode.

Methods Enzymol 1997, 276: 307-326 22

McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC, Read RJ. Phaser

crystallographic software. J APPL CRYSTALLOGR 2007, 40: 658-674 23

Emsley P, Lohkamp B, Scott WG, Cowtan K. Features and development of Coot. Acta

Crystallogr D Biol Crystallogr 2010, 66: 486-501 24

Adams PD, Afonine PV, Bunkoczi G, Chen VB, Davis IW, Echols N, Headd JJ, et al.. PHENIX:

a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr 2010, 66: 213-221 25

Chen VB, Arendall WR, Headd JJ, Keedy DA, Immormino RM, Kapral GJ, Murray LW, et al..

MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr D Biol Crystallogr 2010, 66: 12-21 26

Huang L, Pan CQ, Li B, Tucker-Kellogg L, Tidor B, Chen Y, Low BC. Simulating EGFR-ERK

signaling control by scaffold proteins KSR and MP1 reveals differential ligand-sensitivity co-regulated by Cbl-CIN85 and endophilin. PLOS ONE 2011, 6: e22933 27

Jozic D, Cárdenes N, Deribe YL, Moncalián G, Hoeller D, Groemping Y, Dikic I, et al.. Cbl

promotes clustering of endocytic adaptor proteins. NAT STRUCT MOL BIOL 2005, 12: 972-979 28

Ortega, R. J., Casares, S., Ringkjobing, J. M., Cardenes, N., Bravo, J., Blackledge, M., Azuaga, A.

I., and van Nuland, N. A. (2013) Distinct ubiquitin binding modes exhibited by SH3 domains: molecular determinants and functional implications, PLOS ONE 8, e73018. 29

Haglund K, Shimokawa N, Szymkiewicz I, Dikic I. Cbl-directed monoubiquitination of CIN85 is

involved in regulation of ligand-induced degradation of EGF receptors. Proc Natl Acad Sci U S A 2002, 99: 12191-12196

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Biochemical and Structural Studies of the Interaction between ARAP1

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