Defining the binding interface of Amyloid Precursor Protein (APP) and Contactin3 (CNTN3) by site-directed mutagenesis

Xi Peng,Philip M Smallwood,Jeremy Nathans,John Williams,Madepalli K Lakshmana

doi:10.1371/journal.pone.0219384

Xi Peng, Philip M Smallwood + Show 3 more

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https://doi.org/10.1371/journal.pone.0219384

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Abstract

The Amyloid Precursor Protein (APP) and Contactin (CNTN) families of cell-surface proteins have been intensively studied in the context of neural development and neuropsychiatric diseases. Earlier studies demonstrated both genetic and biochemical interactions between the extracellular domains of APP and CNTN3, but their precise binding interfaces were not defined. In the present study, we have used binding assays between APP-alkaline phosphatase (AP) fusion proteins and CNTN-Fc fusion proteins, together with alanine substitution mutagenesis, to show that: (i) the second Fibronectin domain (Fn(2)) in CNTN3 mediates APP binding; (ii) the copper binding domain (CuBD) in APP mediates CNTN3 binding; and (iii) the most important amino acids for APP-CNTN3 binding reside on one face of CNTN3-Fn(2) and on one face of APP-CuBD. These experiments define the regions of direct contact that mediate the binding interaction between APP and CNTN3.

Highlights

Amyloid Precursor Protein (APP) is an evolutionarily conserved vertebrate protein that is expressed in the developing and adult nervous system, as well as in many non-neural tissues [1]
For in vitro binding assays, fusion proteins consisting of Contactin extracellular domain (ECD) or their derivatives joined to the Fc domain of human IgG (“targets”) were immobilized in Protein-G coated microwells, and the microwells were incubated with soluble fusion proteins consisting of the APP ECD or its derivatives joined to the catalytic domain of human placental alkaline phosphatase (AP) (“probes”)
We focused on CNTN3 for in-depth structurefunction studies and we used the APP-E1-AP probe to assess binding to the CNTN3 derivatives

Summary

Introduction

Amyloid Precursor Protein (APP) is an evolutionarily conserved vertebrate protein that is expressed in the developing and adult nervous system, as well as in many non-neural tissues [1]. APP has a large extracellular domain (ECD), a single trans-membrane region, and a small cytoplasmic tail. The ECD of the major APP isoform (APP695) consists of the following domains (starting from the N-terminus): (i) the E1 domain, which consists of a growth factorlike domain (GFLD) and a copper-binding domain (CuBD), (ii) the extension domain (ED); (iii) the acidic domain (AcD); (iv) a central domain (E2); (v) the juxtamembrane region (JMR); (vi) the transmembrane domain; and (vii) the intracellular domain (AICD) (Fig 1A) [2]. Over the past 25 years, APP has been the object of intense interest because proteolytic cleavage in the juxtamembrane and transmembrane domains generates the beta-amyloid peptide that accumulates in patients with Alzheimer disease (AD) [3].

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