Novel Zinc-binding Site in the E2 Domain Regulates Amyloid Precursor-like Protein 1 (APLP1) Oligomerization

Magnus C Mayer,Daniela Kaden,Linda Schauenburg,Mark A Hancock,Philipp Voigt,Dirk Roeser,Christian Barucker,Manuel E Than,Michael Schaefer,Gerhard Multhaup

doi:10.1074/jbc.m114.570382

Abstract

The amyloid precursor protein (APP) and the APP-like proteins 1 and 2 (APLP1 and APLP2) are a family of multidomain transmembrane proteins possessing homo- and heterotypic contact sites in their ectodomains. We previously reported that divalent metal ions dictate the conformation of the extracellular APP E2 domain (Dahms, S. O., Könnig, I., Roeser, D., Gührs, K.-H., Mayer, M. C., Kaden, D., Multhaup, G., and Than, M. E. (2012) J. Mol. Biol. 416, 438-452), but unresolved is the nature and functional importance of metal ion binding to APLP1 and APLP2. We found here that zinc ions bound to APP and APLP1 E2 domains and mediated their oligomerization, whereas the APLP2 E2 domain interacted more weakly with zinc possessing a less surface-exposed zinc-binding site, and stayed monomeric. Copper ions bound to E2 domains of all three proteins. Fluorescence resonance energy transfer (FRET) analyses examined the effect of metal ion binding to APP and APLPs in the cellular context in real time. Zinc ions specifically induced APP and APLP1 oligomerization and forced APLP1 into multimeric clusters at the plasma membrane consistent with zinc concentrations in the blood and brain. The observed effects were mediated by a novel zinc-binding site within the APLP1 E2 domain as APLP1 deletion mutants revealed. Based upon its cellular localization and its dominant response to zinc ions, APLP1 is mainly affected by extracellular zinc among the APP family proteins. We conclude that zinc binding and APP/APLP oligomerization are intimately linked, and we propose that this represents a novel mechanism for regulating APP/APLP protein function at the molecular level.

Highlights

Oligomeric complexes of amyloid precursor protein (APP), APLP1, and APLP2 contribute to synapse formation and structure
APP E2 and APLP1 E2 bound to the zinc-charged IMAC columns and were eluted, the E2 domain of APLP2 was not retained by immobilized zinc and was detected in the flow-through and the wash fractions (Fig. 1C)
To cross-validate these findings, we performed Surface Plasmon Resonance (SPR) analyses in the reverse orientation; the E2 domains of APP, APLP1, and APLP2 were amine-coupled to SPR sensor chips, and zinc or copper chloride were titrated over all immobilized surfaces (Fig. 1D)

Summary

Introduction

Oligomeric complexes of APP, APLP1, and APLP2 contribute to synapse formation and structure. Results: Zinc binding to the E2 domain of APP and APLPs promotes their oligomerization in the cell, most notably with APLP1. We found here that zinc ions bound to APP and APLP1 E2 domains and mediated their oligomerization, whereas the APLP2 E2 domain interacted more weakly with zinc possessing a less surface-exposed zinc-binding site, and stayed monomeric. Fluorescence resonance energy transfer (FRET) analyses examined the effect of metal ion binding to APP and APLPs in the cellular context in real time. The observed effects were mediated by a novel zinc-binding site within the APLP1 E2 domain as APLP1 deletion mutants revealed. Based upon its cellular localization and its dominant response to zinc ions, APLP1 is mainly affected by extracellular zinc among the APP family proteins. We conclude that zinc binding and APP/APLP oligomerization are intimately linked, and we propose that this represents a

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Conclusion