Competitive Interactions of Ligands and Macromolecular Crowders with Maltose Binding Protein

Andrew C Miklos,Huan-Xiang Zhou,Matthew Sumpter,Paul D Riggs

doi:10.1371/journal.pone.0074969

Abstract

Cellular signaling involves a cascade of recognition events occurring in a complex environment with high concentrations of proteins, polysaccharides, and other macromolecules. The influence of macromolecular crowders on protein binding affinity through hard-core repulsion is well studied, and possible contributions of protein-crowder soft attraction have been implicated recently. Here we present direct evidence for weak association of maltose binding protein (MBP) with a polysaccharide crowder Ficoll, and that this association effectively competes with the binding of the natural ligand, maltose. Titration data over wide ranges of maltose and Ficoll concentrations fit well with a three-state competitive binding model. Broadening of MBP 1H15N TROSY spectra by the addition of Ficoll indicates weak protein-crowder association, and subsequent recovery of sharp NMR peaks upon addition of maltose indicates that the interactions of the crowder and the ligand with MBP are competitive. We hypothesize that, in the Escherichia coli periplasm, the competitive interactions of polysaccharides and maltose with MBP could allow MBP to shuttle between the peptidoglycan attached to the outer membrane and the ATP-binding cassette transporter in the inner membrane.

Highlights

Cellular signaling processes involve a cascade of recognition events that lead from an external stimulus to a cellular response
The decrease in maltose binding affinity of maltose binding protein (MBP) by Ficoll is in distinct contrast to an increase in ATP binding affinity of adenylate kinase, attributed to hard-core repulsion, by the small molecule osmolyte, trimethyl amine Noxide [22]
The present study presented direct evidence for weak association between MBP and the polysaccharide crowder Ficoll, and showed that this weak association is competitive with the binding of the natural ligand maltose

Summary

Introduction

Cellular signaling processes involve a cascade of recognition events that lead from an external stimulus to a cellular response. Each recognition event is the binding of a protein with a partner that can range from a small-molecule ligand to a macromolecular complex. The binding occurs in a complex environment containing high concentrations of proteins, polysaccharides, and other macromolecules [1]. It is recognized that the crowded cellular environment can significantly influence the equilibria of protein binding [2], [3]. The use of macromolecular crowding agents in in vitro studies allows for quantitative characterization of protein binding processes in conditions mimicking the cellular environment, leading to a more complete understanding of recognition and signaling processes inside the cell. We report the effects of a polysaccharide crowder on the binding affinity of maltose binding protein (MBP) for its natural ligand maltose

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Conclusion